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Genomics of secondary metabolite production

by Pseudomonas spp
Article in Natural Product Reports November 2009
DOI: 10.1039/b817075b Source: PubMed
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Genomics of secondary metabolite production by Pseudomonas spp

Harald Gross*a and Joyce E. Loper*b
Received 16th July 2009
First published as an Advance Article on the web 1st October 2009
DOI: 10.1039/b817075b
Covering: up to June 2009
Pseudomonas is a diverse genus of Gammaproteobacteria with more than 60 species exhibiting varied
life styles in a wide range of environments, including soil, water, plant surfaces, and animals. They are
well known for their ubiquity in the natural world, capacity to utilize a striking variety of organic
compounds as energy sources, resistance to a wide range of medically- and agriculturally-important
antimicrobial compounds, and production of a remarkable array of secondary metabolites. Here, we
provide an overview of the astonishing metabolic capacity of the Pseudomonads, summarize the
knowledge of secondary metabolite biosynthesis in this group of organisms, and highlight the
biological significance of these compounds to the diverse life styles exhibited by Pseudomonas spp. A
consistent theme throughout this discussion is the central role of genomics in natural product discovery,
characterization of metabolic gene clusters and patterns of their inheritance, and illuminaton of new
aspects of Pseudomonas biology.
Introduction: An overview of Pseudomonas spp. and
their genomes
1.1
Pseudomonas aeruginosa
1.2
Pseudomonas entomophila
1.3
Pseudomonas fluorescens
1.4
Pseudomonas syringae
2
Biosynthetic enzymes, genes and gene clusters of
Pseudomonads
2.1
Non-ribosomally produced peptides and other amino
acid-derived compounds
2.1.1 Pyoverdines
2.1.2 Pyochelin
2.1.3 Pseudomonine
2.1.4 Paerucumarin and pseudoverdine
2.1.5 Lipopeptides
2.1.6 Safracin
2.1.7 Tabtoxin
2.1.8 Phaseolotoxin
2.1.9 Pyrrolnitrin
2.1.10 Indole-3-acetic acid (IAA)
2.2
Polyketides and fatty acid derived compounds
2.2.1 Mupirocin (pseudomonic acid A)
2.2.2 2,4-Diacetylphloroglucinol (DAPG)
2.2.3 2,5-Dialkylresorcinols
2.3
Hybrid NRPS-PKS compounds
2.3.1 Syringolin
2.3.2 Pyoluteorin
2.3.3 Coronatine
a
Institute for Pharmaceutical Biology, Nussallee 6, 53115 Bonn, Germany.
E-mail: harald.gross@uni-bonn.de; Fax: +49 (0)228/73-3250; Tel: + 49
(0)228/73-2676
b
U.S. Department of Agriculture, Agricultural Research Service, 3420
N.W. Orchard Ave., Corvallis, OR, 97330, USA. E-mail: loperj@science.
oregonstate.edu; Fax: +1 541 738-4025; Tel: +1 541 738-4057
This article is part of a themed issue on genomics.
1408 | Nat. Prod. Rep., 2009, 26, 14081446
2.3.4
2.4
2.4.1
2.4.2
2.4.3
3
3.1
3.2
3.3
3.4
4
5
6
7
Pederin
Other compounds
Phenazines
Quinolones
Hydrogen cyanide
New compounds discovered by genome-guided
strategies in Pseudomonas spp.
Orfamides
Rhizoxins
Enantio-pyochelin
Syringafactins AF
Secondary metabolite gene clusters in the flexible
genome of Pseudomonas spp.
Concluding remarks
Acknowledgements
References
1 Introduction: An overview of Pseudomonas spp.

and their genomes
Pseudomonas spp. entered the genomics era less than ten years
ago when the genome of P. aeruginosa PA01 became available,1
but genomics has since accelerated research in virtually all
aspects of Pseudomonas biology, including secondary metabolism. To date, the complete genomes of at least 22 strains representing seven species have been sequenced, and many more
genomic sequences of Pseudomonas spp. will soon become
available due, in part, to the application of new rapid and
affordable sequencing technologies.2,3 This discussion will focus
on new insights into secondary metabolism of the genus that has
been provided by genomics.
The remarkable ecological and metabolic diversity of Pseudomonas spp. is reflected in the genomes of these bacteria.
Genome size varies from 4.6 to 7.1 Mbases with 4237 to 6396
This journal is The Royal Society of Chemistry 2009
predicted genes, and GC contents range from 57.8 to 66.6%

(Table 1). Genomic diversity is particularly apparent from the
relatively small size of the core genome that is shared among
Pseudomonas species. For example, Mavrodi et al.4 determined
that only 2468 genes are conserved among strains representing
four Pseudomonas species (Fig. 1). Therefore, the percentage of

the proteome shared among the four species varies from 40% for
P. fluorescens Pf-5 (with 6137 predicted protein-encoding genes)
to 46% for P. putida KT2440 (with 5,350 predicted proteinencoding genes). The core genome typically includes
Table 1 Selected strains of Pseudomonas spp. and their genomes

Strain
Source of isolation
Size [Mb]
No. of genes
GC content
Ref.
P. aeruginosa 2192
Chronically-infected cystic fibrosis

patient in Boston,
Massachusetts, USA
Manchester epidemic strain
isolated from cystic fibrosis
patient, UK
Liverpool epidemic strain
isolated from cystic fibrosis
patient, UK
Wound, from culture collection at
University of California at
Berkeley, USA
Burn wound, Melbourne, Australia
Fruit or fruit fly, Guadeloupe
Soil, Texas, USA
Soil, Massachusetts, USA
Leaf of sugar beet, Oxford, UK
Soil, Japan. Cured strain lacking
the TOL plasmid
Endophytic strain isolated from
poplar, Washington, USA
Rice paddy soils in China.
Nitrogen-fixing strain used as
a crop inoculant in China
Rice, China
Bean, Ethiopia
Leaf of bean, Wisconsin, USA
Tomato, Guernsey, UK
Tomato
6.9
6191
66.2
6.2
5578
66.5
6.6
6026
66.3
6.5
5905
66.3
411
6.3
5.9
7.1
6.4
6.7
6.2
5571
5293
6257
5857
6009
5481
66.6
64.2
63.3
60.5
60.5
61.5
1
13
29
40
40
412
5.8
5292
61.4
413
4.6
4237
63.9
414
6.7
6.1
6.1
6.5
6.1
4450
5436
5245
5721
5771
57.8
57.9
59.2
58.3
58.6
2
48
49
47
3
P. aeruginosa C3719
P. aeruginosa LESB58
P. aeruginosa PA14
P. aeruginosa PAO1
P. entomophila L48
P. fluorescens Pf-5
P. fluorescens PfO-1
P. fluorescens SBW25
P. putida KT2440
P. putida W619
P. stutzeri A1501
P. syringae pv. oryzae 1_6
P. syringae pv. phaseolica 1448A
P. syringae pv. syringae B728a
P. syringae pv. tomato DC3000
P. syringae pv. tomato T1
Harald Gross studied pharmacy

at the Friedrich-Alexander
University, Erlangen-Nuremberg, Germany. In 2000, he
started his PhD studies with
Professor K
onig at the University of Bonn, in which he focused
on the chemical analysis of
bioactive metabolites from
marine
invertebrates
with
special emphasis on the elucidation of their absolute configuration.
Harry
then
did
Harald Gross
postdoctoral research, funded by
the German Research Foundation (DFG), in the lab of Professor William Gerwick first at
Oregon State University and later at the Scripps Institution of
Oceanography, San Diego, CA, focusing on algal, cyanobacterial
and bacterial metabolites and their biosynthesis. In 2006, he
returned to the University of Bonn and started his Habilitation. His
current research is devoted to the mining of microbial genomes,
particularly of Pseudomonads and related bacteria for new natural
products.
Joyce E. Loper studied biology

and plant pathology at the
University of California (UC),
receiving her BS and MS
degrees from UC Davis and her
PhD from UC Berkeley in 1983.
She has been a Research Plant
Pathologist in the US Department of Agricultures Agricultural Research Service since
1985. She also holds the position
of Professor in the Department
of Botany and Plant Pathology
Joyce E: Loper
at Oregon State University. Her
research group studies the
ecology, genetics and genomics of plant-associated Pseudomonas
spp., with a focus on secondary metabolite production by strains of
Pseudomonas fluorescens that suppress plant diseases.
Nat. Prod. Rep., 2009, 26, 14081446 | 1409
housekeeping genes and RNAs that are essential for the survival
of the organism, but most genes in individual Pseudomonas sp.
are either species-specific or shared by a subset of the species.
These genes comprise a flexible Pseudomonas genome, which
reflects adaptation of individual strains to a specific life style. The
flexible genome is thought to evolve through horizontal genetic
exchange mediated by a spectrum of mobile elements and sites
for recombination, which enable the acquisition and deletion of
genetic information. It is well known that horizontal gene
transfer (HGT) mediated by conjugation and site-directed
recombination play an important role in the evolution of
bacteria,5 and the core and flexible genomes of Pseudomonas spp.
exhibit a mosaic pattern of conserved and lineage-specific genes
as the remnants of these processes.69 Therefore, one can view
a genomic sequence as a snapshot in the evolution of individual
strains, as they acquire and discard genomic fragments in the
process of developing a genetic repertoire customized to their
ecological niche. Genes conferring secondary metabolite
biosynthesis are one component of this genetic repertoire, which
mediate the bacteriums interactions with plant or animal hosts,
its microbial co-inhabitants, or predators in the environment.
Secondary metabolites play important roles in the diverse life
styles of Pseudomonas spp., functioning in nutrient acquisition,
virulence, and defense against competitors and predators confronted in natural habitats. In this section, we provide a brief
summary of the biology and genomics of P. aeruginosa,
P. entomophila, P. fluorescens and P. syringae. Taken together,
these four species produce an enormous spectrum of secondary
metabolites synthesized from gene clusters distributed between
the core and flexible genomes of each species.
1.1 Pseudomonas aeruginosa
Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is of increasing importance as an opportunistic human
pathogen. The bacterium causes respiratory infections, including
acute pneumonia, in patients breathing through mechanical
respirators or those with neutropenia or immunosuppression.10
P. aeruginosa can also cause persistent respiratory infections in
cystic fibrosis (CF) patients. As life expectancy of these patients
has increased with enhanced treatment, chronic pulmonary
infections, often caused by P. aeruginosa, have emerged as
a leading cause of morbidity and mortality.11 Over the course of
these infections, genetic changes in the bacterial population can
occur, reflected in phenotypes such as the overproduction of
extracellular alginate polysaccharides and loss of motility.12
Fig. 1 Venn diagrams showing the number of proteins shared or unique
among A) four Pseudomonas species, B) five strains of P. aeruginosa, and
C) three strains of P. fluorescens. Protein sequences of four Pseudomonas
genomes were compared, and bidirectional best matches that met specified criteria were scored as shared proteins.4,6 A and C) Criteria were: a
p value less than or equal to 105, identity of 35% or more, and match
lengths of at least 50% of the length of both query and subject sequence.
The number of shared genes is smaller than that reported earlier29 because
a more stringent standard was adopted in order to clearly identify shared
orthologues rather than homologues. B) Criteria included an alignment
over at least 60% of the query and subject sequence, and 90% of the
5021 genes in core P. aeruginosa having more than 98% identity.6
Diagrams were adapted from Mavrodi et al.4 and Mathee et al.6
1410 | Nat. Prod. Rep., 2009, 26, 14081446
Factors contributing to virulence of P. aeruginosa are a subject of

intense study, and the bacterium is known to infect numerous
model host species in experimental settings, including the model
plant Arabidopsis thaliana, the nematode Caenorhabditis elegans,
the insects Drosophila melanogaster and Galleria mellonella, and
the amoeba Dictyostelium discoideum.12
Comparisons of the genomic sequences of P. aeruginosa have
revealed a core genome that is highly conserved within the
species 5021 genes having at least 70% sequence identity are
conserved among the genomes of five strains of P. aeruginosa
that were analysed by Mathee et al.6 (Fig. 1). 90% of these
conserved genes share at least 98% sequence identity. This
analysis conforms well to a study by Spencer et al.,8 who estimated the nucleotide divergence in the core genomes of three
other strains of P. aeruginosa at less than 0.5% (one in 200
nucleotides). To place this in perspective, this level of sequence
diversity is almost an order of magnitude lower than that found
in comparisons between E. coli O157 and K-12.8 Along with this
high level of conservation, each strain of P. aeruginosa has
unique components, which appear to arise primarily from
insertions of gene sets acquired by horizontal gene transfer
(HGT) as well as deletions. These insertions and deletions take
place at a limited number of sites in the genome of P. aeruginosa,
representing hot spots for genetic recombination.6 Sets of genes
inserted in these sites compose the majority of the flexible
genome of each strain, and contribute to the species capacity to
occupy diverse environmental niches characterizing the species.
As detailed below, secondary metabolism is represented in both
the core and flexible genomes of P. aeruginosa.
1.2 Pseudomonas entomophila
Pseudomonas entomophila L48, which was isolated from fruit flies
or decaying fruits on the island of Guadeloupe, was first
described for its capacity to induce a systemic immune response
in larvae of the fruit fly Drosophila melanogaster.13,14 It is among
the few isolates of Gram-negative bacteria that are pathogenic to
insects in both the larval and adult stages of development.13
P. entomophila L48 produces hydrogen cyanide15 and an extracellular protease, which contributes to insect pathogenesis.16
Insect virulence is thought to be conferred by multiple factors,16
and the genomic sequence has identified numerous secondary
metabolite gene clusters that could also contribute to the insecticidal activity of P. entomophila.13
1.3 Pseudomonas fluorescens
Pseudomonas fluorescens is widely distributed on surfaces of
virtually all plant tissues, and is also found in many other natural
habitats, including soil, organic matter, and water. The species is
extremely heterogeneous, and strains are differentiated into five
biovars defined by phenotypic and genotypic variation.17 Certain
plant-associated strains have the capacity to promote plant
growth or function as biological control agents against plant
disease, and some have been developed as commercial products
for management of plant diseases in agricultural settings.18 While
the pathogenic potential of P. fluorescens has been considered
minimal, the species is found in clinical settings and concerns
have been raised regarding the potential of some strains to
function as opportunistic human pathogens.19 Certain members

of P. fluorescens also exhibit toxicity against insects20 and
nematodes21 in laboratory experiments.
The secondary metabolism of P. fluorescens is of particular
interest due its role in biological control of plant disease,22
achieved by applying bacteria to plants or through cropping
practices that increase populations of specific antibiotic-producing
strains of the species.2325 For example, soils that suppress take-all
disease of wheat develop from agronomic practices that promote
phenazine- or 2,4-diacetylphloroglucinol-producing populations
of P. fluorescens in the wheat rhizosphere.23 Wheat planted into
these disease suppressive soils does not develop the take-all disease
even when the fungal pathogen Gaeumannomyces graminis var.
tritici is present. Antibiotic production by rhizosphere-inhabiting
strains of Pseudomonas spp. can also influence the fitness of the
producing strain26 especially in the presence of bacterial predators21 in soil or other natural substrates. Due to the importance of
antibiotics in biological control and microbial ecology, there has
been great interest in determining the structure, biological activity,
and regulation of secondary metabolites produced by
P. fluorescens.22,27,28
The first genomic sequence for the species was obtained from
P. fluorescens strain Pf-5,29 which was isolated from the soil in
College Station, Texas, USA.30 Pf-5 is a well characterized biological control agent3035 that produces a wide spectrum of
secondary metabolites. Prior to genomic sequencing, Pf-5 was
known to produce pyrrolnitrin,30 pyoluteorin,31 2,4-diacetylphloroglucinol,36 hydrogen cyanide,32 a pyoverdine siderophore
of unknown structure, and a second siderophore related to
pyochelin. As described below, the spectrum of known secondary
metabolites produced by Pf-5 has been greatly expanded since
the genomic sequence of the strain became available.3739
The genomic sequences of two additional strains of P. fluorescens, SBW25 and Pf0-1, were published recently.40 Strain
SBW25 was isolated from the leaf surface of a sugar beet plant
grown in Oxfordshire, UK,41 and has become an important
model organism for studies on the ecology and evolution of
environmental bacteria. Pf0-1 was isolated from soil in Sherborn,
Massachusetts, USA,42 and has been the subject of studies
evaluating the molecular basis of bacterial attachment to soil
particles and seeds, environmental fitness, and bacterial gene
expression in natural habitats.
Comparisons between the three sequenced strains of P. fluorescens indicate a high degree of genomic diversity at the species
level 3688 genes are conserved between the three strains, representing 60% to 64% of the genome of each strain (Fig. 1).
Although the percentage of the proteome shared among strains
of P. fluorescens is substantially greater than that shared among
the Pseudomonas species, there is a large fraction of the proteome
(1146 to 1574 genes) unique to each strain of P. fluorescens. This
genomic diversity was also noted by Silby et al.40 who provide
several lines of evidence indicating that P. fluorescens, as
currently defined, is extremely heterogeneous and is likely to be
a species complex.
1.4 Pseudomonas syringae
P. syringae is commonly found in water and on plant surfaces in
natural and agricultural environments.43,44 The bacterium is
Nat. Prod. Rep., 2009, 26, 14081446 | 1411
disseminated by water or in association with a plant host, and

typically establishes epiphytic populations on the plant surface
prior to infection of the host. Within the species, there are at least
50 pathovars, which cause a wide range of plant diseases exhibiting diverse symptoms, such as leaf or fruit lesions, cankers,
blasts, and galls. P. syringae produces several toxins (coronatine,
phaseolotoxin, syringomycin, and tabtoxin45) as well as phytohormones that can function as virulence factors and contribute
to disease symptomatology.
The diverse pathovars of P. syringae are distributed among five
clades,46 and genomic sequences for strains representing four of
the five clades are now available. P. syringae pv. tomato DC3000 is
a pathogen of Brassica spp., Arabidopsis thaliana, and tomato.47
The related strain P. syringae pv. tomato T1 causes bacterial speck
of tomato. P. syringae pv. phaseolicola 1448A causes halo blight
disease of bean, characterized by a yellow halo surrounding leaf
lesions that are associated with the pathogens production of
phaseolotoxin, a chlorosis-inducing phytotoxin.48 P. syringae pv.
syringae B728a49 causes brown spot of bean and also induces frost
injury due to the presence of ice nucleation active proteins in the
bacterial outermembrane.50 P. syringae pv. oryzae 1_6, a pathogen of rice,2 represents a fourth clade of P. syringae.
Comparison of four P. syringae genomes defined a core
genome of 3594 genes, with 78107 variable regions representing
2536% of the proteomes of P. syringae pv. tomato DC3000,
P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae
B728a.9 Ninety-seven genes, representing 2.2% of the genome of
P. syringae pv. oryzae 1_6, are unique to that strain.2 Gene
clusters for the biosynthesis of the phytotoxins coronatine,
phaseolotoxin, and syringomycin are present in the flexible
genomes of specific strains. These clusters are typically present in
blocks of genes that appear to integrate into specific sites in the
P. syringae core genome.9 Therefore, in P. syringae, as for other
species of Pseudomonas discussed herein, the flexible genome
confers traits contributing to the specialized life style of individual strains.
2 Biosynthetic enzymes, genes and gene clusters of

Pseudomonads
Pseudomonas species produce an enormous array of natural
products representing varied metabolic origins and exhibiting
wide-ranging biological activities.45,5155 Although the biosynthetic pathways for the Pseudomonas metabolites have much in
common with those of the well-studied Actinomycetes, they also
exhibit unusual features. Consequently, the study of secondary
metabolism in Pseudomonas spp. has led to the discovery of novel
biosynthetic mechanisms, which are highlighted in the discussion
that follows. Table 2 summarizes the atypical features of Pseudomonas secondary metabolism, highlighting the ingenuity of
this genus regarding natural product biosynthesis. In this section,
we provide an overview of selected secondary metabolism
biosynthetic pathways and gene clusters in Pseudomonas spp.,
omitting those compounds identified through genomic mining
approaches, which are featured in section 3. The characterized
gene clusters and enzymes are presented according to their
metabolic origin. It is noteworthy that neither terpenoid
metabolites nor terpene synthases have been found to date in
Pseudomonas species.
1412 | Nat. Prod. Rep., 2009, 26, 14081446
2.1 Non-ribosomally produced peptides and other amino

acid-derived compounds
Pseudomonas spp. produce a spectrum of structurally-diverse
peptides, and many of these are synthesized by large, multifunctional proteins called non-ribosomal peptide synthases
(NRPSs).45,55 These multienzyme complexes synthesize peptides
in a modular fashion, with each module catalyzing the addition
and modification of a specific amino acid.56,57 The elongating
peptide moves from one module to the next in an assembly-line
manner, and the number and sequence of amino acids in the
peptide product typically correspond to the number and order of
the modules in the NRPS, a phenomenon known as the colinearity rule. Each elongation module has a minimal set of three
domains. An adenylation domain (A) selects a specific amino
acid, activates it as an amino acyl adenylate, and transfers it to
the peptidyl carrier protein (PCP), which tethers the elongating
peptide chain to the module via a thioester bond. The condensation (C) domain catalyzes peptide bond formation between the
amino acid present on the PCP of the same module and the
peptidyl intermediate bound to the PCP of the preceding module.
Because signature sequences within the A domain are specific to
a given amino acid, the amino acid composition of the peptide
product can be predicted bioinformatically.58 In addition to the
core domains (A, C, and PCP), other domains may be present,
including epimerization (E), methyltransferase (MT), and cyclization (Cy) domains, which are responsible for conversion of
regular L-configured amino acids into the corresponding D-form,
N-methylation, and formation of oxazole rings from Ser or
thiazole rings from Cys, respectively. The latter ring systems can
be further reduced by reductase (R) domains, leading to the
corresponding oxazolidine and thiazolidine ring systems,
respectively. A loading module, typically having only A and PCP
domains, and a termination module, containing a thioesterase
(TE) domain required for release and optional cyclization of the
peptide product, typically complete the NRPS assembly line. For
more detailed information on NRPSs, we refer the reader to
several excellent reviews.56,57,5961
2.1.1 Pyoverdines. All fluorescent Pseudomonas spp. secrete
the yellow-green fluorescent, high-affinity strain-specific siderophores termed pyoverdines (also called pyoverdins or pseudobactins).62 They enable the acquisition of Fe(III) ions from the
environment63 and can serve as intracellular signalling
compounds controlling gene expression.64,65 With the exception
of Azotobacter vinelandii, pyoverdines are exclusively produced
by Pseudomonads. Chemically, pyoverdines are composed of
a dihydroxy-quinoline chromophore attached to a variable
peptide chain that comprises 612 amino acids, and a dicarboxylic acid or its amide. Typically, a given strain produces two to five
pyoverdines, differing only in the small dicarboxylic acid side
chain. Genes (pvd) responsible for the biosynthesis of pyoverdines are present in a single locus in some Pseudomonads,
such as P. syringae, or up to five different loci in the genome of
other species, such as P. fluorescens (Fig. 2).66
The pyoverdine biosynthesis gene cluster is best described in
P. aeruginosa PAO167 (Fig. 3). Both the chromophore68 and the
peptide chain69 are synthesized by NRPSs. From incorporation
experiments, 2,4-diaminobutyrate70 and tyrosine71 have been
Table 2 Features of secondary metabolism biosynthetic gene clusters identified from Pseudomonads
Compound
Type
Producer
Unusual features
Pyochelin
Pseudomonine
NRPS
NRPS
Unusual location of the E domain

Nonenzymatic rearrangement during biosynthesis
Paerucumarin
Pseudoverdin
Syringomycin
NRPS
P.
P.
P.
P.
P.
NRPS
P. syringae pv. syringae
Syringopeptin
NRPS
Arthrofactin
Massetolides
NRPS
NRPS
Putisolvin
Orfamides
Syringofactins
NRPS
NRPS
NRPS
Safracin
NRPS
Pseudomonas sp. MIS38

P. fluorescens SS101
Pseudomonas sp. MF-30
P. putida PCL1445
P. fluorescens Pf-5
P. syringae pv. tomato
P. fluorescens A2-2
Tabtoxin
AA-derived
Pyrrolnitrin
AA-derived
Indoleacetic acid
Mupirocin
DAPG
2,5-Dialkylresorcinols
Syringolin
AA-derived
PKS
PKS
PKS
NRPS-PKS
Pyoluteorin
NRPS-PKS
Coronatine
NRPS-PKS
P. aeruginosa
P. fluorescens Pf-5
Pseudomonas sp. M18
P. syringae
Pederin
NRPS-PKS
Pseudomonas sp.
Rhizoxins
NRPS-PKS
P. fluorescens Pf-5
Phenazines
Quinolone
HCN
P. chlororaphis
P. fluorescens
P. aeruginosa
P. aeruginosa
Pseudomonas spp.
aeruginosa
fluorescens AH2
fluorescens WCS374
entomophila L48
aeruginosa
Isonitrile synthase, nonenzymatic conversion

Non-colinear, split modules, combined C/E domains,
non-heme FeII halogenase
Largest linear NRPS system described for
prokaryotes, combined C/E domains, tandem TE
domains
Combined C/E domains, tandem TE domains
Disconnected NRPS cluster, combined C/E domains,
tandem TE domains
Tandem TE domains
Tandem TE domains
Tandem TE domains, linear lipopeptide
P. syringae pv. tabaci,

pv. coronafaciens and pv. garcae
P. fluorescens
P. aurantiaca BL915
Pseudomonas sp.
Pseudomonas spp.
P. fluorescens NCIMB 10586
P. fluorescens
P. aurantiaca BL915
identified as precursors for biosynthesis of the chromophore via

the NRPS PvdL, which consists of four modules. The deduced
amino acid sequence of the first domain in module 1 is similar to
acyl-CoA ligases (ACL) domains, but the function of this
domain has not yet been elucidated. A domains of the remaining
NPRS modules activate and condense the amino acids glutamate, tyrosine and 2,4-diamino-butyric acid. No TE is present at
the C-terminus of PvdL, but a gene encoding a TE is clustered
with pvdL. The NRPSs PvdI, PvdJ, and PvdD, synthesize the
peptide chain of the pyoverdine, extending the tripeptide product
of PvdL by eight amino acids. The peptide chain comprises
L- and D-amino acids, some of which are unusual, such as
N5-hydroxyornithine,
N5-formyl-N5-hydroxyornithine
and
Iterative use of an NRPS module, reductive chain

release
Off-branching product from the lysine-pathway
First example of the FADH2-dependent halogenases
More than one pathway leading to the same molecule
Trans-AT PKS, ACP doublets/triplets
Type III PKS chalcone synthase
Modified fatty acid
Modified amino acids, unknown mechanism for the
incorporation of valine via a urea moiety
Inactive KR and DH domains, lack of loading
modules, iteratively working FADH2-dependent
halogenase, unusual location of TE domain
Unusual starter unit, L-allo-Ile specific A domain,
endotrig cyclization, cryptic halogenation with
a new type of halogenase, the non-heme-Fe2+ketoglutarate dependent halogenase
Split cluster, trans-AT PKS, unusual loading module,
tandem DH domains, MT domains for the
generation of geminal methyl groups, FADdependent monooxygenase involved in the
production of the unusual oxidized single carbon
terminus of pederin, inactive modules (pedH)
Trans-AT PKS, inactive KS/ACP modules, tandem
ACP modules, polyketide-chain branching by an
enzymatic Michael addition
Alkaloid derived from anthranilic acid
Alkaloid derived from anthranilic acid
Toxin derived oxidatively from glycine
hydroxyaspartate.72 During the assembly or upon completion of

the peptide backbone, the peptide chain can be further modified,
particularly to generate the above-mentioned unusual amino
acids. The products of pvdA (ornithine hydroxylase)73 and pvdF
(transformylase)74 perform the respective hydroxylation and
formylation at N-5 of ornithine. For completion of the chromophore, a second ring closure and several hydroxylation and
oxidation steps are required. Several mechanistic proposals for
these steps have been proposed.75 Stintzi et al. suggested that the
products of a separate cluster of four genes (pvcABCD) perform
the corresponding reactions.76,77 At the time of discovery, the
proteins encoded by this operon showed similarity to proteins
involved in spore wall maturation (PvcA), oxygenases (PvcB),
Nat. Prod. Rep., 2009, 26, 14081446 | 1413
Fig. 2 Varied organization of pyoverdine clusters in Pseudomonas spp. Homologous genes are shown in the same color, and genes are not drawn to
scale. Revised from Swingle et al.415 and Ravel and Cornelis.66
Fig. 3 Biosynthesis model of pyoverdines in P. aeruginosa PAO1. ACL: acyl-CoA ligase, Dab 2,4-diaminobutyric acid.
hydroxylases (PvcC) and proteins of the cytochrome c protein

family (PvcD). Chemically, it can be envisioned that PvcB creates
a Da,b double bond in the tyrosine moiety, while PvcC and PvcD
form a catechol structure at the aromatic ring of Tyr. The
resulting intermediate could undergo an intramolecular cyclization to give the characteristic pyoverdine chromophore. The
absence or malfunction of parts of the pvc cluster could explain
the existence of ring-opened pyoverdines missing one double
bond, termed ferribactins or dihydropyoverdines, which have
been found to accompany the pyoverdines.68 Recently, however,
Brady and coworkers demonstrated that the pvc gene cluster
codes for the biosynthesis of paerucumarin and that pvcA, identified as an isonitrile synthase, is not required for pyoverdine
1414 | Nat. Prod. Rep., 2009, 26, 14081446
production by P. aeruginosa.78 Therefore, although P. aeruginosa

may borrow the enzymatic Pvc machinery (PvcBCD) for the
production of the pyoverdine chromophore under some
conditions, this is not always the case. Furthermore, alternative
pathways for completion of the chromophore apparently exist,
because pyoverdines are produced by all fluorescent Pseudomonads whereas pvcABCD is found only in P. aeruginosa, and is not
present in genomes of P. fluorescens, P. putida, or P. entomophila.
Pyoverdines are a defining characteristic of the fluorescent
Pseudomonads, so it is no surprise that the pyoverdine gene
clusters are a component of the core genome common to all
species. Pseudomonas stutzeri, which does not produce pyoverdine, poses an obvious exception to this generality. While the
gene composition is generally conserved, the organization

of pyoverdine biosynthesis genes in the genomes of different
Pseudomonas spp. differ markedly (Fig. 2), as do the nucleotide
sequences of certain genes for pyoverdine biosynthesis and
uptake. In landmark studies evaluating genome divergence in
strains of P. aeruginosa, the pyoverdine gene cluster was found to
be one of most highly divergent regions in the core genome.8
Genes encoding the outer membrane pyoverdine receptor (fpvA)
and the NRPSs specifying the pyoverdine peptide chain (pvdI,
pvdJ, and pvdD) are the most divergent genes in the region,79
indicating a co-evolution that maintains the mutual specificity of
the siderophore for the receptor. While the pyoverdine gene
cluster is a component of the core genome of P. aeruginosa, it
exhibits unusual codon usage and unusual oligonucleotide usage,
both of which suggest a history of HGT, possibly from other
Pseudomonas spp.79 This HGT, coupled with extensive recombination and forces of diversifying selection, provides an explanation for the polymorphism of the pyoverdine gene cluster in
the Pseudomonas spp.79 Therefore, genomic analysis of the
pyoverdine regions of Pseudomonas spp. has revealed an extraordinary level of variation that is in line with the chemical
diversity of the pyoverdine structures themselves.
2.1.2 Pyochelin. Since acquisition of iron is vitally important
for microbes, Pseudomonads created by combinatorial evolution
and shuffling of bacterial assembly lines a range of siderophores.
The iron-scavenging metabolite pyochelin has a very different
scaffold than the pyoverdines. For acquisition of iron, pyochelin
forms a 2:1 complex with ferric iron and acts as a tetradentate
ligand.8082 Pyochelin is the condensation product of salicylic
acid and two molecules of cysteine.83 The structure contains three
chiral centers (C-40 , C-200 , C-400 ) and exists as a mixture of the two
interconvertible diastereomers pyochelin I (40 R, 200 R, 400 R) and
pyochelin II (40 R, 200 S, 400 R).84
In P. aeruginosa, genes required for the biosynthesis of
pyochelin map to a ca. 21 kb genomic region designated as the
pch cluster.8587 As illustrated in Fig. 4, the cluster consists of the

two divergent operons pchDCBA and pchEFGHI, separated by
the regulatory gene pchR. Salicylate is made from chorismate via
isochorismate by the action of the isochorismate synthase PchA
and isochorismate-pyruvate lyase PchB, respectively.88 PchD
activates salicylate as salicyl-AMP in order to start the chain
growth by a thiotemplate mechanism. Each of the two NRPS
modules encoded by pchE and pchF performs an elongation with
cysteine, and the products are heterocyclized by cyclo-dehydration to thiazoline rings. During the formation of the N-terminal
thiazoline ring, the E domain of PchE inverts the absolute
configuration from L-Cys into D-Cys. PchE is noteworthy in the
unusual location of the E domain between the A and PCP
domains, as E domains are typically located directly downstream
of a PCP domain. The C-terminal thiazoline gets reduced by the
NADPH-dependent reductase PchG87 (R-domain), thereby
permitting N-methylation of the generated thiazolidine ring by
the co-substrate S-adenosyl methionine (SAM) and the MT
domain embedded in PchF.89 In a final step, the TE domain
catalyzes the hydrolytic release to give the tandem bisheterocyclic
compound pyochelin. The ABC transporter encoded by pchH
and pchI exports the siderophore, whereas the pyochelin receptor
FtpA functions in uptake of the ferric-pyochelin complex.
Pyochelin was first detected in iron-deficient cultures of
P. aeruginosa ATCC 15692 (strain PAO1) and has since been
isolated from other Pseudomonads and Burkholderia species.9094
Members of the Betaproteobacteria, the genus Burkholderia
encompasses 40 species found in diverse environments, such as
soil, plant surfaces, and water, and associated with insect, fungal,
and mammalian hosts. Like P. aeruginosa, certain Burkholderia
spp. are opportunistic human pathogens that cause respiratory
infections in patients with cystic fibrosis. Prolific production of
secondary metabolites is a hallmark of Burkholderia spp., and
pyochelin is one of many compounds produced by both
Pseudomonas and Burkholderia spp. The organization of the pch
gene cluster is identical in the genomes of Burkholderia spp. and
Fig. 4 Organization of the pch gene cluster in P. aeruginosa PAO1 and corresponding biosynthetic scheme for pyochelin formation. R: reductase
domain, responsible for the reduction of thiazoline to thiazolidine.
Nat. Prod. Rep., 2009, 26, 14081446 | 1415
P. aeruginosa, but is different from that in P. fluorescens Pf-5, as

described below. The conservation and divergence of gene clusters encoding for identical or similar metabolites in the Pseudomonads and Burkholderias is a central theme that emerges
repeatedly throughout this review.
2.1.3 Pseudomonine. Pseudomonine, isolated from P. fluorescens AH2, is another Pseudomonas metabolite that functions
as a siderophore.95 Like pyochelin, the compound consists of
salicylic acid and two heterocyclic amino acid moieties. Despite
the chemical resemblance, the assembly lines of the two molecules exhibit intriguing differences at the genetic and biochemical
levels. The pseudomonine biosynthetic genes were localized by
Bakker and coworkers in the biocontrol strain P. fluorescens
WCS37496 and by Walsh and associates in P. entomophila L48.97
The 21 kb pms-cluster (Fig. 5) is divided into seven genes
encoding three NPRSs (PmsG,D,E) and four precursor biosynthetic enzymes (PmsA,B,C,F). The starting point in the biosynthesis of pseudomonine is the formation of salicylic acid from
chorismate by the isochorismate synthase PmsC and isochorismate-pyruvate lyase PmsB, respectively, followed by its
activation by the A domain of PmsE. The Cy2 domain of PmsD
transfers the salicylic acyl group to L-threonyl-S-pantetheinylPmsG, while Cy1 of PmsD performs a cyclodehydration to
yield the salicylmethyloxazolinyl-PmsG covalent acylenzyme
intermediate. The histidine decarboxylase PmsA and histamineN-hydroxylase PmsF are involved in provision of N-hydroxyhistamine, which serves as a nucleophilic substrate for the C
domain of PmsG to release the corresponding oxazoline
hydroxamate condensation product, termed pre-pseudomonine.
The latter compound is unstable and rearranges nonenzymatically to pseudomonine. An SN2 attack at the b-carbon of the
Thr-derived oxazoline results in the formation of an isoxazolidinone ring. This intramolecular reaction inverts the
absolute configuration at the b-carbon of the Thr side chain,
which explains the presence of L-allo-configured Thr in
pseudomonine. Very recently, it was demonstrated by in vitro

reconstituation experiments that the pseudomonine NRPS
(PmsDEG) shows relaxed substrate specificity.98 In vitro,
PmsDEG can utilize 2,3-dihydroxybenzoate instead of salicylate
and Cys instead of Thr, yielding the siderophores acinetobactin
and anguibactin, which are produced by the Gram-negative
bacteria Acinetobacter baumannii and Vibrio anguillarum,
respectively.
2.1.4 Paerucumarin and pseudoverdine. Paerucumarin is an
isonitrile functionalized dihydroxycumarin that is produced by
the pvc gene cluster, the aforementioned four-gene operon that is
also reported to be involved in the biosynthesis of the pyoverdine
chromophore. Recently, however, Brady and coworkers reported that the pvc cluster is responsible for the production of the
new bicyclic metabolite paerucumarin in P. aeruginosa.78 It is
hypothesized that the isonitrile synthase PvcA generates an isonitrile-functionalized tyrosine that is subsequently oxidized by
the amino acid oxidizing enzyme PvcB (Fig. 6). The resulting
intermediate is then converted by the FAD-dependent monooxygenases PvcC/D into the corresponding catechol structure,
which in turn undergoes an intramolecular cyclization to yield
paerucumarin. Due to the instability of isonitrile-containing
metabolites, paerucumarin can decompose by the addition of
water to its N-formyl adduct pseudoverdine. Therefore, the
observed production ratio of paerucumarin to pseudoverdine
varies with the purification procedure and also from strain to
strain of P. aeruginosa. No biological functions of paerucumarin
and pseudoverdine have been reported.
2.1.5 Lipopeptides. The cyclic lipopeptides (CLPs) are
a structurally-diverse class of compounds containing a fatty acyl
residue ranging from C5C16 in length and chains of 725 amino
acids of which 414 form a lactone ring. Based on their structural
relationships, the CLPs of Pseudomonads can be generally
classified into six groups (Table 3).55 The combination of a polar
Fig. 5 Organization of the pms genes and corresponding biosynthetic scheme for pseudomonin formation.
1416 | Nat. Prod. Rep., 2009, 26, 14081446
Fig. 6 The pvc gene cluster and corresponding biosynthetic scheme for paerucumarin and pseudoverdine formation.
peptide head with a lipophilic fatty acid tail is responsible for the
amphiphilic properties of these compounds, which can lower
surface tension and interact with cellular membranes, thereby
altering their integrity.55 The latter effect is assumed to
contribute to various interactions with other organisms, e.g.
plant pathogenicity,45 antifungal,99101 antibacterial,102 antiviral103 and phosphatidylinositol-specific phospholipase (PIPLC) inhibitory104 activity. Physiologically, this compound class
may be produced by the bacterium for multiple reasons. CLPs
can first increase the bioavailability of water-insoluble
substrates; second, promote cellular swarming, which enhances
the colonization of surfaces;37,105 and third, enhance virulence or
antagonism against other microorganisms.106108
Syringomycin. Characterization of the biosynthesis pathway of
the lipodepsinonapeptide syringomycin,109,110 a virulence determinant of the phytopathogenic strain P. syringae pv. syringae
B301D,111 provided the first insights into the synthesis of Pseudomonas CLPs. Based on mutagenesis and feeding experiments
on the one hand, and due to the presence of D-configured and
unusual amino acids on the other hand, it was hypothesized that
an NRPS mechanism is involved in the biosynthesis pathway of
syringomycin. The syringomycin (syr) gene cluster of P. syringae
pv. syringae strain B301D is about 37 kb in size and show an
unusual and nonlinear genetic organization. It comprises six
genes with structural functions (syrB1, syrB2, syrC, syrE and
syrP), three genes with regulatory functions (syrF, syrG and
salA), and syrD with secretory functions112 (Fig. 7). The
biosynthesis of the syringomycin nonapeptide core is catalyzed
by two NRPSs that do not respect the colinearity rule. The syrB1
gene, responsible for the incorporation of the ninth amino acid, is
located upstream of syrE, which encodes for the NRPS incorporating the first eight amino acids. Additionally, a split-module
phenomenon is present: the C-terminus of SyrE contains the C
and PCP domain of a ninth module but lacks an A domain,
which is present in SyrB1. SyrB1 activates and loads L-Thr
which, while still tethered to the T-domain of SyrB1, is chlorinated to 4-Cl-L-Thr by the non-heme FeII halogenase
SyrB2.113,114 The aminoacyltransferase SyrC shuttles the chlorothreonyl moiety in trans between the PCP domain of SyrB1 and
SyrE, thereby enabling the final elongation to the full-length
nonapeptidyl.115 The TE domain located at the C-terminal end of
SyrE catalyzes the release and cyclization of the mature peptide
chain. Recently, Walsh and coworkers demonstrated that SyrP is
involved in the b-hydroxylation of Asp into L-threo-3-OH-Asp
while it is tethered to the T-domain of the eighth module.116
There is a discrepancy between the encoded genes and the stereoThis journal is The Royal Society of Chemistry 2009
structure of the final peptide. The incorporated serine and aminobutyric acid are D-configured in the CLP product, yet no
E domains were identified in their respective NRPS modules.
Another mechanism by which D-configured amino acids can be
integrated into nonribosomal peptides is the direct selection of
the free D amino acid by a corresponding A domain, as is the case
in for D-Ala1 of cyclosporine synthetase.117 Because SyrE2
recognizes only L-serine, however, the change of the configuration is thought to occur at the peptidyl or at the aminoacyl stage
and to be performed by an external racemase or epimerase.
The mechanism for synthesis and attachment of the lipid
moiety to the peptide chain of syringomycin remains unclear. In
other CLP-producing bacteria, such as Bacillus spp., genes
encoding these functions are commonly clustered with the NRPS
structural genes. The itu gene cluster of Bacillus subtilis RB14,
which codes for the biosynthesis of the CLP iturin A, contains
e.g. a fatty acid synthase,118 while in the srf operon of B. subtilis
ATCC21332, the hydroxy fatty acid moiety of the CLP surfactin,
is contributed by an acyl transferase.119 No evidence for the
presence of an internal or external synthase or transferase system
has been reported to date for the syr cluster. However, the
presence of a C domain in the first module of SyrE provides
a clue for a possible mechanism for incorporation of the lipid
moiety. C domains preceding the first amino acid activation
domains are present in NRPSs having products with an acylated
amino acid in the first position, so C domains with this placement
are thought to be involved in acylation.119122 Thus, it is likely
that this domain catalyzes the attachment of the 3-hydroxy fatty
acid moiety to the N-terminal serine, although a donor ACP
would still be necessary according to our current knowledge.
Little is known about the origin of the incorporated 3-OH-fatty
acids, but they appear to be derived from 3-OH-alkanoates that
are accumulated in Pseudomonads and utilized as carbon and
energy sources.123
Syringopeptin. Directly adjacent to the syr gene cluster in
P. syringae pv. syringae strain B301D is a NRPS gene cluster
coding for the biosynthesis of the phytotoxic and necrosisinducing CLP syringopeptin.124 The syringopeptins form a CLP
class containing either 22 or 25 amino acids, depending on the
bacterial strain,125127 and 3-hydroxydecanoic or 3-hydroxydodecanoic acid as lipid moiety. At 74 kb and carrying
22 NRPS modules, the syringopeptide (syp) cluster represents the
largest linear NRPS system described for prokaryotes.
The syp cluster consists of three large open reading frames,
sypA, sypB and sypC.128 The order and number of the modules of
the syp cluster are colinear to the amino acid sequence of the final
Nat. Prod. Rep., 2009, 26, 14081446 | 1417
1418 | Nat. Prod. Rep., 2009, 26, 14081446
Table 3 Primary structures of representatives of the six classes of cyclic lipopeptides (CLPs) produced by Pseudomonas spp.a
Fig. 7 The syringomycin biosynthetic assembly line from Pseudomonas syringae pv. syringae B301D. Depicted are only the secrectory and structural
genes. The regulatory genes salA, syrF and syrG are not shown but are located downstream of syrE.
syringopeptin SP22. However, analogous to the syr gene cluster,

no E domains are present, despite the presence of multiple
D-configured amino acids in syringopeptin SP22; and no genes
governing the attachment of the lipid moiety have been identified
to date. In contrast to the syr gene cluster, two (instead of one)
internal TE domains (type I) were found at the end of SypC.
Because a single C-terminal TE domain is commonly sufficient
for the hydrolytic cleavage of the peptide from its synthase and
subsequent cyclization, the function of the second TE domain is
unclear. Bioinformatic analysis indicated that the first TE
domain is intact whereas the second TE domain, which lacks
a conserved histidine residue, may be non-functional.128
In P. syringae pv. syringae strains B301D and B728a, the syr
and syp clusters are located on large pathogenicity islands.129,130
At more than 180 kb, the pathogenicity island of strain B728a
accounts for nearly 3% of the genome, and includes genes for
biosynthesis of the siderophore achromobactin, antibiotic resistance, the modification of lipid A with arabinose, and a degenerate locus for phaseolotoxin biosynthesis.49
Arthrofactin. Tandem C-terminal TE domains are also present
in the arthrofactin synthase (arf) from Pseudomonas sp. MIS38,
the third well-investigated CLP biosynthetic pathway of the pregenomic era. The colinear arf operon spans 39 kb and consists of
the three genes arfA, arfB and arfC containing two, four and five
amino activating modules131 (Fig. 8), accounting for all eleven
amino acids found in the arthrofactin structure.132 The
C-terminal end of ArfC contains two internal TE-type I domains,
each being responsible for both chain release and for the cyclization reaction between Asp and the hydroxy group of the
3-hydroxy-decanoic acid, the latter being an unusual reaction in
CLP biosynthesis by Pseudomonas spp. Using site-directed
mutagenesis, Morikawa and coworkers demonstrated recently
that both TE domains are functional and work cooperatively:
a mutation in the first TE domain abolished arthrofactin
production completely, whereas a mutation in the second TE
domain reduced the production of arthrofactin by 95%.133
Similar to the syr and syp gene cluster, the arf gene cluster
contains no genes with putative functions in the fusion of the
lipid moiety or the conversion of L- into D-configured amino
acids. However, Walsh and associates shed light on latter
phenomenon. Using several lines of evidence, they demonstrated
that the epimerization is not catalyzed by external racemases but
rather performed by dual condensation/epimerization (C/E)
domains, i.e. that the epimerase activity is cryptically embedded
with the C domain. These novel C/E domains, which are always
located downstream of the module having the amino acid that is
epimerized in the final product, can be recognized bioinformatically by an elongated His motif conforming to the
sequence HHI/LxxxxGD.134 The occurrence of this pattern was
also identified in the corresponding C-domains of the syringomycin and syringopeptin gene cluster, which may account for the
formation of D-amino acids in these CLPs.
Massetolides. The massetolides consist of a nine amino
acid-containing cyclic peptide moiety linked to either
Nat. Prod. Rep., 2009, 26, 14081446 | 1419
Fig. 8 The arf biosynthesis gene cluster from Pseudomonas sp. MIS38 and resultant chemical structure of arthrofactin.
a 3-hydroxy-decanoic, -undecanoic or -dodecanoic acid. Massetolides AH were first identified in Pseudomonas species isolated from a marine alga and a marine tubeworm, which were
collected near Masset Island, British Columbia, Canada.102
Later, massetolides were isolated from terrestrial Pseudomonads, e.g. the biocontrol strains P. fluorescens SS101135 and
Pseudomonas sp. MF-30.136 In addition to their antimycobacterial properties,102 the massetolides exhibit antifungal
and potent surfactant activities, as well as destructive effects on
zoospores of multiple Oomycete plant pathogens.137 In contrast
to the above-mentioned Pseudomonas CLP gene clusters, genes
for massetolide biosynthesis are not physically linked but are
organized into two separate clusters in the genome of P. fluorescens SS101.138 One gene cluster, spanning about 30 kb,
contains two NRPS genes, designated massBC, regulatory genes
of the luxR-type, and the efflux/resistance genes macA and macB,
respectively (Fig. 9). A second gene cluster containing the NRPS
encoding massA is physically disconnected from the massBC
cluster in the genome of strain SS101. In other respects, the
NRPSs for massetolide biosynthesis are typical of other CLP
biosynthetic pathways: in agreement with the presence of nine
amino acids in the final product, MassABC comprise nine
modules, each with a CAPCP domain structure, and MassC
terminates with two TE domains. Phylogenetic analysis of the
C-domains revealed that they group partially together with
established dual C/E domains, hence explaining the presence of
D-configured amino acids in the molecule despite the lack of

E domains. As described above for other Pseudomonas CLP
clusters, no genes involved in biosynthesis of the lipid side chain
or in acyl transfer were identified. The predominant product of
the gene cluster is massetolide A, but the co-occurrence of the
minor analogs massetolides BH, differing in the amino acid and
fatty acid composition, was ascribed to the relaxed substrate
specificity of the respective A and C domains. In particular, the
flexibility in amino acid selection by A-domains was exploited in
a precursor-directed biosynthesis (mutasynthesis) study to
generate new derivatives, leading to the analogs massetolide IK,
containing nonproteinogenic amino acids at position AA1, AA4
and AA9.102
Putisolvin. The putisolvins I and II were the first known CLPs

to have a peptide chain of 12 amino acids linked to a hexanoic
lipid chain139,140 (Table 3). The two structures differ only at the
second amino acid from the C-terminus, which is Val for putisolvin I and Leu/Ile for putisolvin II. In diverse bioassays,
putisolvins I and II were shown to reduce surface tension,
stimulate swarming motility, inhibit biofilm formation, degrade
existing biofilms and micellate the organic solvent toluene. The
putisolvin-producing strain P. putida PCL1445 was isolated from
soil heavily polluted with polyaromatic hydrocarbons,141 so the
latter capability of putisolvins possibly represents an intriguing
survival strategy. Three NRPS genes, termed psoABC, were
Fig. 9 Organisation of the mass gene cluster in P. fluorescens SS101 encoding the CLPs massetolides AH.
1420 | Nat. Prod. Rep., 2009, 26, 14081446
Fig. 10 The pso biosynthesis gene cluster from Pseudomonas putida PCL1445 and resultant chemical structures of putisolvin I (R1 CH3, R2 R3 H)
and II (R1 R2 CH3, R3 H or R1 H, R2 R3 CH3).
identified in P. putida PCL1445 and shown to be responsible for

the production of both putisolvins (Fig. 10). The structural
NRPS genes are flanked downstream by the genes macA and
macB, which are likely to function in putisolvin export, and
upstream by the luxR-type regulatory gene psoR. PsoABC codes
for 12 typical NRPS modules which obey the co-linearity rule.
Bioinformatic predictions for the substrate specificity of the A
domain of module 11 indicates a preference of Val over Leu or
Ile, which correlates well with the commonly observed ratios of
putisolvin I/II (3:2).140,142 In addition, relative production of the
two putisolvins is influenced by the availability of Val, Leu or Ile
in the culture medium.143
2.1.6 Safracin. Safracins, which exhibit antitumor activity,
have been isolated from P. fluorescens A2-2144 and SC 12695145
and exhibit an extraordinary three-dimensional molecular
architecture based on a tetrahydroisoquinoline skeleton. The
discovery of the safracin biosynthetic pathway was driven by the
structural similarity of safracin to the potent anti-tumor agent
ecteinascidin (ET-743, trabectedin, Yondelis), isolated from
the tunicate Ecteinascidia turbinata. Neither the harvest of
natural populations nor aquaculture could provide sufficient
quantities of ecteinascidin to continue clinical development.
Furthermore, manufacturing or total synthesis was not
economical at an industrial scale. Therefore, a partial synthesis
of ecteinascidin starting from safracin B, produced by Pseudomonads, was envisioned.146,147
The safracin gene cluster (sac) in P. fluorescens A2-2 comprises
ten open reading frames coding for three NPRSs (SacA, SacB,
SacC), three precursor biosynthetic enzymes (SacD, SacF,
SacG), two tailoring enzymes (SacI, SacJ), one resistance protein
(SacH) and one protein of unknown function148 (Fig. 11). The
unusual amino acid derivative 3-hydroxy-5-methyl-O-methyltyrosine (3h5mOmTyr) is synthesized from L-Tyr by two methyltransferases, SacF and SacG, and the hydroxylase SacD. Two
molecules of 3h5mOmTyr, together with Ala and Gly, are
selected and loaded on specific NPRS modules to form the
safracin tetrapeptide basic skeleton. The NRPS pathway exhibits
several unusual features. While the peptidic backbone of safracin
contains a stretch of four amino acids, sacABC encode only three
NRPS modules. The mechanism by which four amino acids are
incorporated by three modules was revealed in the homologous

saframycin A gene cluster (sfm) from Streptomyces lavandulae
NRRL 11002, where SacA and SacB recognize and activate Ala
and Gly, respectively, and SacC acts in an iterative fashion,
activating the tyrosine derivative 3h5mOmTyr twice to produce
the required tetrapeptide intermediate.149 In addition to the
iterative use of one module, the NRPS assembly line composed
of SacABC is also conspicuous due to substitution of the TE
domain, typically found as the C-terminal domain of NRPSs, with
a terminal reductase (R) domain, suggesting a reductive chain
release similar to that in saframycin,150 myxochelin151 or nostocyclopeptide152 biosynthesis. In nostocyclopeptide biosynthesis,
the R domain performs dual functions, catalyzing the reductive
release of the matured peptide chain as an aldehyde and triggering
the spontaneous formation of an imino head-to-tail linkage.
Similarly, the R domain in SacC may catalyze the chain release of
the matured peptide chain as an aldehyde and then trigger the
spontaneous formation of the imino head-to-tail linkage, leading
to the closure of ring C of safracin. Before or during the subsequent cyclization reactions, one of the two 3h5mOmTyr moieties
is additionally hydroxylated by the monoxygenase SacJ, which
leads to a quinone ring structure, while the methyltransferase SacI
performs N-methylation at the amine of the remaining
3h5mOmTyr residue. In a final step, the resulting safracin B is
converted by SacH into safracin A, which lacks a hydroxyl-group
at C-21. Because safracin A exhibits lower antibiotic and antitumoral activity than safracin B, sacH is considered to be a resistance gene functioning in detoxification.
2.1.7 Tabtoxin. Tabtoxin represents another example of
phytotoxic peptides from P. syringae. The compound consists of
tabtoxine-b-lactam (TbL) and threonine. The small dipeptide is
known to be secreted by three pathovars of P. syringae: tabaci,
coronafaciens and garcae.153 The phytotoxin is associated with
the symptoms of wildfire disease on tobacco and halo blight of
oats. However, the chlorosis-inducing effects occur only after
hydrolysis of the dipeptide with aminopeptidases present in
either the bacteria or the plant, yielding the actual toxin TbL.
The mode of action of TbL is the irreversible inhibition of
glutamine synthase, which catalyzes the synthesis from glutamic
acid to glutamine in amino acid metabolism. This inhibition
Nat. Prod. Rep., 2009, 26, 14081446 | 1421
Fig. 11 Structural organization of the sac gene cluster of P. fluorescens A2-2 and proposed safracin biosynthesis mechanism. R: reductase domain,
responsible for reductive chain release.
results in reduced availability of Gln, which blocks the only

efficient ammonia detoxification pathway in plants, leading to
ammonia toxicity.154,155
The biosynthetic enzymes of the virulent isolate P. syringae BR2
are encoded by the 15 kb tab/tbl gene cluster156 (Fig. 12). Several
genes of the cluster (e.g. tabA and tabB) show a high resemblance
to genes of the bacterial lysine biosynthesis pathway.
Labeling157,158 and knockout159 studies demonstrated that tabtoxin
biosynthesis proceeds along the lysine pathway (dabABCDE),
branching off after tetrahydropicolineate and before diaminopimelate formation. From this branch point, tabtoxin is
assembled by the action of TblS (putative b-lactam synthetase),
TblC (putative clavaminic acid synthase), TblD (putative GNAT
acyltransferase), and TblE in conjunction with TblF (putative

membrane protein, forming a functional pair with a D-Ala D-Ala
ligase). It has been shown that these enzymes are essential for
tabtoxin biosynthesis, but details about the enzymatic mechanisms
remain to be elucidated.160,161 Upon assembly, the completed
tabtoxin can be converted by the metallopeptidase TabP into the
toxin TbL, which is subsequently exported by the transporter
TblR. The cleavage of tabtoxin occurs only in the presence of zinc,
which is required for peptidase activity.162,163
Evidence for HGT is found in sequences surrounding the tbl/
tab cluster in P. syringae pv. tabaci.156 The cluster is bordered by
a lysC tRNA gene, which is a common site for integration of
genomic islands in bacteria including Pseudomonas spp.,164166
Fig. 12 Structural organization of the tab/tbl gene cluster and proposed biosynthetic pathway to tabtoxin.
1422 | Nat. Prod. Rep., 2009, 26, 14081446
Fig. 13 Pht gene cluster of Pseudomonas syringae pv. phaseolicola NPS3121 and its resulting structure phaseolotoxin. In planta, phaseolotoxin is
processed by a peptidase to create the active toxin PSOrn, also known as octicidine.
and a tyrosine recombinase, which is also associated with HGT

of the phaseolotoxin gene cluster, described below. The tabtoxin
biosynthetic gene cluster has been reported to excise readily from
the chromosome of P. syringae pv. tabaci,167 and the locus
exhibits a patchy distribution among the major groups of
P. syringae.46 Certain genes in the region are also present in the
PAPI-1 pathogenicity island of P. aeruginosa PA14.156 Together,
these data provide convincing evidence for HGT in the evolution
of tabtoxin-producing strains of P. syringae.
2.1.8 Phaseolotoxin. Phaseolotoxin is a phytotoxic tripeptide
consisting of ornithine, alanine and homoarginine, linked to an
inorganic sulfodiaminophosphinyl moiety,168,169 that is produced
by P. syringae pvs. phaseolicola and actinidiae,170 pathogens
causing halo blight of bean and canker of kiwi fruit, respectively,
as well as by P. syringae pv. syringae CFBP3388.171 Phaseolotoxin exerts its phytotoxic effects by competitive inhibition of
ornithine carbamoyl transferase (OCTase), a key enzyme in the
urea cycle that converts ornithine and carbamoyl phosphate to
citrulline. As a result, ornithine accumulates and a deficiency in
intracellular pools of arginine occurs. Although phaseolotoxin is
a reversible inhibitor of OCTase, it is hydrolyzed in planta by
peptidases to Nd-(N0 -sulfodiamino-phosphinyl)ornithine (syn.
PSorn or octicidine), which is a strong irreversible inhibitor of
OCTase.172 The 28 kb phaseolotoxin gene cluster, termed the
argKtox cluster, is composed of 23 genes (Fig. 13).173175 At
different stages of the discovery process, the designation of the
genes changed, and the following text conforms to a recently
proposed nomenclature.175 Despite long-standing efforts reaching back more than two decades, the identities and roles of most
of the gene products of the phaseolotoxin cluster remain to be
determined. The unambiguous genes identified so far are argK,
amtA and desI. By the production of ArgK, a resistant OCTase
isoform, P. syringae pv. phaseolicola, protects itself against an
impairment of its own prokaryotic urea cycle.176178 The product
of the desI gene shows similarity to a fatty acid desaturase, a class
of enzymes that commonly generate unsaturated fatty acids for
the synthesis of phospholipids in the cytoplasmic membrane.179 It
has been hypothesized that DesI may facilitate the export of
phaseolotoxin across the bacterial membrane at the low
temperatures (1820 C) conducive to phaseolotoxin production.180 AmtA encodes an amidinotransferase and is demonstrably involved in the formation of the amino acids homo-Arg
and Orn, which are synthesized by transamination from Arg and

Lys, respectively.181 Regarding the assembly of the tripeptidic
backbone of phaseolotoxin, the involvement of a non-ribosomal
thiotemplate mechanism was always assumed, but corresponding
NRPS genes have not yet been identified. Very recently, experimental work by Kino and Arai gave indications of another
possible biosynthesis strategy for the tripeptide moiety. In the
genome of P. syringae pv. phaseolicola 1448A, Kino et al. identified phtU as a novel L-amino acid ligase-encoding gene. The
protein encoded by phtU (PSPPH_4299) was proven to synthesize various hetero-dipeptides, but not a tripeptide, in an ATPdependent manner.182 Therefore, it can be envisioned that the
peptide backbone might be constructed by PhtU and an additional, currently unidentified L-amino-acid ligase instead of the
NRPS machinery. However, a growing body of evidence is
pointing to the fact that at least one gene outside the argKtox
gene cluster, possibly encoding an NRPS, is partly involved in
the synthesis and assembly of the tripeptide moiety (S. Aguilera
and A. Alvarez-Morales, personal communication).
In phaseolotoxin-producing strains of P. syringae pv. actinidiae and P. syringae pv. phaseolicola, argKtox clusters have
been associated with genomic islands integrated into the genomes
in a site-specific manner.183 These clusters are flanked by insertion sequences or genes encoding for members of the tyrosine
recombinase family, which facilitate integration of horizontallyacquired DNA into the genome.183 The genomic sequence of the
phaseolotoxin-producing strain P. syringae pv. phaseolicola
1448A contains an argKtox cluster, as expected, as well as
a second cluster containing homologs (4080% similarity) to
a subset of the argKtox genes.48 The same degenerate cluster
was found in the genome of P. syringae pv. syringae B728a,
which does not produce phaseolotoxin, where it exists on the
large genomic island also containing genes for syringomycin,
syringopeptin and achromobactin biosynthesis.49 In both B728a
and 1448A, the degenerate cluster appears to define a lateraltransfer hotspot that exhibits evidence of multiple HGT
events.48 Thus, the phaseolotoxin gene cluster provides another
example of secondary metabolite gene cluster acquisition
through HGT events.
2.1.9 Pyrrolnitrin. Pyrrolnitrin is a chlorinated phenylpyrrole
compound and its production by Pseudomonas pyrocinia was first
discovered in 1964.184 Since that time, production of pyrrolnitrin
Nat. Prod. Rep., 2009, 26, 14081446 | 1423
Fig. 14 The genetic organization of the prnABCD gene cluster from P. fluorescens BL915 and biosynthetic route to pyrrolnitrin.
has been reported from other Pseudomonas spp., as well as

Enterobacter agglomerans, Myxococcus fulvus, Burkholderia
spp., and Serratia spp.185187 Pyrrolnitrin is an inhibitor of fungal
respiratory chains188 and, due to its strong antifungal activity,
pyrrolnitrin was developed as a topical antimycotic for human
use.189,190 A derivative of pyrrolnitrin exhibiting greater environmental stability is used as an agricultural fungicide.191,192 A
four-gene cluster (prnABCD) for the biosynthesis of pyrrolnitrin
was first described in Pseudomonas aurantiaca (previously identified as P. fluorescens) BL915.193,194 The order of the four prn
genes corresponds to the order of their protein products in the
biosynthetic pathway for pyrrolnitrin (Fig. 14). L-Tryptophan
serves as the basic precursor,195199 which is chlorinated by the
tryptophan halogenase PrnA to form 7-chloro-Trp. PrnB catalyzes the rearrangement from the indole to the phenylpyrrole
skeleton including a decarboxylation reaction to give monodechloro-pyrrolnitrin, which is in turn halogenated at position 3
of the pyrrol ring by the halogenase PrnC. In the last step, the
amino group of the resulting intermediate metabolite aminopyrrolnitrin is converted into a nitro group by the action of the
oxidase PrnD to yield pyrrolnitrin. Especially notable are the two
halogenases PrnA and PrnB, which exhibit high substrate specificity and regioselectivity and provided the first examples of the
FADH2-dependent halogenases.200
The four pyrrolnitrin biosynthetic genes (prnABCD) are highly
conserved among strains of P. fluorescens that produce pyrrolnitrin.187,193,194,201 To date, P. fluorescens Pf-5 is the only pyrrolnitrin-producing Pseudomonad whose complete genomic
sequence is available, but the highly-conserved prnABCD cluster
is also present in the completed genomes of several Burkholderia
spp.39,186 Whereas a syntenic prnABCD cluster is conserved, the
composition and organization of flanking genes differ markedly
between the two genera, and even among certain of the Burkholderia spp. Flanking the four known biosynthetic genes in the
Pf-5 genome are genes with regulatory, transport, and biosynthetic functions that could play a role in pyrrolnitrin production.
Especially notable is the flavin reductase PrnF that, when
expressed in Escherichia coli, provides reduced FADH2 to the
PrnD oxygenase.202 Genes having putative regulatory, transport
and flavin reductase functions are also found near the prnABCD
operon in several (but not all) of the Burkholderia genomes.186
Because pyrrolnitrin is produced by heterologous host bacteria
such as Escherichia coli harboring the prnABCD operon alone,193
the flanking genes are not strictly required for pyrrolnitrin
biosynthesis.
Burkholderia spp. and Pseudomonas spp. are coinhabitants of
soil and other natural habitats, and it is likely that the presence of
the highly conserved prnABCD cluster in these distantly related
bacteria is a function of HGT. In P. fluorescens Pf-5, the
prnABCD cluster is in a large (ca. 70 kb) lineage-specific region of
1424 | Nat. Prod. Rep., 2009, 26, 14081446
the genome lacking REP regions, as described in Section 4. In

three B. pseudomallei genomes, prnABCD is flanked by putative
transposases, which could mediate insertion of transferred DNA
sequences into the genomes. A closely-related strain of
B. pseudomallei lacks only prnABCD but retains the transposases
and flanking genes, perhaps representing a predecessor of a HGT
event or a successor of an excision event. Notably, however, the
regions lack certain hallmarks of genomic islands, exhibiting no
abrupt alterations in G + C content or dinucleotide or trinucleotide skew from the surrounding genomic regions.186
2.1.10 Indole-3-acetic acid (IAA). Indole-3-acetic acid is
a naturally occuring indole-based phytohormone that influences
virtually every aspect of plant development and physiology. IAA
is produced by many microorganisms, including plant pathogens
such as P. syringae203 and non-pathogenic strains of Pseudomonas spp.204207 IAA production is a well established virulence
factor of plant pathogens that cause hyperplasia, such as Pseudomonas savastanoi, which causes gall formation on olive and
oleander.208 P. savastanoi, many pathovars of P. syringae203 and
certain strains of other plant-associated Pseudomonas spp.204
produce high concentrations of IAA, whereas low levels are
produced by rhizosphere-inhabiting strains that are beneficial to
plants.206 The effects of exogenous IAA on plant growth and
development are concentration-dependent, so the concentrations
of IAA produced by bacteria on plant surfaces are thought to be
key to the plant response.206 The relationship between IAA
production and plant response is further complicated, however,
by the fact that certain IAA-producing Pseudomonas sp. also
catabolize IAA.209 Auxin signaling pathways are important
components of plant defense strategies, and recent evidence
indicates that pathogens such as P. syringae can interfere with
these pathways to overwhelm host defense.210212 Although it is
commonly assumed that IAA production evolved in bacteria due
to its importance in plantbacteria interactions, IAA can also
function as a signaling molecule in prokaryotes, effecting many
aspects of bacterial physiology206 including secondary metabolism in P. syringae.213
In bacteria, the biosynthesis of IAA proceeds from L-tryptophan to IAA via at least five possible biosynthetic routes, classified in terms of their intermediates.206,214,215 Pseudomonads
utilize the indole-3-acetamide (IAM) pathway and the indole-3pyruvic acid (IPyA) pathway, including its shortcut, the tryptophan side chain pathway (Fig. 15).214,215 A single strain of
Pseudomonas spp. may utilize two of the three pathways for IAA
biosynthesis.213,216 The plant pathogen P. savastanoi and certain
strains of P. syringae assemble IAA primarily via the IAM
pathway,217 but may also use the IPyA pathway. In general, the
IAM pathway is constitutively expressed whereas IPyA pathways are expressed only in the presence of tryptophan.218220
In the IPyA pathway, tryptophan is converted by a transaminase to the unstable intermediate indole-3-pyruvic acid,
which is decarboxylated to yield indole-3-acetaldehyde (IAAld).
In a final step, IAAld dehydrogenase transforms IAAld into
IAA.214 The tryptophan side chain pathway, in which tryptophan
side chain oxidase (TSO) converts tryptophan directly to IAAld,
represents a shortcut to the IPyA pathway (Fig. 15)232 that is
preferred for IAA production in at least some strains of P. fluorescens.216 Surprisingly, almost nothing is known about the
genetics that underlie the IPyA/trytophan side chain pathways.
An indolepyruvate decarboxylase gene (ipdC) sequence was
reported for P. putida GR12-2,233 but has since been shown to
originate from a strain of Enterobacter cloacae.
2.2 Polyketides and fatty acid derived compounds
Fig. 15 A) Pathways leading from Trp to IAA in Pseudomonads: [I]

Indolacetamide pathway, catalyzed by Trp-2-mono-oxygenase and IAM
hydrolase. [II] Tryptophan side chain pathway, utilizing Trp side chain
oxidase (TSO) and IAAld-dehydrogenase. [III], indolepyruvic acid
pathway [III], initiated by Trp transaminase and followed by decarboxylation to produce IAAld and subsequently IAA. B) Organization of the
iaaMH and iaaL genes in the plasmid plIAA1 of P. syringae subsp.
savastanoi (P. savastanoi).
The beneficial plant growth-promoting Pseudomonas strains,

which produce lower amounts of IAA, use primarily the IpyA
pathway or its shortcut, the side chain pathway.216,221,222
The gene cluster directing IAA biosynthesis via the IAM
pathway, which was first identified in P. savastanoi,223 comprise
two genes organized in one operon, iaaH and iaaM.224 The latter
gene encodes a tryptophan 2-monooxygenase, catalyzing the
oxidative decarboxylation of tryptophan to indole-3-acetamide
(Fig. 15).225,226 iaaH, coding for indoleacetamide hydrolase,
subsequently hydrolyzes the intermediate indole-3-acetamide to
IAA and ammonia. In P. savastanoi, the iaaMH operon is
commonly accompanied by iaaL, which is located upstream and
in the opposite orientation. This gene encodes N-(indole-3acetyl)-L-lysine synthetase, which is responsible for the production of biologically inactive IAAlysine conjugates227229 with
proposed roles in regulation of the IAA pool size and virulence.229 In strains of P. savastanoi, the iaaLiaaHM cluster can
be located on the chromosome or on conjugative plasmids,230
such as those in the pPT23A family,231 which are widely
distributed in phytopathogenic Pseudomonas spp. and are known
to carry genes required for virulence and ecological fitness.
Although IAA production is common in many pathovars of
P. syringae, the iaaMH operon is found in only a subset of IAAproducing strains,203 indicating that other IAA biosynthetic
pathways (such as IPyA) are more common. The iaaMH operon
is present in only one (B728a) of the strains of P. syringae with
published genomic sequences. Although iaaL appears to be more
widely distributed in the species,203 it is also present in only one
(DC3000) of the strains of P. syringae sequenced to date.
Polyketides are a large class of structurally-diverse natural

products that include compounds with antibiotic, chemotherapeutic, and phytotoxic activities.51,234,235 The formation of polyketides can be envisioned as a series of decarboxylative Claisen
thioester condensations of acetic acid units.236 In this way, polyketide and fatty acid biosynthesis are quite similar, but important
distinctions differentiate the polyketide synthases (PKSs) from
fatty acid synthases (FASs).237 PKSs use a broader range of
biosynthetic building blocks, exhibit wider variation in chain
length of the final product, and do not fully reduce each acyl unit
following every round of chain elongation as done by FASs.
Historically, PKSs have been categorized in three major types:
type I PKSs are large, modular proteins;237 type II PKSs are
composed of complexes of monofunctional proteins;236 and type
III PKSs238 are most commonly associated with chalcone and
stilbene synthases in plants, enzymes containing a single activesite cysteine and lacking acyl carrier protein (ACP) functionalities found in type I and type II PKSs. Collectively, polyketide
biosynthesis in Pseudomonas spp. involves each of the three types
of PKSs.
Like the NRPSs described above, Type I PKSs are large multifunctional proteins organized into modules, each of which
functions in the addition and modification of an extender unit
(usually acetyl-CoA or malonyl-CoA).234,237 PKS modules are
composed of three core domains: An acyltransferase (AT)
domain, which selects the extender unit and transfers it to an acyl
carrier protein (ACP) domain, having a phosphopantetheine
prosthetic group that is linked to the extender unit via a covalent
thioester bond, and a ketosynthase (KS) domain, which catalyzes
the condensation reaction between the extender unit and the
polyketide intermediate on the preceding module. The three core
domains are present in all elongating modules, whereas the
loading module lacks a KS domain, and the terminal module
contains a TE domain, responsible for the release of the polyketide product from the PKS. Modules may also contain
domains for tailoring reactions that contribute to polyketide
diversity. For background and perspectives on PKS biosynthesis,
we refer the reader to excellent reviews on the subject.236240
2.2.1 Mupirocin (pseudomonic acid A). Pseudomonic acids
are a mixture of antimicrobial compounds isolated from P. fluorescens consisting of four components: pseudomonic acids A,239
B,240 C241 and D.242 The four compounds share certain features,
Nat. Prod. Rep., 2009, 26, 14081446 | 1425
including a dihydroxylated tetrahydropyran ring bearing functionalized side chains in position 2 and 5. Pseudomonic acid A,
the major metabolite in the pseudomonic acid mixture, exhibits
the highest level of antibacterial activity towards Gram-positives243 and has been developed as a topical antibiotic (Bactroban, Centany or Eismycin, INN: mupirocin). It is used
today clinically for dermal staphylococcal infections, particularly
those caused by methicillin-resistant Staphylococcus aureus
(MRSA).244 The use of mupirocin is limited to topical application
because it is inactivated rapidly by esterases in the human serum
and bound with high affinity to serum proteins, resulting in poor
bioavailability. Mupirocin has been shown to interfere with
RNA, proteins and, to a lesser extent, with cell wall biosynthesis.245,246 Mupirocin is a competitive inhibitor of the bacterial
isoleucyl-tRNA synthetase, and this inhibition leads to reduced
protein biosynthesis. The methyl end of mupirocin mimics an Ile
moiety and interacts with the amino acid binding site of IletRNA synthase, while the remaining molecule interacts with the
respective ATP binding site.247 Pseudomonic acid-producing
Pseudomonads protect themselves from the inhibitory effects of
the antibiotic by altering their own isoleucyl-tRNA synthetase.248
Biosynthetically, mupirocin is the product of an esterification of
the polyketide monic acid and the fatty acid 9-hydroxy-nonanoic
acid (Fig. 16). Feeding studies indicated that the molecule is
derived from acetate units, except for C-16 and C-17, which are
introduced by SAM biomethylation, and C-15, which has been
proposed to be derived from acetate via 3-hydroxy-3-methylglutarate (HMG).249251
In 1995, Thomas et al. identified the genomic region of
P. fluorescens NCIMB 10586 that is required for mupirocin
production.252 Eight years later, the same group provided the
nucleotide sequence of the complete 74 kb cluster253 (Fig. 16). It
codes for six multi-domain enzymes (MmpAF) with sequence

similarity to type I PKSs or fatty acid synthases and 29 additional proteins (MupAX and MacpAE), resembling type II
PKSs, auxiliary, tailoring and resistance enzymes. The cluster
exhibits two extraordinary features. First, the mupirocin cluster
was one of the early examples of trans-AT PKS gene clusters:254,255 these PKS modules lack AT domains, but genes
encoding ATs are present in the cluster, acting iteratively
in trans.255 Second, the pathway contains several tandem ACP
doublets or triplets, which are hypothesized to increase the
throughput rate or to bind multiple substrates simultaneously.
The biosynthesis of monic acid starts with the production of
a pentaketide moiety by MmpD (Fig. 16). Starter units could be
activated by MupU or MupQ, which show similarity to acylCoA synthases, and one of the AT domains from MmpC
transfers an activated acetyl group from acetyl-CoA to the first
ACP domain. Due to a mutation in the conserved active site
region, the dehydration domain of module 1 of MmpD is inactive, but all other domains are functional and catalyze the first
four condensations. The assembly of monic acid is continued by
the transfer of the pentaketide product of MmpD to MmpA,
which performs further extension with two malonyl-CoA units.
Gene knockout studies indicated that the first KS domain of
MmpA is essential for mupirocin production, but does not act as
a classic ketosynthase; instead, it facilitates the transfer of the
pentaketide intermediate between MmpD and MmpA. The
incorporation at C-15 has been proposed to be performed by the
action of the putative HMG-CoA-synthase MupH. In order to
complete the structure of monic acid, several oxidation and
reduction post-PKS tailoring reactions are required to create the
epoxide group and the tetrahydropyran ring system. Candidate
enzymes for these steps are MupC (NADH oxidoreductase),
Fig. 16 The mupirocin gene cluster and biosynthesis model. Asterisks indicate an inactive domain.
1426 | Nat. Prod. Rep., 2009, 26, 14081446
MupO (a cytochrome P450), MupT (ferredoxin dioxygenase)

and MupW (dioxygenase). Further gene knockout experiments
indicated that MupW is actually involved in the formation of the
tetrahydrapyran ring and that MupO,Q,S,T,U,V and MacpE are
essential for the production of pseudomonic acid A.256 In
contrast, MupO, MupU, MupV and MacpE are not essential for
production of pseudomonic acid B, which bears an additional
hydroxyl group at C-18. These results indicate that pseudomonic
acid B is not simply produced by hydroxylation of pseudomonic
acid A, but represents either a precursor of pseudomonic acid A
or a shunt product. Upon completion of the tailoring reactions, if
not earlier, monic acid is fused to the lipid 9-hydroxy-nonanoic
acid, which is proposed to be synthesized by MmpB with
a 3-hydroxy-propionate starter unit and three malonyl-CoA
extender units. Alternatively, since MmpB possesses an enoyl
reductase domain, the putative enoyl reductase MupE could
possibly catalyze the final reduction step in the 9-hydroxy-nonanoic acid biosynthesis. Apart from the discussed structural
genes, mupM has been identified as a resistance gene, encoding
the modified mupirocin-resistant Ile-tRNA-synthase, which is
essential for survival. The expression of mupirocin is controlled
by MupR and MupI, which are members of the LuxR and LuxI
family of quorum sensing regulators.253 Several genes of the
mupirocin cluster have not yet been characterized in detail and
their functions warrant future investigation.
2.2.2 2,4-Diacetylphloroglucinol (DAPG). DAPG is a
primary factor contributing to biological control of plant disease
by many plant-associated Pseudomonas strains.36,257260
The phenolic molecule is toxic to a wide range of plant pathogenic fungi259262 and also exhibits antibacterial,262 antihelmintic263 and, in high concentrations, phytotoxic
properties.262 DAPG also triggers systemic resistance of plants
against disease,264 and is a primary determinant of the diseasesuppressive properties of certain soils against the take-all pathogen of wheat.265 The 6.5 kb DAPG biosynthetic locus (Fig. 17)
has been identified and analyzed in P. fluorescens strains
Q2-87,266 CHA0,267 and F113.268 The gene cluster is highly
conserved among DAPG producers258 and comprises genes for
biosynthesis (phlACBD),266 efflux (phlE),269 degradation (phlG)270
and regulation (phlF and phlH).267,268,271 Quite rare and unusual
for microorganisms,272 the biosynthesis of DAPG is mediated by
PhlD, a type III PKS chalcone synthase. Frost and coworkers
proved that PhlD first condenses three molecules of malonylCoA to form an activated 3,5-diketoheptanedioate, which is in
turn cyclized via a Claisen condensation upon decarboxylation to
yield phloroglucinol272,273 (Fig. 17). Stepwise acetylation performed by PhlABC leads to monoacetylphloroglucinol (MAPG)
and 2,4-DAPG, respectively, but only the conversion of MAPG
to DAPG has been experimentally demonstrated to date.274 The

level of produced DAPG is regulated mainly by degradation
through the specific hydrolase PhlG, which converts DAPG into
MAPG, and by the regulatory proteins PhlF and PhlH. PhlF
represses the expression of the phlABCD operon by binding to
two conserved sites in the phlA leader region. DAPG itself is able
to dissociate the repressor PhlF from the phlA promotor, hence
acting as an autoinducer of it own biosynthesis.267 Since DAPG is
exported and diffusible, it can function as a signaling
compound.275 PhlH, the second pathway-associated transcriptional regulator, is hypothesized to antagonize the repressive
effect of PhlF.28
DAPG production is characteristic of a subset of strains of
P. fluorescens, which include the fully-sequenced strain Pf-5.
Moynihan et al.276 recently reported that the cluster is ancestral
in P. fluorescens, based upon its presence in a single phylogenetically-defined branch of the species. To date, the phl cluster
has been found in three distinct locations, defined by flanking
genes, in the genomes of different strains of P. fluorescens. Along
with the known phl biosynthetic genes, an additional gene was
found adjacent to the cluster only in the DAPG-producing
strains.276 While the role of this gene in DAPG production
remains to be explored, it provides an excellent example for the
value of genomics in delimiting biosynthetic gene clusters. The
phl cluster is flanked by conserved genes of the core P. fluorescens
genome in Pf-5, whereas the cluster is present in a large lineagespecific region in the genome of another DAPG-producing
strain, P. fluorescens F113.
2.2.3 2,5-Dialkylresorcinols. A series of three 2-n-hexylresorcinols bearing in position 5 either an n-propyl, n-pentyl or
n-heptyl side chain are produced by specific strains of Pseudomonas spp.277280 The 2,5-dialkylresorcinols, like the structurallyrelated acetylated phloroglucinols, also exhibit antifungal and
antibacterial activity. Despite the structural resemblance,
however, the 2,5-dialkylresorcinols and acetylated phloroglucinols have completely different biosynthetic pathways.
Several labeling experiments and genomic analysis suggested that
dialkylresorcinol biosynthesis represents a unique offshoot of
fatty acid metabolism in which medium-chain-length fatty acidderived precursors are further modified. The 4-kb dar gene
cluster was identified in Pseudomonas aurantiaca (previously
identified as P. fluorescens) BL915, which produces 2-hexyl-5propyl-alkylresorcinol as the predominant analog of several
dialkylresorcinols.281 As Fig. 18 illustrates, the dar cluster
consists of five genes; darABC are sufficient for 2,5-dialkylresorcinol biosynthesis, whereas darS and darR encode
regulatory proteins. DarA catalyzes an unusual head-to-head
condensation between two b-ketoacyl thioester precursors
Fig. 17 The phl gene cluster and proposed biosynthesis pathway of 2,4-diacylphloroglucinol (2,4-DAPG).
Nat. Prod. Rep., 2009, 26, 14081446 | 1427
Fig. 18 The dar gene cluster of P. aurantiaca and proposed biosynthesis pathway of 2,5-dialkylresorcinols, exemplified with 2-hexyl-5-propylresorcinol.
R ACP or acyl-CoA.
(3-ketohexanoate and 3-ketodecanoate) to form, after subsequent dehydration, a dioxocyclohexene intermediate. The two
b-ketoacyl thioesters originate from the medium-chain-length
fatty acid octanoate, which is partially degraded by b-oxidation
to give the shorter-chain precursor 3-ketohexanoate. The longer
chain precursor, 3-ketodecanoate, is generated via adol condensation by DarB, a b-ketoacyl-acyl carrier protein synthase III,
and DarC, an acyl carrier protein. Upon thioester hydrolysis of
the dioxocyclohexene intermediate, a decarboxylation reaction
at C-6 is thought to occur. Aromatization of this decarboxylated
structure through ketoenol tautomerism leads to the formation
of 2-hexyl-5-propyl-alkylresorcinol.
2.3 Hybrid NRPS-PKS compounds
2.3.1 Syringolin. Syringolins constitute a family of structurally-related phytotoxic cyclic peptide-polyketides produced by
certain strains of P. syringae pv. syringae.282,283 Syringolins can
activate defense-related genes in plants, thereby reducing the
severity of plant disease282,284 and their production is a virulence
factor in P. syringae pv. syringae.285 The mode-of-action was
recently attributed to irreversible inhibition of the eukaryotic
proteasome.285 As a consequence, unneeded and damaged
proteins cannot be degraded, which effects the regulation of the

cell cycle, gene expression and responses to oxidative stress,
leading ultimately to cell death. In human cancer cells, the
interaction of syringolins with the proteasome leads to the inhibition of cell proliferation and apoptosis.286 The major derivative, syringolin A, consists of a 12-membered ring structure,
formed by the two non-proteinogenic amino acids 5-methyl-4amino-2-hexenoic acid and 3,4-dehydrolysine. The a-amino
group of the latter is joined by a peptide bond to a valine residue
that, in turn, is linked to a second valine via a urea moiety. Other
members of the syringolin family differ from syringolin A by the
substitution of 3,4-dehydrolysine by lysine or of one or both
valine residues by isoleucine residues.
The biosynthetic genes for syringolin A have been described by
Dudler and coworkers.287 This biosynthetic locus is also assumed
to be responsible for the biosynthesis of the minor structural
variants syringolins BF, whose appearance can be attributed to
the relaxed substrate specificity of A domains and to the alterable
efficacy of an involved desaturase. The syringolin (syl) gene
cluster spans about 28 kb and consists of the five open reading
frames sylABCDE (Fig. 19). SylA is a transcriptional regulator
and SylE, which shows similarity to an efflux pump, may be
involved in the efflux of syringolins. Together, sylC and sylD
Fig. 19 The syl biosynthesis gene cluster encoding syringolins.
1428 | Nat. Prod. Rep., 2009, 26, 14081446
encode three typical NRPS modules that account for the

assembly of the tripeptide Val-Lys-Val. The latter valine residue
represents an activated precursor, which gets further extended by
two carbon atoms through a classical type I PKS. Subsequent
release and ring closure lead to a mixed peptide-polyketide
intermediate. SylB exhibits similarity to a fatty acid desaturase
domain and is hypothesized to be responsible for the desaturation of lysine. However, it is not known at which stage of the
biosynthesis this reaction may occur. Furthermore, the mechanism by which the second valine is linked via the ureido group to
the core structure is unknown.
The syringolins are structurally related to the glidobactins,
a group of antifungal, cytotoxic, acylated tripeptides produced
by Burkholderia spp.288 Homologs of sylB, sylC, and sylD are
present in the genomes of several Burkholderia spp. and Photorhabdus luminescens, an endosymbiont of entomopathogenic
nematodes in the genus Heterorhabditis.288 Structural differences
between the syringolins and glidobactins have been attributed to
variations in the A domains of the three modules of the NRPSs
present in all three bacterial genera, as well as the activities
conferred by auxiliary genes specific to each cluster. The syringolins and glidobactins provide additional examples of gene
clusters that are likely to have been acquired via HGT between
Pseudomonas spp. and Burkholderia spp.
2.3.2 Pyoluteorin. Pyoluteorin was first isolated in the late
1950s from cultures of the P. aeruginosa strains T359 and IFO
3455,289 and later from many other strains of Pseudomonads.51,290 This hybrid NRPS/PKS natural product is toxic
against Oomycetes,31 certain bacteria and fungi289 and, at high
concentrations, exhibits phytotoxicity against certain plants.291
The compound is best known for its toxicity against the Oomycete Pythium ultimum,31 an important plant pathogen causing
broad-scale economic losses to agriculture. Chemically, pyoluteorin is composed of a resorcinol ring attached to a bichlorinated pyrrole moiety. Feeding experiments revealed that the
resorcinol ring is derived from polyketide biosynthesis292,293 and

that L-proline is the primary precursor of the dichloropyrrole
moiety.294 Initially, L-proline is activated to L-prolyl-AMP by the
aminoadenylation domain of PltF and is then transfered to the
phosphopantetheinyl arm of carrier protein PltL295 (Fig. 20).
While tethered to PltL, the prolyl moiety is desaturated by the
dehydrogenase PltE and subsequently dichlorinated first at
position 5 and then at position 4 by the FADH2-dependent
halogenase PltA.296 The dichloropyrrolyl residue is most likely
passed on to the type I polyketide synthases PltB and PltC,
which add three malonyl-CoA monomers. Subsequent cyclization to resorcinol and release by the thioesterase PltG yields
pyoluteorin.
The biosynthetic gene cluster (plt) for pyoluteorin production,
regulation and efflux was discovered in P. fluorescens
Pf-5,293,294,297 where it encompasses 17 genes spanning a total of
about 30 kb (Fig. 20). The plt cluster shows several unusual
characteristics. The PKSs PltB and PltC lack a loading module
and a terminal TE; pltG, which encodes a TE, is located three
ORFs downstream of pltC. In addition, PltB contains a nonfunctional KR and PltC a defective DH domain. The formation
of the dichloropyrrolyl moiety in pyoluteorin is unique in several
aspects. A single halogenase (PltA) appears to be solely responsible for the chlorination at both positions of the pyrrole ring,
despite the presence of two additional genes encoding putative
halogenases in the plt cluster, i.e. pltD and pltM. Mutations in
pltD and pltM abolish pyoluteorin production by P. fluorescens
Pf-5,294 indicating an essential role of these genes in pyoluteorin
biosynthesis. On the other hand, PltD lacks a conserved NADH
binding site, which is essential to the function of other halogenases. Therefore, the functions of PltM and PltD in pyoluteorin biosynthesis remain unclear. Furthermore, it is unusual
that the pyrrolyl substrate is recognized and accepted only when
tethered to the carrier protein scaffold, whereas the free pyrrole2-carboxylate is not accepted for halogenation by PltA. Apart
from the structural genes pltABCDEFGLM, genes functioning in
Fig. 20 The plt gene cluster and model of pyoluteorin biosynthesis. Asterisks indicate an inactive domain.
Nat. Prod. Rep., 2009, 26, 14081446 | 1429
efflux of pyoluteorin (pltIJKNOP) and regulation (pltZ and pltR)

are present in the biosynthetic gene cluster.294,297299 The pyoluteorin biosynthetic locus of Pseudomonas sp. M18 is identical to
that of P. fluorescens Pf-5, although the gene nomenclature
deviates slightly from that used in Pf-5.298 A pyoluteorin
biosynthesis cluster of identical synteny is also present in
a genomic island of P. aeruginosa LESB58.7 Intriguingly, this
cluster is adjacent to a putative chalcone synthase most closely
related to phlD of P. fluorescens Pf-5.
2.3.3 Coronatine. The chlorosis-inducing non-host-specific
phytotoxin coronatine is produced by several pathovars of
P. syringae, including atropurpurea, glycinea, maculicola,
morsprunorum, and tomato.300302 Xanthomonas campestris pv.
phormiicola also produces analogs of coronatine.303,304 Coronatine acts as a virulence factor,305307 promotes entry of the
bacteria into the plant host by stimulating the opening of
stomata308 and suppresses salicylic acid-dependent host
defenses.309,310 The toxin is composed of the ethylcyclopropyl
amino acid moiety coronamic acid (CMA), which is linked to
a bicyclic hydrindane ring-based polyketide moiety termed
coronafacic acid (CFA). Due to the close resemblance of CMA
and CFA to precursors of the endogenous plant hormones
ethylene and jasmonic acid, respectively, coronatine is thought to
impact signaling in host plants via the ethylene and jasmonic
acid311 pathways.
Genes required for coronatine production were first identified
by Bender and coworkers in P. syringae pv. glycinea PG4180, in
which the 32.8 kb cor cluster is borne on the plasmid p4180.312

The intriguing biosynthesis of coronatine proceeds by formation
of CMA and CFA, which are then linked to produce coronatine.313,314 The structural genes for CFA and CMA biosynthesis
are located at opposite ends of the gene cluster (Fig. 21), separated by three regulatory genes (corPSR) involved in the transcriptional control of the two regions.315317
The nonproteinogenic character of CMA suggested the
involvement of a thiotemplate mechanism and the work of Parry
et al. demonstrated that CMA was derived from L-allo-isoleucine.318,319 Initially, a set of four genes, cmaABTU was identified.314 Three genes thereof, cmaABT, were proven to be essential
for coronatine production, while the role of cmaU remained
unknown. Later on, the genes cmaC, D and E were recognized in
the cma cluster and added by the Walsh group. The biosynthesis
of CMA starts with the didomain protein CmaA which catalyzes
the adenylation of L-allo-isoleucine and the attachment of this
activated amino acid to the CmaA PCP domain.320 The A
domain was proven to prefer L-allo-Ile over other branched
amino acids and represents the first example of an A domain
specific for this amino acid. How L-allo-Ile is synthesized remains
unknown. The aminoacyltransferase CmaE transfers the aminoacyl group from the PCP-domain of CmaA to a second PCP
domain, CmaD.321 While tethered to CmaD, the formation of the
cyclopropyl ring system occurs by cryptic halogenation. CmaB,
a member of the non-heme Fe2+a-ketoglutarate-dependent
enzyme superfamily, chlorinates the g-position of L-allo-Ile113 in
a regiospecific manner, thereby activating this position for
Fig. 21 Biosynthesis of coronatine. ACL: acyl-CoA ligase, CCR: crotonyl-CoA reductase.
1430 | Nat. Prod. Rep., 2009, 26, 14081446
further chemical reactions. CmaC then catalyzes the formation

of a kinetically stabilized a-C carbanion, which displaces the
g-Cl group in an intramolecular cyclization reaction to form
a cyclopropyl ring.322 CmaB was the founding member of a new
type of halogenases, the non-heme Fe2+ketoglutarate-dependent halogenases. Along with PrnA and B, CmaB provides
another example in which novel mechanisms of biohalogenation
were discovered through the analysis of secondary metabolite
biosynthesis pathways of Pseudomonas spp.
The polyketide portion of coronatine, CFA, is synthesized by
the cfa gene cluster consisting of nine open reading frames, cfa1
through cfa9323 (Fig. 21). The translation products of the first
three genes, cfa123, are related to ACPs, fatty acid dehydratases,
and b-ketoacyl synthetases, respectively. The role of cfa4 could
not be predicted, while Cfa5 showed sequence similarity to acylCoA ligases (ACL). Cfa6 and Cfa7 represent a two modular type
I PKS system responsible for CFA production. Cfa8 is also
required for CFA biosynthesis and represents a crotonyl-CoA
reductase (CCR) thought to be involved in the formation of
butyryl-CoA, which is in turn commonly used as a four-carboncontaining extender unit in polyketide synthesis. Cfa9, which
exhibits similarity to TE domains, has proven to be dispensable
for CFA production.324 With the exception of Cfa6 and Cfa7,
little is known about the interplay of the cfa gene products and
their role in CFA biosynthesis. The current proposed route to
CFA starts with a cyclopentenone compound, commonly
synthesized by coronatine producers,325 which is shuttled to the
first modular PKS, Cfa6 (Fig. 21). The starter unit is at first
elongated with ethyl malonate via the action of Cfa6. The
resulting intermediate is then transferred to the downstream PKS
Cfa7, where it is condensed with a malonate unit, yielding
a latent reactive product. Upon the latter elongation step, a
reactive b-keto thioester intermediate is formed that undergoes
a rapid conformationally controlled 6-endo-trig cyclization
before the tethered intermediate is reduced and dehydrated on
the assembly line by the remaining DH and KR domains of
Cfa7.326 These domains act subsequently on the enzyme-bound
bicyclic hydrindane skeleton to complete the structure of CFA.
The final step in the coronatine pathway is presumed to be the
ligation of CFA and CMA via formation of an amide bond.327,328
Small quantities of coronatine congeners were isolated from
P. syringae pv. glycinea when CMA was replaced by norcoronamic acid, Leu, Val, Ile, allo-Ile, Ser or Thr, indicating that
the responsible enzyme(s) catalyzing this reaction lacks rigid
specificity.329331
In many strains of P. syringae, the cor biosynthetic gene cluster
is carried on large (80110 kb) indigenous plasmids in the
pPT23A family that are transferred readily via conjugation
between strains of the pathogen.231 In other strains, such as
P. syringae pv. tomato DC3000, the cor genes are borne on the
chromosome where they are co-localized with other genes
involved in virulence, including two clusters of effector genes.47
Plasmid-encoded cor genes are typically in a single cluster
whereas chromosomal cor genes can be dispersed, as in the
genome of DC3000 where 26 kb separates the cfa and cma loci.
The region of the DC3000 genome containing the cfa and cma
gene clusters is enriched in mobile genetic elements of several
classes, which could provide a mechanism for HGT of the
region.47 The presence of a full complement of cfa genes on
a genomic island in the genome of another plant pathogen,

Pectobacterium carotovora pv. atroceptica, provides further
evidence for HGT of the locus. As in P. syringae, the cfa genes
contribute to virulence of P. carotovora pv. atroceptica,332
providing intriguing insights into the role of HGT in the evolution of pathogenesis in these bacteria.
2.3.4 Pederin. Pederin is a highly active cytotoxic agent,333
isolated originally from rove beetles in the species Paederus and
Paederidus, which use this structurally unique compound as
a chemical weapon against predators. Intriguingly, the occurrence of pederin was correlated to the presence of a bacterial
symbiont of the beetle, so the biosynthesis of pederin was
ascribed to a non-cultivated Pseudomonas species.334,335 Using
a metagenomic approach, Piel and co-workers confirmed this
hypothesis by cloning the pederin biosynthesis cluster from total
DNA isolated from the beetle Peaderus fuscipes.336,337 The ped
cluster and the pederin biosynthetic pathway have several
unusual and fascinating features. Whereas genes for the
biosynthesis, efflux, and regulation of secondary metabolites are
commonly clustered in a single site in Pseudomonas genomes, the
ped cluster is distributed on three distinct genomic regions,
designated pedABCDEFGH, pedIJK, and pedLMNOPQR
(Fig. 22 and Fig. 23). Due to the presence of transposase gene
fragments flanking each ped region, it has been hypothesized that
translocation of DNA to different genome regions may have
caused the observed fragmentation of a formerly intact gene
cluster. Furthermore, the modules are devoid of AT domains.
Instead, PedCD encode two acyl transferases, which act in trans
to acylate each PKS module in the cluster.
Pederin biosynthesis starts with the assembly of the pederin
tetraketide performed by the tetramodular PKS PedI (Fig. 22).
An acetyl starter unit is first selected and incorporated by
a GCN5-related N-acetyltransferase (GNAT). The three
Fig. 22 Organization of the pedIJK and pedLMNOPQR fragments of

the ped cluster. CR: crotonase domain, EST: esterase, GNAT: GCN5related N-acetyltransferase, HMGS: 3-hydroxy-3-methylglutaryl-CoA
synthase, OR: oxidoreductase.
Nat. Prod. Rep., 2009, 26, 14081446 | 1431
Fig. 23 Organization of the pedABCDEFGH fragement of the ped cluster and proposed biosynthetic scheme for pederin formation. OR: oxidoreductase.
remaining PKS modules of PedI catalyze the extension of the

backbone by three acetate units and trigger the cyclization of the
resultant tetraketide intermediate. The formation of the unusual
exomethylene group at the C-4 position in pederin is assumed to
be generated by the two crotonase domains (CR) in the third
module of PedI. As a mechanism, a nucleophilic attack of acetylCoA on a keto group, followed by a decarboxylative Grob-type
fragmentation of the resulting b-hydroxy-acid, has been
proposed.338 Since this reaction requires the involvement of an
HMG-CoA synthase, a KS, an ACP, and an additional CR
domain, it was hypothesized that pedLMNPQ, which encode
proteins possessing these catalytic properties, might participate in
exomethylene bond formation. The tetraketide intermediate
assembled by PedI is then transferred to mixed PKS/NPRS
megaenzyme PedF (Fig. 23). In a canonical fashion, the tetraketide is at first elongated with glycine and subsequently in four
cycles with acetate units. Noteworthy are the methyltransferase
domain in module four, presumably attaching the geminal
dimethyl group, and the tandem dehydratase domain in the
subsequent module, which is most likely responsible for pyran
ring formation. Finally, pedG, which encodes a FAD-dependent
monooxygenase, catalyzes oxidative cleavage of a two-carbon
extension unit in a BaeyerVilliger fashion in order to produce the
unusual and rare oxidized single-carbon terminus of pederin. The
additional PKS/NRPS gene pedH has no structural counterpart,
but the corresponding enzyme would hypothetically extend the
pederin structure to the skeleton of the sponge-derived compound
onnamide A. In addition to the previously-described biosynthetic
route, it can be also envisioned that PedH produces an onnamide1432 | Nat. Prod. Rep., 2009, 26, 14081446
like intermediate, which is then cleaved by PedG to form the

proposed intermediate 7,10-didehydroxy-6,10,17,18-tetrademethoxypederin. Upon the assembly of the basic carbon skeleton of
pederin, several hydroxylations and methylations occur as
tailoring reactions. Recently, PedO was shown to selectively
methylate the terminal 18-OH group.339 The additional methoxy
groups are presumably provided by the O-methyltransferases
PedA or PedE. Since pederin carries four methoxy groups, but
only three MT genes were identified, PedA or PedE could act
repeatedly. Alternatively, the fourth methylation could be performed by the beetle host. Bioinformatic analyses indicate that
PedR has regulatory functions, PedQ functions as an esterase,
and PedB and PedJ show similarity to oxidoreductases.
2.4 Other compounds
2.4.1 Phenazines. Phenazines are a large family of colorful
nitrogen-containing tricyclic molecules with antibiotic,
antitumor, and antiparasitic activity.54,340 The unusually broadspectrum activity of phenazines relies on interactions with polynucleotides, topoisomerase inhibition, and the generation of free
radicals.54 Among the Pseudomonads, strains of P. aeruginosa,
P. chlororaphis, and P. fluorescens are the most prominent producers
of phenazines.54,341 Pyocyanin (5-N-methyl-1-hydroxyphenazine)
is a pathogenicity factor produced by P. aeruginosa,342,343 whereas
phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic
acid, and phenazine-1-carboxamide are antifungal compounds
produced by rhizosphere isolates of P. fluorescens and
P. chlororaphis.54 These redox-active compounds function as
Fig. 24 Comparison of phenazine biosynthetic loci from different Pseudomonads and current biosynthesis scheme.
intercellular signals influencing transcriptional regulation of the

producing cell and having broad effects on bacterial physiology
and fitness, including biofilm formation.344346 In addition to
Pseudomonas spp., strains of Burkholderia, Brevibacterium,
Streptomyces, Pectobacterium, and Pantoea produce phenazines.54,340,347 More than 50 naturally-occurring phenazine
compounds have been described and a few strains produce ten
different congeners at the same time. The basic skeleton is usually
extended by hydroxyl or carboxylic acids groups on the benzene
ring moiety and by oxides and methyl groups on the nitrogen atoms.
The core phenazine biosynthetic locus (phzABCDF) is highly
conserved among phenazine-producing strains of Pseudomonas
spp., with some some species (such as P. aeruginosa) having two
copies (Fig. 24). Chorismic acid is converted to 2-amino-4-deoxychorismic acid (ADIC) by the anthranilate synthase homologue
PhzE. PhzD, a member of the isochorismatase enzyme family,
cleaves ADIC to generate trans-2,3-dihydro-3-hydroxyanthranilic
acid (DHHA). The isomerase PhzF performs a 1,5-prototropic
shift, yielding an enol that converts to the corresponding ketone
6-amino-5-oxocyclohex-2-ene-1-carboxylic acid. Subsequently,
two of these amino-cyclohexenone molecules undergo a symmetrical head-to-tail double condensation reaction, yielding a double
imine. While this bimolecular key step is spontaneous and does not
require enzyme catalysis in vitro,348 it is catalyzed by the dimeric
protein PhzA/B in vivo.349 Together, PhzA and its duplicate PhzB
form a small dimeric protein of the D5-3-ketosteroid isomerase/
nuclear transport factor 2 family, which acts as an acid/base catalyst and significantly increases the reaction rate by orienting two
substrate molecules and by neutralizing the negative charge of
tetrahedral intermediates through protonation. Possibly catalyzed
also by PhzA/B, the condensation product rearranges further to
prevent back-hydrolysis. The resultant rearranged condensation
product is prone to oxidation and undergoes oxidative decarboxylation in a nonenzymatic reaction. The final aromatization of the
ring system is performed by the FMN-dependent oxidase PhzG,
leading to PCA.350 PhzC, the remaining gene of the core operon,
encodes a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP)
synthase, which catalyzes the first step of the shikimate pathway. It
is hypothesized that PhzC acts to redirect intermediates from
primary metabolism into phenazine biosynthesis. The genes
responsible for the generation of the various known congeners of

PCA are either linked to the core operon (e.g. phzM encoding
a SAM-dependent N-methyltransferase in P. aeruginosa PAO1) or
reside elsewhere in the genome (e.g. phzH performing the conversion of carboxylic acids into carboxamides in P. aeruginosa
PAO1).351 A linked pair of regulatory genes in the luxR/luxI family
control phenazine production in a density-dependent manner, and
this system serves as a model for the role of quorum sensing in the
ecology of bacteria in natural environments.352
The phenazine gene cluster provides an excellent example of the
modification of secondary metabolites to a range of diverse
functions by auxiliary genes that complement a common set of
core genes. Across diverse bacterial genera, the core biosynthetic
genes (phzB, phzD, phzE, phzF and phzG) are conserved among all
phenazine-producing strains, indicating that they are essential for
synthesis of the phenazine scaffold.353,354 Variations in the core
phenazine cluster include the absence of the DAHP synthaseencoding gene phzC in some bacterial genera and presence of
redundant phzA/B genes in the Pseudomonads and Streptomyces
cinnamonensis.353,354 In P. aeruginosa PA01, the phenazine
biosynthesis genes are in a region of the chromosome having GC
content, codon usage patterns, and dinucleotide frequencies
typical of the genome at large. The phz gene clusters in other
Pseudomonas spp. are also located on the chromosome,54 but phz
genes are plasmid-borne in at least one bacterium, Pantoea
agglomerans Eh1087.355 In other species, such as the plant pathogen Pectobacterium carotovora subsp. atroseptica, phz genes are
located within a defined genomic island.332 In Mycobacterium
abscessus and Burkholderia cepacia, transposases and insertion
regions adjacent to the phz gene cluster could have played a role in
HGT of the region.353,354 Analysis of the fully sequenced genomes
available to date indicates that the phenazine locus has a complex
evolutionary history that includes HGT.353,354
2.4.2 Quinolones. Quinolone derivatives are small antibacterial alkaloids, isolated from several P. aeruginosa strains.356
The basic skeleton of 4-quinolone is typically substituted with an
alkane or alkene chain at position 2. Occasionally, the structures
are also formulated in the tautomeric forms and referred to as
4-hydroxy-2-alkylquinolines (HAQs). Additionally, N-hydroxy
Nat. Prod. Rep., 2009, 26, 14081446 | 1433
derivatives can be observed.341 The antibacterial activity of

P. aeruginosa or its preparations was discovered in the late 19th
century,357359 but it was not until 1945 that the bioactivity was
attributed to the presence of the quinolones Pyo I-IV.360 In
addition to their antibacterial roles, 4-quinolones also serve as
signal molecules for cell-to-cell communication. The model
compound 2-heptyl-3-hydroxy-4-quinolone was therefore
termed the Pseudomonas quinolone signal (PQS). PQS regulates
diverse target genes including those coding for elastase, rhamnolipid, the PA-IL lectin and pyocyanin, as well as diverse
phenotypes such as biofilm development and cellular fitness.
Chemical investigations and labeling experiments indicate that
PQS and other 4-quinolones originate from a head-to-head
condensation of anthranilate and b-keto-(do)decanoic acids.361363
The genes directing the biosynthesis of PQS were found by
serendipity during a random transposon mutagenesis screen in
P. aeruginosa PAO1 for genes involved in the regulation of the
phenazine pyocyanin synthesis. The screening approach hit the
phnAB region, which was thought previously to be involved in
phenazine biosynthesis, and led to the identification of the 9 kb
cluster364 (Fig. 25). It comprises seven structural genes encoding
PqsA, PqsB, PqcC, PqsD, PqsH, PhnA, and PhnB, and two genes
encoding regulatory functions, PqsE and PqsR. PhnA and PhnB
presumably synthesize the anthranilate precursor of PQS from
chorismate. Alternatively, in other P. aeruginosa strains, anthranilic acid can also be provided by the kynurenine pathway via
tryptophan degradation (Fig. 25).365 In the next steps, anthranilic
acid is condensed with b-ketodecanoic acid and subsequent
decarboxylative cyclization yields the PQS precursor 4-hydroxy-2heptylquinoline (HHQ). PqsABCD have been shown to be
essential for these biosynthetic steps. PqsA exhibits similarity to
benzoate A ligases, possibly involved in anthranilate activation,
whereas PqsB,C and D may be b-keto-acyl-ACP synthetases.
Fig. 25 The PQS biosynthetic operon of P. aeruginosa and biosynthetic

route to PQS.
1434 | Nat. Prod. Rep., 2009, 26, 14081446
However, the enzymatic functions of PqsABCD remain to be

elucidated. HHQ represents an interim end-product of the
biosynthesis gene cluster because, as a messenger of cell-to-cell
communication, it is released into the extracellular environment
immediately upon synthesis. HHQ can then be taken up by
neighboring cells und converted into PQS by the action of the
FAD-dependent monoxygenase PqsH.366 Subsequently, PQS is
exported and functions as an intercellular signal molecule.367
Homologs of the pqsABCDE cluster are present in the fullysequenced genomes of several Burkholderia spp.368,369 As in
P. aeruginosa, certain HAQs function as intercellar signaling
molecules in Burkholderia spp. The predominant HAQs
produced by Burkholderia spp. are methylated and saturated; the
less common unsaturated forms have a double bond at the
2-position, in contrast to the 1-position found in the unsaturated
HAQs produced by P. aeruginosa. Differences in the prevalence
of unsaturated vs. saturated forms of HAQs may reflect differences in the pool of fatty acid intermediates between Burkholderia spp. and P. aeruginosa. In addition to the core genes
hmqABCDE (also called hhqABCDE), the clusters in the Burkholderia genomes contain an additional two genes: hmqG, which
encodes a putative SAM-dependent methyltransferase, and
hmqF. HmqF, which is predicted to contain two acyl-CoA
dehydrogenase domains and an attachment site for 3-phosphopantetheine, could be responsible for the double bond at the
2 position in the unsaturated HAQs produced by Burkholderia
spp.,369 a possibility that has not yet been verified experimentally.
Alternatively, differences in the biosynthesis of the 3-keto fatty
acid intermediates carried out by pqsBCD vs. hmqBCD may
account for the altered position of the double bond in the
respective HAQ products. HmqG appears to be responsible for
the presence of the methyl group on the HAQs produced by
Burkholderia spp.369 Therefore, the HAQ gene cluster, like the
phenazine gene cluster described above, provides an additional
example of the modification of a secondary metabolite structure
by auxiliary genes that complement core genes of the biosynthetic gene cluster. Further chemical diversity may be afforded
by variations in the availability of primary precursors for
secondary metabolism, represented by pool of saturated vs.
unsaturated fatty acids in the case of HAQ.
2.4.3 Hydrogen cyanide. Hydrogen cyanide (HCN, syn.
prussic acid) is extremely poisonous to most organisms due to its
effective inhibition of cytochrome c oxidase and other metalloproteins. At physiological pH and in the absence of complexing
ions, HCN is largely undissociated, volatile (boiling point:
25.6 C) and colorless. Biological HCN production has been
demonstrated in many insects,370 higher plants371 and fungi,372
but in only a few species of bacteria in the genera Chromobacterium,373 Bacillus,374377 Burkholderia,378 and particularly
Pseudomonas.374,379383 HCN production by Pseudomonads
inhabiting the rhizosphere can be beneficial to the host plant
because it contributes to the suppression of various plant
diseases.384,385 In cystic fibrosis patients infected by P. aeruginosa,
elevated HCN concentrations were detected in the sputum, thus
suggesting that HCN production is a virulence factor.386
Early experiments indicated that Pseudomonads synthesize
HCN from glycine,387,388 which is converted by oxidative reactions
into HCN and CO2. Several reaction mechanisms have been
Fig. 26 The hcn gene cluster and current working hypotheses for
cyanide formation from glycine by Pseudomonads.
proposed for bacterial HCN biosynthesis,389 but the instability of

the produced intermediates and of the HCN synthase itself
impeded early efforts to define the biosynthetic pathway. It was not
until the HCN biosynthetic gene cluster of P. fluorescens CHA0
was described that the biochemistry of cyanogenesis was illuminated.390,391 Three contiguous structural genes, hcnABC, which
together encode a membrane-bound HCN synthase complex, were
shown to be sufficient for cyanogenesis. The amino acid sequences
of HcnB and HcnC are similar to amino acid oxidases (HcnB and
HcnC), an observation that supports the dehydrogenase biosynthesis model proposed by Wissing.383 According to this mechanism,
glycine is oxidized in the first dehydrogenase reaction to iminoacetic acid (Fig. 26). Subsequently, with the help of a second
dehydrogenase reaction, iminoacetic acid is converted into an
unstable nitrile derivative which undergoes in a third step a rapid

CC bond cleavage, yielding HCN and CO2.383,389 Because HcnA
showed similarity to formate dehydrogenases, LaVille et al.
proposed that HCN and formic acid are formed from the intermediate iminoacetic acid, whereby formic acid is further oxidized
to CO2 by HcnA.390 However, the exact functions and the interplay
of the HcnABC subunits remain to be elucidated.
The hcnABC operon is highly conserved in sequence and
organization among the cyanogenic strains of Pseudomonas
spp.,15,39,384 although the genomic context of the operon differs
among species. hcnABC is also present in the genomes of Chromobacterium violaceum and many species of Burkholderia,378
providing another example of shared metabolic capabilities of
the Pseudomonads and Burkholderias.
3 New compounds discovered by genome-guided

strategies in Pseudomonas spp.
From a natural product chemistry point of view, Pseudomonads
were known for decades only for the production of siderophores,
phenazines and small molecular weight antibiotics or phytoxins.341 This picture changed fundamentally as it became evident
that the genus has a tremendous capacity to assemble exciting
metabolites using intriguing biochemistry. Appreciation for the
Fig. 27 Circular representation of the genome of Pseudomonas fluorescens Pf-5. The outer scale designates coordinates in base pairs (bp), with the origin
of replication at 1 bp. The first circle (outermost circle) shows predicted coding regions on the plus strand, and the second circle shows predicted coding
regions on the minus strand, color-coded by role categories. The third circle shows the set of 1489 P. fluorescens Pf-5 genes that are not found in the
genomes of P. aeruginosa PAO1, P. syringae pv. tomato DC3000, and P. putida KT2440 (see Fig. 1A). The fourth circle shows the set of 1472 genes that
are not found in the genomes of P. fluorescens SBW25 or PfO-1 (See Fig. 1B). The fifth circle shows nine secondary metabolite gene clusters. The sixth
circle shows REP repeat elements. The seventh circle shows the PFGI-1 mobile island in olive, and phage regions. The eighth circle shows trinucleotide
composition. The ninth circle shows percentage G + C in relation to the mean G + C in a 2000-bp window. The tenth circle shows rRNA genes in green,
tRNA genes in blue.
Nat. Prod. Rep., 2009, 26, 14081446 | 1435
metabolic potential of the Pseudomonads increased further once

the prevalence of secondary metabolite gene clusters in their
genomes was discovered. In addition to illuminating the genomic
context and the comparative analysis of known metabolic clusters, as described above, genomic sequence data highlighted the
presence of many new genetic loci with the hallmark sequences
typical of NRPSs or PKSs in Pseudomonas genomes.9,29,49,61 It
quickly became evident that most Pseudomonads are capable of
producing, in addition to pyoverdines, an additional siderophore
(i.e. pyochelin, pseudomonine, achromobactin, or yersiniabactin),47,1,29,49 lipopeptides,392 and numerous non-ribosomally
derived peptides, polyketides, or hybrids thereof, many of
unknown structure.9,29,61 Indeed, bioinformatic analyses revealed
the presence of orphan biosynthetic gene clusters, i.e. loci
encoding secondary metabolites whose product is yet unknown,
in the genomes of almost all Pseudomonas spp. In P. fluorescens
Pf-5, for example, secondary metabolic gene clusters, including
those with known and unknown products, were estimated to
occupy approximately 6% of the genome29 (Fig. 27). Therefore,
the identified orphan gene clusters in Pseudomonas spp. are
comparable in number, novelty and diversity, to the prolific
Actinomycetes.
One of the most exciting outcomes of Pseudomonas genomics
is the discovery of novel traits, including secondary metabolite
production, exhibited by these bacteria. Here, we highlight the
novel compounds discovered through genome mining strategies
applied to Pseudomonas genomes.
3.1 Orfamides
The first new compounds to be identified from mining of Pseudomonas genomes were the orfamides discovered by the groups
of Gerwick and Loper37 from P. fluorescens Pf-5. Following
a bioinformatic analysis of the Pf-5 genome for genes encoding
NRPSs and PKSs, three orphan gene clusters were identified.29
One of the orphan gene clusters contained three contiguous genes
termed ofaABC, whose deduced amino acid sequences are similar
to NRPSs. Together, ofaABC comprise ten modules with the
typical CAPCP architecture of NRPSs synthesizing a decapeptide (Fig. 28). With OfaA lacking a typical initiation module
and OfaC having two TE domains near the C-terminus, the
cluster exhibits characteristics of NRPSs synthesizing CLPs in
other Pseudomonas spp. (see section 2.1.5). In silico analysis of
the substrate specificity of the A domains allowed the prediction
of the amino acid sequence of the resulting peptide product,
which indeed resembled that of CLPs of the viscosin group
(Table 3). The product of the ofa gene cluster was isolated using
a traditional bioassay-guided as well as a new genomisotopic
approach, in parallel. The latter approach uses an isotopicallylabeled amino acid, predicted to be a precursor of the considered
peptide, to guide the fractionation and purification process. In
the case of the ofa cluster, leucine was predicted to be present
four times in the final peptide and not to be present in other
metabolites. Hence, 15N-labeled L-leucine was chosen as the
labeled precursor and a 1H15N HMBC NMR experiment for its
detection. Both the genomisotopic and the bioassay-guided
Fig. 28 Organization of the ofa gene cluster and corresponding biosynthetic scheme for orfamide A formation.
1436 | Nat. Prod. Rep., 2009, 26, 14081446
strategies led to the isolation of orfamides A, B and C, which

represent founding members of a new class of CLPs characterized by a 3-hydroxy-dodecanoic or tetradecanoic (myristic) acid
connected to the N-terminus of a 10 amino acid containing cyclic
depsipeptide. The order and identity of the amino acids forming
the peptide chain of orfamide A, the dominant CLP produced by
Pf-5, conforms exactly to that predicted in silico. Due to flexibility in amino acid and fatty acid selection by the NRPS system,
Pf-5 produces orfamides B and C that differ in the amino acid
composition and in the length of the lipid side chain. As observed
for many CLPs produced by Pseudomonas spp., the stereochemistry of the amino acids and the generation of the fatty acid
moiety are not reflected in the ofa gene cluster. In the orfamides,
the occurrence of D-configured amino acids can not be fully
explained by the presence of C/E domains134 because the six of
the ten C domains that contain the characteristic C/E sequence
motif are not strictly associated with D-amino acids in the
resulting peptide. Regarding its biological significance, orfamide
A was shown to function as an antifungal agent and a biosurfactant, influencing swarming motility of Pf-5 and lysing
zoospores produced by an Oomycete plant pathogen.37
3.2 Rhizoxins
An orphan NRPS/PKS gene cluster was the second subject of
genomic mining approaches employed to identify novel metabolites produced by P. fluorescens Pf-5. Six of the nine genes in the
79 kb cluster (Fig. 29) have predicted functions as PKS or mixed
NPRS/PKS (rzxA-F), whereas the remaining genes have putative
functions as a tandem acyl transferase (rzxG), a methyltransferase (rzxI) and a P450 monooxygenase (rzxH). All PKS modules
lack integral AT domains and the tandem AT encoded by rzxG
provides a third example of ATs acting in trans in the Pseudomonads. The products synthesized from this pathway were
identified using a genomics-guided strategy involving insertional
mutagenesis and subsequent metabolite profiling.38,393,394 In this

way, five analogs of rhizoxin, one of which had not been
described before,38 were correlated with this cluster (Fig. 30).
Rhizoxins are 16-membered polyketide macrolides that exhibit
significant phytotoxic,395 antifungal396 and antitumoral397 properties by binding to b-tubulin,398 thereby interfering with
microtubule dynamics during mitosis. They were originally isolated from the fungus Rhizopus microsporus,396 but it is now
recognized that the compounds are not produced by the
fungus, but by the endosymbiotic bacterium Burkholderia
rhizoxinica.399,400
Except for some inactive KS-ACP modules and one twin ACP,
the order and number of the modules in the rzx cluster are
colinear with the backbone of the isolated structures. Intriguingly, the polyketide chain is branched at C-5 by an enzymatic
Michael addition involving the addition of an acetyl building
block to an acryloyl precursor.401 In general, the rzx cluster
resembles the rhi cluster in Burkholderia rhizoxinica, encompassing genes for the biosynthesis rhizoxin itself (Fig. 30).402
Noteable differences between the two clusters are the gene order
and the absence of a rhiJ equivalent, coding for an oxygenase, in
the rzx cluster. This oxygenase could be responsible for the
epoxide functionality at position C-2 and C-3 of rhizoxin, which
is absent in the rhizoxin analogs produced by P. fluorescens Pf-5.
Compound WF-1360 F, the predominant rhizoxin derivative
produced by Pf-5, and the new compound 22Z-WF-1360 F are
highly antifungal, anti-oomycetal, differentially phytotoxic and
cytotoxic.
In Pf-5, the rhx cluster is located in a very large lineage-specific
region of the genome that also includes fitD, a gene encoding
a functional insect toxin related to the Mcf toxin of Photorhabdus
luminescens.20 Rhizoxin provides another example of a biosynthetic locus shared among specific strains of Pseudomonas and
Burkholderia spp., and the fit cluster is the second example (along
with syringolin) of a locus shared with P. luminescens. Again,
Fig. 29 Biosynthetic gene cluster for rhizoxin analogs (rzx) including putative functions of the genes in P. fluorescens Pf-5 and biosynthetic pathway for
rhizoxin (rhi) in B. rhizoxinica. OXY: oxygenase, HC: condensation-heterocyclization, P450: cytochrome P-450 monooxygenase, B: domain involved in
b-branching, GNAT: N-acetyltransferase. Asterisks indicate domains with motifs deviating from consensus sequences of functional domains and
indicate therefore possibly inactive domains.
Nat. Prod. Rep., 2009, 26, 14081446 | 1437
Fig. 30 Rhizoxin and rhizoxin analogs obtained from P. fluorescens Pf-5.
HGT is a likely mechanism for the presence of these closely related

loci in diverse bacterial genera.
3.3 Enantio-pyochelin
The sequenced genome of P. fluorescens Pf-5 continued to be an
inspiration for the isolation of new compounds from Pseudomonads by genome-guided approaches. In the case of enantiopyochelin, Reimmann and coworkers discovered that the pch
cluster of Pf-5 (pchPf-5) deviated from the cluster in P. aeruginosa
PAO1 (pchPAO1).403 Three major differences were noticed
(Fig. 31). First, in contrast to the two operon-structure of
pchPAO1, all genes of pchPf-5 are organized in one operon. Second,

PchGPf-5 showed limited sequence similarity to its counterpart in
PAO1 and was therefore renamed to PchK. However, a motif
search indicated that PchK could have a reductase and/or
epimerase function and therefore could fulfill the reduction task
of PchGPAO1. Third and most strikingly, pchE of Pf-5 lacked, in
comparison with pchEPAO1, the E domain required to generate
the R configuration at the asymmetric center, C-40 (see section
2.1.4 and Fig. 31).403 According to the colinearity rules, the new
40 S enantiomers enantio-pyochelin I (40 S, 200 S, 400 S) and II (40 S,
200 R, 400 S) were predicted products of this biosynthetic pathway.
Enantio-pyochelin was isolated from P. fluorescens strain CHA0,
Fig. 31 Comparison of the two divergent pch gene cluster of P. aeruginosa and P. fluorescens Pf-5/CHA0 and the resultant stereoisomers of pyochelin.
1438 | Nat. Prod. Rep., 2009, 26, 14081446
a strain closely related to Pf-5 that contains the same modified

pch cluster, and its chemical structure and absolute configuration
were determined using a combination of spectroscopy and
chemical synthesis.403 The modification of PchE and therefore
the production of the optical antipode of pyochelin represent
a physiological advantage for the bacterium. Strain CHA0
utilizes ferric-complexes of enantio-pyochelin, but not pyochelin
itself, whereas P. aeruginosa utilizes pyochelin, not enantiopyochelin, for iron nutrition.403 By producing enantio-pyochelin,
strains Pf-5 and CHA0 have evolved a mechanism for sequestering iron in a form that is available to themselves, but not to
competing bacteria. It is hypothesized that this strategy provides
a competitive advantage for Pf-5 and CHAO in natural environments where concentrations of biologically-available iron are
limited.
3.4 Syringafactins AF
In silico mining of the genomes of P. syringae pv. tomato
DC300047 and P. syringae pv. syringae B728a49 by several
research groups led to the identification of an orphan biosynthetic gene cluster involved in the production of an octalipopeptide.61,392,404 The cluster, dubbed syf, consisted of two NRPS
genes (syfA and syfB) that are flanked by syfR, encoding a LuxRlike transcriptional regulator, and syfC and syfD, whose products may be involved in the export of the resultant lipopeptide.
Together, the two structural genes syfA and syfB encode eight
NRPS modules that are terminated by tandem TE domains
(Fig. 32). Inspection of the A domains of the NRPS for their
amino acid substrate specificities, as well as the bioinformaticsbased investigation of the corresponding C domains for hidden
epimerase activity, allowed the prediction of the resulting peptide
sequence:
lipid/L-Leu/D-Leu/D-Gln/D-Leu/L-Thr/D-Val/L-Leu/
404
D-Leu (Fig. 32).
Typical of the NRPSs for lipopeptides, the
N-terminal module of SyfA includes a C domain, which was
expected to recognize and condense a thioesterified fatty acid
from primary metabolism to the first amino acid of the peptide.
To test the hypothetical structure developed by bioinformatics,
Thomas and coworkers proceeded with metabolite analysis
applying the prediction and screening approach393 to P. syringae pv. syringae DC3000. After identification of optimal culture
conditions, six related octalipopetides, syringafactins AF, were

obtained.393 Their planar chemical structures were proposed
based on mass-spectrometry data and amino-acid analysis. With
the exception of one amino acid, the peptide sequence correlated
well with the bioinformatic prediction. The analogues syringafactin AF vary either in the length of the attached 3-hydroxy
fatty acid (C10 vs. C12) or in the amino acid residue integrated
into the molecule at position AA6 (Val, Leu, Ile). Both variations
can be readily attributed to a recognition flexbility of the A
domain of the NRPS module 6 towards branched amino acids
and of the initiating C domain of SyfA, respectively.
Surprisingly, in contrast to the CLPs produced by many
Pseudomonads, all isolated syringafactins are linear rather than
cyclic. The linear octalipopeptide corrugatin is produced by
Pseudomonas corrugata,405 but corrugatin possesses a non-functionalized lipid side chain (n-octanoic acid) and a peptide
(OH-His/Dab/Dab/Ser/Ser/OH-Asp/Dab/OH-Asp) containing
a high number of polar amino acids due to its physiological role
as a siderophore. In contrast, the syringafactins are not siderophores, but function, like CLPs, in swarming motility. Therefore, the syringafactins are related to the classical Pseudomonas
CLPs, forming a new group within this compound family.
4 Secondary metabolite gene clusters in the flexible

genome of Pseudomonas spp.
In the Pseudomonas spp., most secondary metabolites are
produced only by specific lineages, contributing to their
specialized associations with plant or animal hosts or altering
their competitive interactions with other organisms in the environment. Accordingly, the genetic loci for the biosynthesis of
these metabolites are, by definition, in the flexible genome of
Pseudomonas spp. Biosynthetic gene clusters are considered to be
among lifes most diverse and rapidly evolving genetic
elements,406 and their diversity is thought to be mediated
primarily via HGT followed by mutation, recombination, and
duplication or excision events.407,408 Gene clusters for secondary
metabolism are therefore considered to be prototypic members
of the prokaryotic mobilome, which consists of bacteriophages,
plasmids, transposable elements and associated genes.5 Indeed,
secondary metabolite biosynthetic clusters (coronatine or
Fig. 32 The syf biosynthesis gene cluster from P. syringae pv. tomato DC3000 and resultant chemical structures of syringafactins. The indicated
stereochemistry is deduced only by bioinformatics but not experimentally proven, and was therefore not included in the resultant structure.
Nat. Prod. Rep., 2009, 26, 14081446 | 1439
phytohormones such as indole acetic acid) have long been

associated with plasmids in pathovars of P. syringae,409
providing an obvious mechanism for HGT. In the vast majority
of strains, however, secondary metabolite gene clusters are
located on the chromosome, in regions that vary from species to
species and from strain to strain.
As described above, bacterial genomes are mosaics composed
of relatively stable core regions interspersed with more variable
regions that diverge from even closely-related strains with respect
to both the composition and organization of genes.5 Variable
regions are commonly described as genomic islands, defined as
horizontally acquired genomic regions. The sequence composition of genomic islands often deviates from that of the core
genome, and this deviation can sometimes be quantified from
sequence properties. Features reported to be associated with
genomic islands include the presence of flanking repeats,
mobility genes (e.g., integrases and transposases), proximal
transfer RNAs (tRNAs), atypical guanine and cytosine content,
and skews in dinucleotide or trinucleotide composition. Some
genomic islands are associated with mobile genetic elements or
integrate preferentially into regions flanked by tRNAs. The
structures and sequences of genomic islands or their flanking
regions do not always clearly reflect a mechanism for their
transmission or integration into the genome, however, because
variable regions continue to be shaped by a range of processes,
including excision, recombination, duplication, or mutation,5
and these obscure the evidence of HGT. Over time, the compositional variation between the core genome and GIs lessens, and
consequently, more ancient HGT events are detected with less
sensitivity.
Comparative genomic analysis of Pseudomonas spp. provides
convincing evidence that the remarkable diversity of secondary
metabolism in Pseudomonas spp. is commonly a consequence of
the acquisition of genomic islands containing biosynthetic loci.
By comparing the genomic sequences of different Pseudomonas
spp. or strains, genes unique to a given strain can be identified
(Fig. 1), and the distribution of these unique genes provides
a pictoral view of the mosaic nature of the bacterial genome.
Using P. fluorescens Pf-5 as an example, the non-random
distribution of the unique genes is striking (Fig. 27): blocks of
genes unique to Pf-5, whether defined at the strain or species
level, coincide on the genomic map. Therefore, the distribution of
lineage specific genes, whether defined at the strain or the species
level, provided one criterion used to identify genomic islands in
the Pf-5 genome.29 Other criteria were atypical trinucleotide
composition and the distribution of 1052 copies of a 34-bp REP
sequence. REP elements are repetitive and palindromic
sequences, varying in length between 21 and 65 bases, that are
found the in the extragenic regions of some bacterial genomes.410
The REP elements are clustered in the Pf-5 genome, with distinct
gaps that often correspond to regions of atypical nucleotide
content, the presence of prophages, and genes unique to Pf-5.29
In short, they segregate preferentially into the core genome
shared with other species of Pseudomonas and other strains of
P. fluorescens.
Strain Pf-5 produces at least nine secondary metabolites, and
biosynthetic gene clusters for many of these are located in
genomic islands identified using the criteria described above:
distribution of genes unique to the strain, atypical trinucleotide
1440 | Nat. Prod. Rep., 2009, 26, 14081446
skew, and the lack of REP sequences (Fig. 27).29 Therefore,

analysis of the genome of Pf-5 provides strong evidence for HGT
as a fundamental mechanism for the inheritance of metabolic
gene clusters, which is consistent with the conclusions from
studies focused on many of the individual compounds discussed
previously in this review.
Concluding remarks
Pseudomonas spp. exhibit enormous metabolic capabilities and

versatile biochemistry through their production of structurally
diverse, bioactive chemical structures. The metabolic pathways
for the biosynthesis of these compounds often reveal surprising
sophistication, as well as divergence from the model systems
studied in the Gram-positive actinomycetes. Some Pseudomonas
biosynthetic pathways have functionally combined modular,
dissociated, or chalcone synthase-like polyketide synthases with
adenylating enzymes or with components of fatty acid synthases
(2,4-diacetylphloroglucinol, pyoluteorin, mupirocin, coronatine). The common occurance of trans-AT polyketide synthases
in PKS biosynthetic gene clusters (mupirocin, pederin, rhizoxins)
is striking in the genus. Intriguingly, Pseudomonads also employ
cryptic halogenation in order to activate alipathic groups (coronatine). Many Pseudomonas pathways are composed of
enzymes that rearrange and modify primary metabolic products
to produce secondary metabolites (pyrrolnitrin, phenazine
1-carboxylic acid). Genomic analysis has highlighted major
themes contributing to chemical diversity in the Pseudomonads,
including variations in the numbers and composition of structural domains in the modular NRPSs and PKSs, the complementation of core genes by auxiliary genes (phenazines,
syringolins, and rhizoxin), and relaxed substrate specificities of
biosynthetic enzymes. Through genomic comparisions between
Pseudomonas and other Proteobacteria, notably Burkholderia
spp., it is clear that HGT is a major process leading to chemical
diversity operate across the phylum.
One of the contributions of genomics to prokaryotic biology is
a deepened recognition for the prevalence of HGT and its
consequences as a driving force enhancing microbial diversity.5
Comparative genomics provides ample evidence for the movement of biosynthetic gene clusters among microorganisms,
which, along with subsequent alterations, can translate into
novel chemical structures. This is certainly the case for Pseudomonas spp., and many of the structures discussed herein provide
clear examples of this phenomenon. In addition to the discovery
of novel structures, the identification of new biosynthetic loci in
Pseudomonas spp. has provided new direction for studies
exploring the biological significance of compounds, and in some
cases has revealed new aspects of the biology of the producing
strain. We predict that future exploration of novel gene clusters
in this heterogeneous genus will greatly expand the alreadyremarkable spectrum of secondary metabolites known to be
produced by Pseudomonas species.
Acknowledgements
We are grateful to Marcella Henkels, Rachel Blumhagen,

Edward Davis, and Kedy Shen for assistance in preparation of
this article. We express sincere thanks to D. Mavrodi,
L. Thomashow, S. Aguilera and A. Alvarez-Morales for sharing

unpublished data, and to I. Paulsen, S. Lory, and B. Swingle for
permission to revise figures. Research in the laboratory of J.L. is
supported by USDA-CSREES projects #2008-35600-18770
and #2006-35319-17427. H.G. gratefully acknowledges
financial support by the Deutsche Forschungsgemeinschaft (GR
2673/2-1).
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Genomics of secondary metabolite production

All in-text references underlined in blue are linked to publications on ResearchGate,

Available from: Joyce Elizabeth Loper

www.rsc.org/npr | Natural Product Reports

Genomics of secondary metabolite production by Pseudomonas spp

1408 | Nat. Prod. Rep., 2009, 26, 14081446

1 Introduction: An overview of Pseudomonas spp.

predicted genes, and GC contents range from 57.8 to 66.6%

four Pseudomonas species (Fig. 1). Therefore, the percentage of

Table 1 Selected strains of Pseudomonas spp. and their genomes

Chronically-infected cystic fibrosis

Harald Gross studied pharmacy

Joyce E. Loper studied biology

Nat. Prod. Rep., 2009, 26, 14081446 | 1409

1410 | Nat. Prod. Rep., 2009, 26, 14081446

This journal is The Royal Society of Chemistry 2009

Factors contributing to virulence of P. aeruginosa are a subject of

function as opportunistic human pathogens.19 Certain members

disseminated by water or in association with a plant host, and

2 Biosynthetic enzymes, genes and gene clusters of

2.1 Non-ribosomally produced peptides and other amino

Unusual location of the E domain

P. syringae pv. syringae

P. syringae pv. syringae

Pseudomonas sp. MIS38

Isonitrile synthase, nonenzymatic conversion

P. syringae pv. tabaci,

identified as precursors for biosynthesis of the chromophore via

Iterative use of an NRPS module, reductive chain

hydroxyaspartate.72 During the assembly or upon completion of

hydroxylases (PvcC) and proteins of the cytochrome c protein

production by P. aeruginosa.78 Therefore, although P. aeruginosa

gene composition is generally conserved, the organization

pch cluster.8587 As illustrated in Fig. 4, the cluster consists of the

This journal is The Royal Society of Chemistry 2009

Nat. Prod. Rep., 2009, 26, 14081446 | 1415

P. aeruginosa, but is different from that in P. fluorescens Pf-5, as

pseudomonine. Very recently, it was demonstrated by in vitro

1416 | Nat. Prod. Rep., 2009, 26, 14081446

This journal is The Royal Society of Chemistry 2009

1418 | Nat. Prod. Rep., 2009, 26, 14081446

This journal is The Royal Society of Chemistry 2009

syringopeptin SP22. However, analogous to the syr gene cluster,

D-configured amino acids in the molecule despite the lack of

Putisolvin. The putisolvins I and II were the first known CLPs

1420 | Nat. Prod. Rep., 2009, 26, 14081446

This journal is The Royal Society of Chemistry 2009

identified in P. putida PCL1445 and shown to be responsible for

incorporated by three modules was revealed in the homologous

results in reduced availability of Gln, which blocks the only

acyltransferase), and TblE in conjunction with TblF (putative

1422 | Nat. Prod. Rep., 2009, 26, 14081446

This journal is The Royal Society of Chemistry 2009

and a tyrosine recombinase, which is also associated with HGT

and Orn, which are synthesized by transamination from Arg and

has been reported from other Pseudomonas spp., as well as

the genome lacking REP regions, as described in Section 4. In

Fig. 15 A) Pathways leading from Trp to IAA in Pseudomonads: [I]

The beneficial plant growth-promoting Pseudomonas strains,

Polyketides are a large class of structurally-diverse natural

codes for six multi-domain enzymes (MmpAF) with sequence

1426 | Nat. Prod. Rep., 2009, 26, 14081446