Antonie van Leeuwenhoek 64: 85-107,1993.

© 1993KluwerAcademic Publishers. Printedin the Netherlands.

Genetics of lactobacUli: plasmids and gene expression

Peter H. Pouwels & Rob J. Leer

Department Molecular Genetics and Gene-Technology, T N O Medical Biological Laboratory, Postbox 5815,
2280 H V Rijswijk, The Netherlands

Received1July1993;accepted31 August1993

Key words: Lactobacillus, plasmid vector, DNA replication, structural stability, segregational stability,
chromosomal integration, transcription, promoter, antisense RNA, translation, codon usage,
protein secretion, heterologous gene expression

Abstract

This paper reviews the present knowledge of the structure and properties of small (< 5 kb) plasmids present in
Lactobacillus spp. The data show that plasmids from Lactobacillus spp., like many plasmids from other Gram-
positive bacteria, display a modular organization and replicate by a mechanism of rolling circle replication.
Structurally, plasmids from lactobacilli are closely related to plasmids from other Gram-positive bacteria.
They contain elements (plus- and minus origin of replication, element(s) for control of plasmid replication,
mobilization function) showing extensive similarity to analogous elements in plasmids from these other orga-
nisms. It is believed that lactobacilli have acquired such elements by intra- and/or intergenic transfer mecha-
nisms. The first part of the review is concluded with a description of plasmid vectors with a Lactobacillus
replicon and integrative vectors, including data concerning their structural and segregational stability.
In the second part of this review we describe the progress that has been made during the last few years in
identifying and characterizing elements that control expression of genetic information in lactobacilli. Based
on the sequence of eleven identified and twenty presumed promoters, some preliminary conclusions can be
drawn regarding the structure of Lactobacillus promoters. A typical Lactobacillus promoter shows significant
similarity to promoters from E. coli and B. subtilis. An analysis of published sequences of seventy genes
indicates that the region encompassing the translation start codon A U G also shows extensive similarity to that
of E. coli and B. subtilis. Codon usage of Lactobacillus genes is not random and shows interspecies as well as
intraspecies heterogeneity. Interspecies differences may, in part, be explained by differences in G + C content
of different lactobacilli. Differences in gene expression levels can, to a large extent, account for intraspecies
differences of codon usage bias. Finally, we review the knowledge that has become available concerning
protein secretion and heterologous gene expression in lactobacilli. This part is concluded with a compilation
of data on the expression in Lactobacillus of heterologous genes under the control of their own promoter or
under control of a Lactobacillus promoter.

Introduction for centuries in the preparation and processing of

foods and beverages (Rose 1982; Kandler 1984;
The genus Lactobacillus belongs to the family of Chassy 1985, Chassy 1987). These rod-shaped, an-
Lactobacteriaceae also known as lactic acid bacte- aerobic to micro-aerophilic, non-pathogenic bacte-
ria, a group of microorganisms that have been used ria, which produce lactic acid as their major end-
86
product, are nowadays used in numerous fermenta-
tion processes, either alone or together with other
organisms such as streptococci, pediococci, lacto-
cocci, leuconostocs and yeasts (Kilara & Treki
1984). Particularly in dairy industry these bacteria
are of vital importance. Fermented dairy products
represent about 20% of the total economic value of
fermented foods worldwide (Sharpe 1979). In addi-
tion, lactobacilli are used in the production and
preservation of sausages and meat, in fermentation
of olives and vegetables, in baking, and in the prep-
aration of silage. To maintain products of high qual-
ity and to guarantee reproducibility of production
processes, cultures of bacteria with selected and
predictable properties are used as starters to inoc-
ulate food or feed.
Some fermented milk products for many years
are believed to have certain health promoting prop-
erties for humans. This assumption was originally
largely based upon Metchnikoff's theory (Metchni-
koff 1908) that harmful effects of undesired bacteria
can be overcome by establishing a new balance be-
tween intestinal bacteria, through ingestion of lac-
tobacilli or fermented products made by these orga-
nisms. Research carried out during the last few dec-
ades has resulted in additional claims of health and/
or nutritional benefits for humans and animals as-
sociated with the consumption of fermented milk
products. Such claims include: control of intestinal
infections, improved nutritional value of some
foods, control of serum cholesterol levels, improve-
ment of lactose metabolism, induction of a-specific
and specific immune responses, and anti-carcino-
genic activity (Fernandes et al. 1987; Gurr 1987;
Perdigon et al. 1988; Gerritse et al. 1991a, b). Some
of these - assumed - properties of certain Lactoba-
ciUus species have also focused interest on their use
as probiotics to improve the growth potential of
livestock.
Due to its great economic importance for the
agro-feed sector and its alleged importance for hu-
man and animal health, research on selection and
characterization of Lactobacillus strains, on metab-
olism, physiology and genetics of these organisms,
and investigations aimed at the improvement of the
characteristics of such strains have increased pro-
gressively over the last decade. Based on knowl-
edge and methods applied to the development of
genetic manipulation systems for other microbes, in
particular Escherichia coli, Bacillus subtilis and
Lactococcus, rapid progress has been made in de-
veloping the methodology for genetic manipulation
of lactobacilli by recombinant DNA techniques.
Initially, research was focused on the characteriza-
tion of Lactobacillus DNA by cloning and expres-
sion of DNA fragments in E. coli, and on the isola-
tion and characterization of phages and plasmid
DNA molecules. Aided by the development of a re-
producible system to transform Lactobacillus
strains (Chassy & Flickinger 1987; Aukrust & Nes
1988; Luchansky et al., 1988), methods became
available to (re)introduce DNA into these orga-
nisms. This has prompted studies of more funda-
mental processes like DNA replication and control
of gene expression, as well as studies aimed at the
improvement of specific properties of lactobacilli.
As a result of the efforts carried out in a number of
laboratories, Lactobacillus research has come of
age. Methods for the introduction and stable main-
tenance of DNA into Lactobacillus are routine now
and can be applied to almost any Lactobacillus spe-
cies. Both broad host-range and narrow host-range
multi-copy plasmid vectors based on a variety of
replicons have become available for the introduc-
tion and expression of homologous and heterolo-
gous genes. Finally, methods have been developed
to insert genes at specific sites of the chromosome
allowing genes to be mutated at will.
No attempts will be made in this review to be ex-
haustive with respect to the genetics of lactobacilli.
In this review the state of the art will be presented of
our knowledge concerning plasmid structure and
plasmid replication. Secondly, a description will be
given of our present knowledge of gene structure
and gene expression of lactobacilli. For further de-
tails on the chromosomal organization of the Lacto-
bacillus genome, on the genetics of phages and
phage resistance, and gene-transfer systems the
reader is referred to recent review articles (Chassy
& Murphy 1993; Le Bourgeois et al. 1993; Mercenier
et al. 1993; Pouwels et al. 1992).

Plasmids
Occurrence
Plasmids have been found in many Lactobacillus
spp. since they were first discovered in 1976 by
Chassy and co-workers (Chassy et al. 1976; Hofer
1977; Chassy et al., 1978; Smiley & Fryder 1978; Ishi-
wa & Iwata 1980; Klaenhammer & Sutherland 1980;
Vescovo et al. 1981; Vescovo et al. 1982; Morelli et al.
1983a, b; Klaenhammer 1984; Nes 1984; West &
Warner 1985; Jewell & Collins-Thompson 1989;
Rinckel & Savage 1990). Several studies concerning
plasmid contents of lactobacilli isolated from plant
material (Daeschel et al. 1987), meat (Ahrn6 et al.
1989; Ahn & Stiles 1990), silage (Hill & Hill 1986),
sour dough (Spicher & L6nner 1985; L6nner et al.
1990) and the gastro-intestinal tract (Klaenhammer
& Sutherland 1980; Vescovo et al. 1982; Lin & Sav-
age 1985) have been reported. From these studies it
has become clear that many, but not all species, har-
bour one or more plasmids. Only a fraction of L.
plantarum strains isolated from different sources
was reported to contain plasmids (Klaenhammer
1984; Nes 1984; West & Warner 1985; Hill & Hill
1986; Von Hysby & Nes 1986; Mayo et al. 1989). Dif-
ferences in plasmid content of Lactobacillus strains
reported by different investigators, may in part be
explained by differences in plasmid extraction
methods. However, also differences in source of
strains can account for such differences. In contrast
with most of the aforementioned studies, Ruiz-Bar-
ba et al. (Ruiz-Barba et al. 1991) detected a variety
of plasmids in each of the 35 L. plantarum strains
from olives and many of the plasmids were found to
be of considerably higher molecular weight than
those previously observed. In Lactobacillus strains
isolated from the gastro-intestinal tract from chick-
ens and mice, plasmids were detected in a minor
fraction of strains analyzed (Lin & Savage 1985;
Posno, pers. comm.). Plasmids are also rarely found
in L. bulgaricus strains (Chassy, pers. comm.). L.
bulgaricus strain 10 (Culture Collection at North
Carolina State University) and L. bulgaricus strain
M-878 (Meiji Institute of Health Science, Japan) ap-
pear to form the exception (Chagneaud et al. 1992;
Sasaki, pers. comm.). Plasmids with variable de-
87
grees of sequence homology, as revealed by DNA-
DNA hybridization and/or nucleotide sequence
analysis, are found in several species (Bringel et al.
1989; Josson et al., 1989; Vogel et al. 1991; Leer et al.
1992), suggesting that horizontal transfer of plas-
mids and/or recombination between plasmids are
rather frequent events.
Although their ubiquitous presence is well estab-
lished, little is known about the function of plasmids.
Unlike in lactococci, where the occurrence on plas-
mids of genes involved in essential functions like
proteolysis (Kok 1990) or sugar metabolism (Gasson
1990), and other traits like phage resistance and con-
jugal transfer (Sanders et al. 1986; McKay & Baldwin
1984; de Vos et al. 1984) and bacteriocin production
(Klaenhammer 1993) is quite common, few such
plasmid-borne traits have been found in lactobaciUi.
During the last few years extensive research has
been carried out to link plasmid content with specific
bacterial characteristics. Except for cases where
plasmid content could be correlated with such phe-
notypical properties as drug resistance (Ishiwa &
Iwata 1980; Vescovo et al. 1982; Morelli et al. 1983a, b;
Axelsson et al. 1988), N-acetyl-glucosamine and slow
acid formation (Smiley & Fryder 1978), glucidic me-
tabolism (Liu et al. 1988), carbohydrate metabolism
(Chassy et al. 1978; Chassy & Rokaw 1981; Shimizu-
Kadota 1987; Kanatani et al. 1992), aminoacid me-
tabolism (Shay et al. 1988), bacteriocin production
and immunity (Muriana & Klaenhammer 1987;
Schillinger & Lficke 1989; Van der Vossen et al., in
preparation) or slime production (Ahrn6 et al. 1989),
most plasmids in Lactobacillus, in particular small
plasmids, are cryptic.
Segregational stability
Most indigenous Lactobacillus plasmids are segre-
gationally stable. A number of strains have already
been propagated under experimental conditions
for many years without noticeable changes in plas-
mid content. Some exceptions have, however, been
found. For example, maltose utilization which is a
plasmid-borne trait in some lactobacilli present in
meat, was found to be unstably inherited (Liu et al.
1988). Three consecutive transfers in the presence
88
of acriflavine resulted in an almost complete loss of
the plasmid. Similar phenomena where observed
for plasmid-linked galactose utilization markers
from a strain of L. acidophilus (Kanatani et al. 1992)
and plasmid-borne lactose markers (Chassy et al.
1978). In these cases the markers were found to be
present on large plasmids (40-80 MDa). The rela-
tive instability of large plasmids contrasts that of the
small cryptic plasmids. A number of plasmids found
in strains of e.g.L, plantarum or L. pentosus cannot
be eliminated by cultivation of the bacteria at sub-
lethal temperatures in the presence of acriflavine or
novobiocin (Bringel et al. 1989; Leer, unpublished
observations), conditions that are effective in
curing of most other bacterial strains of endoge-
nous plasmids. The presence of these small plas-
mids might confer a selective advantage over
strains lacking them under conditions used outside
the laboratory, although they appear not to carry
essential genes.
Plasrnid structure
The nucleotide sequence of eight small cryptic plas-
mids from Lactobacillus spp. has been determined.
Plasmids pLB4 (Bates & Gilbert 1989), pC30il
(Skaugen 1989), pLPI (Bouia et al. 1989), p8014-2
(Leer et al. 1992) and pAl (Vujcic & Topisirovic
1993) are from L. plantarum, p353-2 (Leer et al.
1992) from L. pentosus, pLJ1 (Takiguchi et al. 1989)
from L. helveticus and pLAB1000 (Josson et al.
1990) from L. hilgardii. The organization of the
plasmids, which vary in size from 1.9-3.5 kb, is very
similar. All plasmids, except pLJ1, code for a pro-
tein showing similarities to the replication proteins
(Rep) of plasmids from Gram-positive bacteria.
The involvement of this protein in replication has
been demonstrated by in trans complementation
assays (Bringel et al. 1989; Josson et al. 1990). Based
on the functionality of these proteins as replication
proteins and their structural similarity to replica-
tion proteins from other plasmids, it was assumed
that small plasmids from Lactobacillus spp. repli-
cate by a mechanism of Rolling Circle Replication
(RCR). Direct proof for this hypothesis has been
obtained for pLAB1000 and p353-2, by showing the
accumulation of single-stranded DNA replication
intermediates (Josson et al. 1990; Leer et al. 1992).
The minimal replication region has been deter-
mined for pLAB1000 in Bacillus, Enterococcus and
Lactobacillus strains as being 1.5 kb (Josson et al.
1990). Based on the similarity of the structure of
pLAB1000 and other plasmids from Lactobacillus
spp. with that of other RCR plasmids from Gram-
positive bacteria, it seems fair to assume that the
minimal replication region for all other Lactobacil-
lus plasmids is comparable in size.
Replication protein
The replication proteins of RCR plasmids have
nicking-closing activity at a specific site of the plus-
DNA strand, called plus-origin of replication, dso
(double-stranded DNA origin, previously called ori
(+)) as was first demonstrated for pT181 (Koepsel et
al. 1987). RCR plasmids have been grouped into
four families based on structural similarities of the
replication proteins and cognate dso (Gruss & Ehr-
lich 1989; Bron 1990; Ilyina & Koonin 1992), exem-
plified by pT181 (Khan & Novick 1983), pUBll0
and pC194 (McKenzie et al. 1986, 1987; Horinouchi
& Weisblum 1982b), pLS1 and pE194 (Lacks et al.
1986; Horinouchi & Weisblum 1982a) and pSN2
(Khan & Novick 1982). All plasmids from Lactoba-
cillus spp. either belong to the second or third cate-
gory, as judged by this criterium (Table 1). Although
Rep proteins and dso sequences from pLAB1000
and pUB110 display extensive similarity, no trans-
complementation is observed (Josson et al. 1990),
indicating that Rep proteins show strict specificity
for their target nicking site, even towards members
of the same family. A similar phenomenon was ob-
served for plasmids from the pT181 family (Iorda-
nescu & Projan 1988).
The Rep proteins from pAl and pLB4 are similar
to pE194 and other members of the pLS1 family of
plasmids. Plasmid pLJ1 is a 3.3 kb plasmid contain-
ing one open reading frame (ORF) which could
code for a protein of 41 kDa, presumed to be the
replication protein. No sequence similarity with
other known (replication) proteins has been found,
suggesting that the Rep protein of pLJ1 might rep-
 89

T a b l e 1. Characteristic features of L a c t o b a c i l l u s plasmids.

Plasmid Strain Size (kb) dso sso Mob Repressor References

pLJ1 L. h e l v e t i c u s 3.3 ? palL - - Takiguchi et al. 1989

pLAB1000 L. hilgardii 3.3 pC194 ? + - Josson et al. 1990
p353-2 L. p e n t o s u s 2.4 pC194 paiL - - Leer et al. 1992
pAl L. p l a n t a r u m 2.8 pE194 ? - + Vujcic & Topisirovic 1993
pLPI L. plantarum 2.1 pC194 pal L - - Bouia et al. 1989
p8014-2 L. p l a n t a r u m 1.9 pC194 palL - - Leer et al. 1992
pC30il L. p l a n t a r u m 2.1 pC194 palL - - Skaugen 1989
pLB4 L. p l a n t a r u m 3.5 pE194 palL + + Bates & Gilbert 1989

resent a new class of RCR replication proteins. Al-
ternatively, pLJ1 might replicate by a different
mechanism. A comparison of the structure of repli-
cation proteins of a variety of plasmids from Sta-
phylococcus, Bacillus, Streptococcus and Lactoba-
cillus indicates that the Rep proteins from Lactoba-
cillus plasmids pLAB1000, pC30il, pLP1, p8014-2
and p353-2 form a cluster that is closely related to
the Rep protein of pUB110 but more distantly relat-
ed to that of pC194 (Alonso pers. comm.).
Minus-origin of replication
The formation of complementary DNA on single-
stranded DNA intermediates is initiated at a specif-
ic site of the plus-DNA strand, the minus-origin of
replication, sso (single-stranded DNA origin), dis-
playing complex secondary DNA structure (Del
Solar et al. 1987; Gruss et al. 1987; Boe et al. 1989;
Devine et al. 1989). Minus-origins of replication are
usually not essential for plasmid replication, but in
their absence the amount of single-stranded DNA
intermediates increases, which may result in a de-
creased copy number and segregational instability
(Del Solar et al. 1987; Gruss et al. 1987; Deng et al.
1988). Their functioning is host dependent (Del So-
lar et al. 1987; Gruss et al. 1987; Boe et al. 1989). Ex-
cept for the minus-origin of pUBll0, which is func-
tional in more than one host, most minus-origins
present on staphylococcal plasmids like pT181,
pC194 and pE194 are not functional in B. subtilis
(Gruss et al. 1987). Based on sequence compari-
sons, at least three families of minus-origins of rep-
lication have been found, designated palA, palT
and palU (Bron 1990).
The presence of a minus-origin of replication in
plasmid pLAB1000 from L. hilgardii was predicted
from an analysis of the secondary structure (Josson
et al. 1990). For plasmid p353-2 the functionality of
an inverted repeat sequence in conversion of single-
stranded to double-stranded DNA was determined
by mutation deletion analysis. The inverted repeat
in p353-2 shows strong similarity to similar inverted
repeats in pLB4, p8014-2, pLP1, pC30il and pLJ1,
but not to any of the minus-origin sequences found
in plasmids from other bacterial genera, and thus
represents a new class of minus-origins of replica-
tion, named palL (Leer et al. 1992). The high level
of similarity (74-98 %) among minus-origins of rep-
lication of Lactobacillus plasmids mentioned above
is even more striking, because these plasmids show
otherwise no sequence similarity. The finding that
the same type of minus-origin of replication is
found in plasmids with different types of replication
protein and cognate dso, suggests that also plasmids
from Lactobacillus spp. have a modular organiza-
tion, comprising sequence elements that are hori-
zontally transferred from and to other Lactobacil-
lus strains and possibly even other genera. Such a
phenomenon has already been observed for DNA
cassettes from other plasmids (Projan & Novick
1988; Gruss & Ehrlich 1989).
The observation that minus-origins of replica-
tion, which are virtually identical (> 98% similari-
ty), are found in two plasmids (p8014-2 and pLJ1)
from different Lactobacillus species, suggests that
the palL type of minus-origin is functional in differ-
ent hosts, unlike most other minus-origin of repli-
90
cation. Conversely, the palU type of minus origin
from pUBll0, which is functional in both Bacillus
and in Staphylococcus, was found not to be func-
tional in L. casei (Shimizu-Kadota et al. 1991).
These authors also reported that the palA type of
minus origin from pC194 and phage M13 decreased
the accumulation of ss-DNA intermediates in L.
casei, indicating that, besides palL, also other types
of minus origin may be functional in Lactobacillus.
Mobilization functions
Plasmids pLB4 and pLAB1000 carry a gene coding
for a 42-46 kDa protein which is presumably in-
volved in plasmid mobilization. The protein encod-
ed by pLAB1000 was shown not to influence DNA
replication in Lactobacillus, Bacillus and Staphylo-
coccus strains (Josson et al. 1990). The mob gene
product is thought, by analogy with similar proteins
from pE194, pT181 and pUB110, to fuse plasmids at
a specific co-integration site called, RSa, allowing
mobilization of non-conjugative plasmids. An R S A
site showing considerable similarity to that present
in pE194 and pT181, has been found in plasmids
pLB4 and pLAB1000 (Bates & Gilbert 1989; Josson
et al. 1990), as well as in pAl (Vujcic & Topisirovic
1993). interestingly, pAl does not code for a Mob
protein but contains an ORF which could code for a
protein of 103 amino acids showing extensive simi-
larity to the N-terminal region of Mob proteins
from other plasmids. This truncated mob gene is
probably not expressed as no expression signals
were found 5' to the gene and no protein of the ex-
pected size could be demonstrated when the plas-
mid was expressed in vitro (Vujcic & Topisirovic
1993). The R S A site in pLAB1000 overlaps the pro-
moter of the mob gene as was found for plasmid
pT181 (Gennaro et al. 1987).
Control of D NA replication
Plasmid DNA replication is regulated through neg-
ative control mechanisms by plasmid-borne repres-
sors. These trans-acting repressors bind to plasmid-
specific targets, controlling plasmid replication in
an indirect way (Novick 1987; Thomas 1988). For
many plasmids antisense RNA (also called counter-
transcript RNA or CT-RNA) has been implicated
in the mechanism of control of plasmid DNA repli-
cation. These CT-RNAs, which are complementary
to the 5' end of Rep protein encoding RNA, inter-
fere with the expression of the rep gene, by a mecha-
nism of transcription attenuation (Novick et al.
1989) or by inhibition of translation initiation
(Alonso & Taylor 1987; Maciag et al. 1988; Del Solar
& Espinosa 1992).
In only one of the plasmids from Lactobacillus
spp., pLB4 from L. plantarum, has a gene been
found which might encode a repressor protein. The
protein shows similarity to repressors encoded by
members of the pLS1 family of plasmids. By anal-
ogy with pLS1, the pLB4 repressor might repress
the synthesis of rep RNA and, in addition, control
its own synthesis (Del Solar & Espinosa 1992).
These authors suggested that control of DNA repli-
cation in pLB4 is also exerted at the level of trans-
lation initiation by a CT-RNA which sequesters the
Shine-Dalgarno sequence of rep RNA (Shine &
Dalgarno 1974).
Plasmid p353-2 of L. pentosus codes for two CT-
RNAs of ~ 75 and ~ 250 nucleotides transcribed
from the region encoding the 5' end of the rep gene
and in the opposite direction (Pouwels et al. 1993).
CT-RNA negatively controls plasmids DNA repli-
cation. Control of plasmid replication is exerted by
a mechanism involving transcription attenuation of
rep RNA at a site just in front of the rep gene. In the
presence of CT-RNA (wild-type plasmid) more
than 90% of transcription initiated at an upstream
promoter is prematurely terminated at this atten-
uator, whereas in the absence of CT-RNA nearly all
transcripts reach a size corresponding to that of the
rep gene (Fig. 1). A computer-assisted analysis of
RNA secondary structures indicates the presence
near the 5' end of rep RNA of a sequence which can
hybridize to part of the attenuator sequence, pre-
cluding the formation of the attenuator stem-loop
structure. Secondary structure predictions also sug-
gest that CT-RNA induces a conformational
change of the 5' untranslated region of rep RNA,
resulting in the formation of the transcription ter-
minator (Pouwels et al. 1993).

Promoter region of repA gene of plasmid p353-2
~ 190hi
PrepA
Pml I Pml I P CT
Fig. 1. Promoter region of repA gene of plasmid p353-2 from L. pentosus, repA RNA synthesis is controlled by an antisense RNA
(CT-RNA) which induces the formation of a transcription attenuator, just in front of the ATG codon. As a result, more than 90% of all
transcripts initiated at the promoter of repA are arrested at the attenuator. Deletion of the promoter of CT-RNA (Pm/I fragments) results
in readthrough at the attenuator which is accompanied by a 5-10-fold increase of the plasmid copy number.
The dissimilarity of the replication proteins but
similarity of the organization of the replication reg-
ulatory region and mode of regulation of DNA rep-
lication of p353-2 and members of the pT181 family
of plasmids is of importance with respect to the evo-
lutionary relationship between the two plasmids. In
contrast to p353-2 and pT181, but similar to plasmid
pLS1, control of replication of pC194 and pUB110
takes place post-transcriptionally and might in-
volve sequestration of the translation-initiation sig-
nals (Alonso & Taylor 1987; Maciag et al. 1988).
These data indicate that the mode of control of
DNA replication is different for different members
of the pUB110 family of plasmids. Apparently, rep-
lication proteins and replication control region are
found in different combinations, presumably as a
result of horizontal transfer of DNA elements (Pro-
jan & Novick 1988; Gruss & Ehrlich 1989; Ilyina &
Koonin 1992; Pouwels et al. 1993).
Plasmid vectors
General properties
Broad-host-range vectors like the lactococcal plas-
mid pGK12, which has been shown to replicate in a
variety of bacterial strains (Kok et al. 1984), can also
replicate in different Lactobacillus species (Bringel
et al. 1989; Posno et al. 1991a). These vectors have
been instrumental in the development of gene-
transfer systems for Lactobacillus, at the time that
91
~900n|
~ - m l l - . -
ATG
Pml !
vectors based on Lactobacillus replicons were not
yet available. To date a spectrum of plasmid vectors
with Lactobacillus replicons has been constructed,
allowing genetic manipulation of a wide variety of
Lactobacillus species. Most plasmid vectors are de-
rived from small cryptic plasmids of different Lac-
tobacillus species, which replicate through a mecha-
nism of RCR. This may affect the stability of recom-
binant plasmids, as will be shown in a subsequent
section. Table 2 presents a list of plasmid vectors
currently in use in various laboratories. Vectors
contain either the erythromycin-resistance (ery)
gene from pE194 or pAM~I, or the chloramphen-
|col-resistance (cml) gene from pC194 or pBR328 as
selection marker. Since lactobacilli are intrinsically
resistant to relatively high concentrations of kana-
mycin/neomycin, these markers cannot be used for
vector construction. Also ampicillin resistance can-
not be used as selection marker in Lactobacillus
(Shimizu-Kadota et al. 1991).
Most vectors, as for example pLP825 and
pLPE323, display a wide-host-range phenotype,
since they can be propagated in a wide variety of
Lactobacillus species (Posno et al. 1991a). More-
over, their copy number in different Lactobacillus
strains does not significantly differ, indicating that
also control mechanisms for DNA replication in
different host bacteria operate in a similar way. The
average copy number of these plasmid vectors is es-
timated at 30-50 copies per cell. Copy-number
mutants, showing a 3-5 fold elevated copy number,
can arise spontaneously during vector construction,
92 /

probably as a result of selection pressure (Fig. 2).
The mutations, which give rise to the cop pheno-
type, have not been mapped. A cop mutant of
pLPE323, which was purposely made by deletion of
the replication repressor gene, shows a 5-10-fold in-
crease of the copy number (Pouwels et al. 1993).
Some of the Lactobacillus vectors lacking E. coli
sequences can nevertheless replicate in this host or-
ganism. For example, plasmids of the pPSC series
and pAl-derived vectors are promiscuous plasmids
that can be propagated in different Lactobacillus
species, in Bacillus and in E. coli (Cocconcelli et al.
1991; Vujcic & Topisirovic 1993). Similar findings
have been reported for lactococcal plasmids like
pSH71 and pWV01 (Kok et al. 1984; Gasson & An-
derson 1985; de Vos 1987). Replication proteins of
RCR plasmids generally are expressed in E. coli,
suggesting that replication of these plasmids in E.
coli depends on the presence of host factors that sta-
bilize ss-DNA intermediates and/or initiate replica-
tion at the minus origin. Recently, a series of broad-
host-range vectors was constructed for Lactococcus
based on the replication functions of the theta-type
plasmid pAM[31 (Simon & Chopin 1988; O'Sullivan
& Klaenhammer 1993). They might be useful for
cloning in Lactobacillus as plasmids replicating by a
theta-type mechanism show structural and segrega-
tional stability (Swinfield et al. 1991; Br0ckner
1992).
Recently, an improved version of vector
pLPE323, named pLPE23M (Fig. 3) was obtained
by introduction of a multi-linker region with 19
unique restriction enzyme sites. The usefulness of
the vector has been demonstrated by cloning and
overexpression of several proteins in Lactobacillus.
Also a vector with narrow host-range has been de-
scribed. Plasmid pLUL631 from L. reuteri carrying
an erythromycin-resistance gene was found to rep-
licate in L. reuteri and in a strain of L. fermentum
among several lactobacilli and other Gram-positive
bacteria tested (Ahrn6 et al. 1992). Similarly, a
3.6 kb plasmid replicon from L. crispatus was found
to replicate only in the host strain from which it was
derived (Posno pers. comm.). The latter type of vec-
tors offers attractive properties with regard to safe-
ty aspects associated with the use of live recombi-
nant DNA organisms in e.g. food products. Vectors
with a narrow host range are less likely to be hori-

Table 2. Plasmid vectors with Lactobacillus replicon.

Plasmid Replicon Origin E. coli Size Marker Cloning sites References

DNA (kb)

pLE16 pLB10 L. bulgaricus pBR328 7.6 cml HindlII Chagneaud et al. 1992
pBG10 pLJ1 L. helveticus pBR329 6.0 lacZ BamHI, PstI Hashiba et al. 1992
pLP3537 p353-2 L. pentosus pUC19 6.3 ery BamHI, HindIII, KpnI, PstI,
SphI Posno et al. 1991a
pLP317 p353-1 L. pentosus no 2.9 ery (SphI) Posno et al. 1991a
pLP317cop p353-1 L. pentosus no 2.9 ery (SphI) Posno et al. 1991a
pLPE323 p353-2 L. pentosus no 3.6 ery EcoRI, XbaI Posno et al. 1991a
pLPE350 p353-2 L. pentosus ** 3.9 ery HindIII, KpnI, PstI, Smal, SphI Leer et al. 1992
pLPE23M p353-2 L. pentosus no 3.7 ery MCS* This paper, Fig. 3
pLEP24Mcop p353-2 L. pentosus no 3.7 ery MCS This paper
pLP3537xyl p353-2 L. pentosus pUC 6.3 xyl, ery BamHI, KpnI, SmaI Posno et al. 1991b
pLP825 p8014-2 L. plantarum pBR322 5.8 cml SalI, Sph! Leer et al. 1987;
Posno et al. 1991a
pLP82H p8014-2 L. plantarum pUC 5.8 ery BamHI, SacI, Sphl, XbaI Posno et al. 1991a
pLPC37 p8014-2 L. plantarum ** 3.7 cml EcoRV, SphI Leer et al. 1992
pULP8/9 pLP1 L. plantarum pUC 6.6 ery HindIII Bouia et al. 1989
pSC10 L. plantarum no 3.0 ery Cocconcelli et aL 1991
pPSC20 L. plantarum no 5.5 cml, ery Cocconcelli et al. 1991
pPSC22 L. plantarum no 4.3 cml, ery Cocconcelli et al. 1991
pLUL634 pLUL631 L. reuteri no 5.1 ery ClaI, Hpal Ahrn6 et al. 1992

* MCS, multi-cloning site. ** 0.3 kb pBR

 93

Incompatibility

Lactobacillus strains are cured from the endoge-

Fig. 2. Effect of plasmid copy number on the activity of the bacte-
riocin acidocin B. The acidocin B activity is visualized using Clos-
tridium sporogenes as indicator bacteria, immobilized in agar. To
the wells were applied neutralized supernatants of (A) parent
strain L. acidophilus M46 with acidocin B gene on a low-copy
number plasmid, (B) L. plantarum with low-copy vector
(pGKV21) containing acidocin B gene, (C) L. plantarum with
high-copy mutant (pLPE24M) containing acidocin B gene. In L.
acidophilus M46 and L. plantarum/pGKV12-acidocin B, the
copy number is 5-15. The copy number in L. plantarum/
pLPE24M-acidocin B is 50-100.
nous plasmid when transformed by a vector with a
replicon from that plasmid (Bringel et al. 1989; Pos-
no et al. 1991a; Leer et al. 1992). This finding, which
can be easily explained by selective advantage of
vector DNA over the resident plasmid, can be ex-
ploited to cure strains from plasmids that are other-
wise difficult to eliminate. Also functional relation-
ships between replication functions in different
plasmids can be assessed in this way. For example,
the lactococcal plasmid pGK12 was found to cure L.
pentosus MD353 from an 1.7 kb plasmid, indicating
that the plasmids share replication functions, al-
though they show little DNA homology (Posno et
al. 1991a). L. plantarum ATCC8014 and L. pentosus
MD353 were reciprocally cured of the endogenous
plasmids p8014-2 and p353-2, when they were trans-
formed by vectors based on these plasmids. The as-
sumption that the two plasmids have similar repli-
cation functions was verified by sequence analysis,
showing that the two plasmids encode replication
proteins displaying 94 % similarity and have identi-
cal target sites for these proteins (Leer et al. 1992).

0
zontally transferred to other bacterial species than
vectors based on broad-host-range replicons, and
J
are, consequently, intrinsically more safe.
Vectors have also been described which, poten- 3000

l
tially, are useful for the development of food-grade
Pst l
vectors. Plasmid pBG10, which carries the L. bul-
garicus gene encoding J3-galactosidase under con-
trol of the promoter of the ery gene from pAM[31,
I BspM I

Bgl ]1
Stu 1

Cla 1
- Sph ]
- Xho I
might be useful for the selection of transformants in - Spo l
- Bal I
milk where lactose is the sole energy source (Hashi- - Eae [
ba et al. 1992). Plasmid pLP3537xyl, which contains II
- Sal I
- Pvu
genes from L. pentosus involved in the catabolism - Bbv I

of xylose, is capable of conferring to lactobacilli the 2000 I "'i',,,,,,,,, - Fsp I

Ill
capacity to utilize xylose as sole energy source, a
EcoR I ",, - Hind
- Kpn l
- Sma t
Barn H I
trait which is infrequently found in lactobacilli
(Posno et al. 1991b). Fig. 3. Structure of plasmid pLPE23M. Plasmid pLPE23M is
constructed by insertion of an erythromycin-resistance gene
from pE194 and a multi-cloning oligonucleotide into the XbaI
site of plasmid p353-2 from L. pentosus. In plasmid pLPE24M
(see Fig. 2) the erythromycin-resistance gene was cloned in the
opposite orientation.
94
Structural stability
For application in industrial fermentation process-
es, it is essential that the vector remains structurally
intact (structural stability) and can be maintained in
the host cell in the absence of selective pressure
during the fermentation process (segregational sta-
bility). Most plasmid vectors remain structurally in-
tact when the bacteria are cultivated in the presence
or absence of the selective agent. No structural in-
stability in L. casei or L. pentosus was observed af-
ter insertion into a Lactobacillus vector of DNA
fragments derived from bacteriophage )~varying in
size from 2 to 9 kb, irrespective of whether the bac-
teria were cultivated in the presence or absence of
the selective agent (Leer et al. 1992). Complete
structural stability was also observed when a 4 kb
DNA fragment coding for a hybrid protein consist-
ing of a foot-and-mouth disease virus epitope and
E. coli [3-galactosidase or a 7.2 kb DNA fragment
from L. pentosus comprising genes involved in xy-
lose catabolism were cloned in a Lactobacillus vec-
tor (Posno et al. 1991b). However, attempts to clone
the Hepatitis delta surface protein under control of
a Lactobacillus promoter in L. casei did result in
transformants with plasmids of the expected size
only, when the cloned gene lacked proper transla-
tion-initiation signals (Jore, pers. comm.). Appar-
ently, the expression of a protein which is harmful to
lactobacilli can be obviated by the occurrence of de-
letions. Recombinant DNA could also be stably
maintained without structural changes when DNA
was inserted into the chromosome (Scheirlinck et
al. 1989; Bhowmik & Steel 1993; Leer et al. 1993). It
appears that except for cases where expression of
the cloned gene results in a deleterious protein,
cloning of homologous and heterologous DNA into
Lactobacillus offers no serious problems. Even rel-
atively large fragments can be stably maintained
without the occurrence of detectable deletions or
rearrangements.
Segregational stability
Accurate copy number control is required for sta-
ble plasmid maintenance. Since RCR plasmids ap-
pear to lack a partitioning function, plasmids most
probably are randomly distributed over daughter
cells (Novick 1987). Large fluctuations in copy num-
ber as a result of a disturbance of copy number con-
trol systems may lead to daughter cells receiving no
plasmid molecules. For the development of vectors
that can be stably maintained, knowledge about
plasmid or host factors that contribute to copy num-
ber control thus is of paramount importance.
Most vectors are rapidly lost (50-> 95 % loss after
100 generations) when lactobacilli are cultivated in
the absence of the selective agent (Bringel et al.
1989; Posno et al. 1991a; Shimizu-Kadota et al. 1991).
Vectors with a replicon from Lactococcus or Sta-
phylococcus are even less stable in Lactobacillus,
showing segregation rates of several percent per
generation (Posno et al. 1991a; Shimizu-Kadota et
al. 1991). Some vectors with Lactobacillus replicons
can, however, be stably maintained for more than
100 generations in Lactobacillus in the absence of
selective pressure (Posno et al. 1991a; Cocconcelli et
al. 1991; Leer et al. 1992). Of particular interest is
plasmid pLPE323 which was found to be segrega-
tionally fully stable in all except one Lactobacillus
strain (Leer et al. 1992). Plasmid pLP3537, which is
rapidly lost under non-selective conditions, har-
bours the same replicon as pLPE323 but differs
from it by the presence of E. coli vector sequences.
The difference in stability between the two plas-
mids can be fully accounted for by E. coli sequenc-
es. After removal of ~ 95% of the E. coli sequences
from pLP3537, the resulting vector had become
segregationally completely stable. A similar result
has been obtained for pLP825 which comprises
pBR322 sequences and a replicon from L. planta-
rum. Segregational instability increases as the size
of the inserted DNA fragment increases (Leer et al.
1992), like a phenomenon also observed in B. sub-
tilis for plasmids derived from pUB110 (Bron &
Luxen 1985; Bron et al. 1988a, b).
In studies with RCR plasmids from S. aureus
(Gruss et al. 1987), Streptococcus pneumoniae (Del
Solar et al. 1987), Streptomyces lividans (Deng et al.
1988) and B. subtilis (Bron 1990), a functional minus
origin has been implicated in plasmid segregational
stability, probably because of inefficient synthesis
of the minus strand of the plasmid. A plasmid with a

deletion removing one-half of the stem-loop, which
is involved in the conversion of ss-DNA to ds-DNA,
is considerably less stable than the parent plasmid
in two Lactobacillus species, indicating that also in
Lactobacillus a functional minus-origin of replica-
tion is important for segregational stability (Leer et
al. 1992). Likewise, Shimizu-Kadota and coworkers
have observed that introduction of a palA-type or
M13 minus origin into a pUB110-derived vector in-
creases the segregational stability of the vector in L.
casei (Shimizu-Kadota et al. 1991). Increased segre-
gational stability was positively correlated with a de-
crease of the amount of ss-DNA intermediates (Shi-
mizu-Kadota et al. 1991; Leer et al. 1992), indicating
that inefficient conversion of ss-DNA into ds-DNA
in Lactobacillus disturbs copy number control and,
consequently, the segregational stability.
Chromosomal integration
Segregational stabilization of cloned genes by inte-
gration into the chromosome offers an alternative
to replicating plasmid vectors, especially in cases
where recombinant vectors are highly unstable, as
was observed in other bacteria (Raibaud et al. 1984;
Prozorov et al. 1987; Chopin et al. 1989; Leenhouts
et al. 1991). Chassy has demonstrated that a plasmid
encoding the Lactobacillus Lac-PTS is integrated
into the chromosome upon transformation of a lac-
tose-negative L. casei strain (Chassy 1987). Clostri-
dium thermocellum endoglucanase and B. stearoth-
ermophylus a-amylase genes have been inserted at
an unknown site of the L. plantarum chromosome
by means of a homologous single-cross-over recom-
bination event. The genes introduced could be sta-
bly maintained and expressed under non-selective
conditions (Scheirlinck et al. 1989). Replacement of
chromosomal genes by mutant alleles was demon-
strated in L. helveticus and L. plantarum after trans-
formation with vectors that do not replicate in these
organisms and contain an internal part of the target
gene or a gene in which an antibiotic-resistance
marker was spliced into the target gene (Bhowmik
& Steele 1993; Leer et al. 1993). The mutant pheno-
type is stably maintained for more than 100 gener-
ations under non-selective conditions (Leer et al.
95
1993). To obtain transformants with an insertion in-
to the chromosome, a high efficiency of transforma-
tion with replicating vectors is required, as the effi-
ciency of transformation is three to four orders of
magnitude lower than with replicating vectors. Al-
ternatively, vectors may be used carrying a thermo-
sensitive replicon. The broad-host-range plasmids
pGhost (Maguin et al. 1992) and pE194ts (GrycZan
et al. 1982) carrying a thermo-sensitive replicon de-
rived from pGK12 and pE194, respectively, have
been used for insertion of DNA fragments into the
chromosome of Bacillus and Lactococcus. Since
these vectors are also able to replicate in lactobacil-
li, they might be used for gene-tagging in these orga-
nisms as well.
Gene expression
Transcription
To date more than sixty-five Lactobacillus genes
have been cloned and sequenced. They originate
from more than ten different species, which are
evolutionary related but significantly distinct. This
is, amongst others, reflected by considerable differ-
ences in overall G + C content among different lac-
tobacilli (L. divergens 33-35%; L. fermentum
52%). This evolutionary difference should be kept
in mind when comparing expression signals such as
promoters, terminators and ribosome binding sites.
Evidence has been obtained indicating that expres-
sion of Lactobacillus genes takes place when such
genes are transferred to other Lactobacillus species
or to E. coli (Lerch et al. 1989a, b; Natori et al. 1990;
Toy & Bognar 1990; Posno et al. 1991b). These re-
sults have allowed a preliminary analysis of Lacto-
bacillus transcription and translation signals that
are sufficiently similar to be recognized in other
hosts, even although control of gene expression
might be different.
Promoter sequences
To date, transcription start sequences have been
identified for eleven Lactobacillus genes originat-
ing from seven different species (Table 3). All pro-
moters show typical - 35 and - 10 sequences which,
96

i:~ t,q
•5 .v.

s~

~4--~a,--I
S

V
~ gA

o &a o
='~,~ ~ ~ ~v ~

r; ~s~ < ~

q--4

l"q
r~

when aligned, allow to define a consensus sequence.
This consensus sequence resembles that of promo-
ters in E. coli and Bacillus. Also, - 35 and - 10-like
sequences with a spacer of ~ 18 nucleotides have
been identified upstream of the coding region of
nineteen other chromosomal and plasmid-borne
Lactobacillus genes (Table 3). Conclusions con-
cerning promoter structure of these genes should as
yet be taken with some caution, since the - 35 and
- 1 0 sequences were assigned on the assumption
that they would conform to a consensus sequence.
The length of the untranslated region of most tran-
scripts is relatively small (< 100 nucleotides), com-
parable to that found in E. coli and Bacillus. The
region 5' to the - 35 sequence is rich in A residues,
as has previously been observed for Bacillus pro-
moters that are recognized by (y-70 factors (Moran
et al. 1982; Graves & Rabinowitz 1986). The spacer
region between the two conserved hexanucleotides
is rich in T and A residues. A conserved dinucleo-
tide, TG was found 5' to the - 10 region as was ob-
served for B. subtilis promoters (Moran et al. 1982).
The distance between the - 10 sequence and actual
startsite of transcription varies considerably and no
specific sequence element can as yet be observed.
Several Lactobacillus promoters have been
shown to drive expression of genes fused to such
promoters. Expression of the cat gene (encoding
chloramphenicol acetyl transferase) was demon-
strated in L. casei for the L-ldh promoter of L. casei
and the a-amy promoter from L. amylovorus, and
for the L. pentosus xyIR and xylA promoters in L.
pentosus. Considerable differences in expression
levels were observed, suggesting differences in
'strength' of the promoters (Table 4). Expression of
the cat gene driven by Pamy is regulated in a similar
fashion as is the amy gene in L. amylovorus. The
results indicate that these promoters are expressed
in heterologous hosts, and are subjected to the same
regulation mechanism in lactobacilli as in their
chromosomal environment in the original host
when the promoter is placed on a multi-copy plas-
mid. Constitutive expression of the cat gene with
these promoters could be demonstrated in E. coli.
The promoters Pamy and PxylR have also been
used succesfully to express foreign genes in L. casei
(see Section 5.5).
97
Terminator sequences
Rho-independent-like terminators have been ob-
served at the 3' end of most Lactobacillus genes.
The results of Northern blot analyses of several
transcripts are consistent with the notion that these
sites function as true terminators of transcription
(Copeland et al. 1989; Lokman, pers. comm.). The
conclusion that these sequences do indeed function
as transcription terminators is reinforced by the ob-
servation that insertion of the presumed terminator
sequence of the xylA/B operon of L. pentosus
downstream (3') of a marker gene results in a tran-
script that is terminated at the site of the terminator.
In the hdcA/B operon of Lactobacillus 30a (Van-
derslice et al. 1986) and the xylA/B operon of L.
pentosus (Lokman et al. 1991), palindromic se-
quences have been found in the intergenic region
that might function as transcription attenuators
and/or RNA processing sites. The function of such
elements might be to reduce the efficiency with
which the promoter-distal gene is transcribed rela-
tive to that of the promoter-proximal gene, or to al-
low for a difference in RNA turnover rates by dis-
section of the two transcripts (Higgins et al. 1982;
Belasco & Higgins 1988; McCormick et al. 1991).
Palindromic structures believed to function as sites
for transcription attenuation and transcription anti-
termination, have been found in the 5' untranslated
region of the replication protein (repA) encoding
gene of the L. pentosus plasmid p353-2 and up-
stream of the lacEGF genes from L. casei, respec-
tively (Pouwels et al. 1993; Alpert & Siebers, pers.
comm.).
Regulation of transcription
Knowledge concerning regulation of gene expres-
sion in lactobacilli is scarce and is mainly limited to
lactose and xylose fermentation routes. In L. casei
the lacEGF genes are preceded by a sequence
which has been tentatively identified as the promo-
ter. This promoter is followed (50 nt) by a sequence
capable of forming a stable stem-loop structure in
the RNA that resembles a rho-independent tran-
scription terminator. Downstream (25 nt) of the
terminator starts an 879nt open reading frame
98
which is terminated 120 nt before lacE. Northern
blot experiments suggest that the ORF is tran-
scribed together with the genes lacEGF as a 4.4 kb
polycistronic mRNA, which is initiated at the pro-
moter located in front of the ORE The data also
suggest that the transcriptional unit does not com-
prise genes coding for tagatose fermentation, as is
the case in Lactococcus lactis and Staphylococcus
aureus (Oskouian & Stewart 1990; Van Rooijen et
al. 1991a; Van Rooijen et al. 1991b). The putative
translation product of the ORF (34 kDa) shows sig-
nificant similarity to the anti-terminator proteins,
SacT, SacY, BglG and ArbB (Debarbouill6 et al.
1991). These data may indicate that transcription of
the lac-PTS genes in L. casei is at least partly regu-
lated by an anti-termination mechanism similar to
that observed for the ~-glucoside operon in E. coli.
The enzymes involved in catabolism of D-xylose
in L. pentosus and L. brevis are induced by xylose.
Control of xyl gene expression in L. pentosus has
been shown to take place at the level of initiation of
transcription, which is induced by xylose and nega-
tively controlled by a repressor protein. The N-ter-
minal region of the repressor protein displays a he-
lix-turn-helix motif characteristic for DNA-binding
proteins. In the intergenic region between xyIR and
xylA a palindromic structure was found immediate-
ly after the transcription-initiation site. The ele-
ment renders expression of the xyl genes constitu-
tive, when introduced into L. pensosus on a multi-
copy plasmid, indicating that the element can func-
tion as an operator that titrates the repressor (Lok-
man et al., in preparation).
The xyl genes in L. pentosus and L. brevis appear
to be subject to catabolite repression as no enzyme
synthesis is observed in a medium containing glu-
cose. During growth on a medium containing xy-
lose, high levels of xylP/Q mRNA and xylA/B
mRNA are found in L. pentosus, however these lev-
els are greatly reduced when the bacteria are culti-
vated in a glucose-containing medium or when both
xylose and glucose are present in the medium (Lok-
man et al., in preparation). In the intergenic region
between xylR and xylA, a second palindromic struc-
ture is present which overlaps the - 35 element of
the promoter PxylA/B. This element shows similar-
ity to the catabolite responsive element found near
Table 4. Expression of cat gene under control of different promo-
ters in different lactobacilli.
Promoter Species L. casei L. pentosus
Inducer
- q- - -I-
xylA L. pentosus n.d.* n.d. - xx
xylR L. pentosus n.d. n.d. x x
L-ldh L. casei xxx xxx n.d. n.d.
amyA L. amylovorus - xx n.d. n.d.
* n.d., not determined; the inducer of the promoters of xylA and
xylR was xylose, that of promoter amyA, cellobiose. Bacteria
with a plasmid carrying the promoter of the L-ldh gene were cul-
tivated in glucose (-) or cellobiose (+) containing medium.
x-xxx, weak to strong expression.
the 5' end of a number of genes in Bacillus (Weicker
& Chambliss 1990). The hypothesis that the ele-
ment is also involved in catabolite repression in L.
pentosus is supported by the finding that glucose re-
pression is partially alleviated when the element is
introduced on a multi-copy vector. Presumably, a
protein which interacts with the element is titrated,
resulting in levels of expression of xylA/B in the
presence of glucose that are comparable to those
found in the presence of xylose (Lokman et al., in
preparation).
Translation
The nucleotide sequences around the translation-
initiation sites of approximately seventy Lactoba-
ciUus genes are known. Despite the fact that the
genes originate from more than ten species it is evi-
dent that the sequences show some common fea-
tures. The preferred startcodon is AUG. While four
genes start with GUG and three with UUG, all
others start with AUG. A highly conserved se-
quence (AGGAGG), resembling the Shine-Dal-
garno motif is found in most of the genes at a dis-
tance of 6-10 nucleotides 5' to the startcodon (Table
5). No specific motifs were found in the spacer re-
gion, nor was a significant deviation from the A/T
content or the Pu/Py ratio observed in the spacer
region. The A/T content of the region immediately
downstream from the translation startcodon, how-
 99

Table 5. Translation initiation region of Lactobacillus genes. gene (Toy & Bognar 1990), the trpF (N-5'-phospho-
Species No. of genes RBS Spacer (nt)
ribosylanthranilic acid isomerase) gene (Natori et
al. 1990) and the purL (phosphoribosylformylglyci-
L. acidophilus 6 GGAGG 5-11 namidine synthetase II) gene (Gu et al. 1992) from
L. bulgaricus 6 AGGA 4- 9
L. casei. Interestingly, the lacL/lacM (Chassy &
L. casei 17 AGGAGG 3-11
L. helveticus 7 AGGAG 5- 8 Flickinger 1993), trpB/A (Natori et al. 1990) and
L. pentosus 9 GGAGG 6- 9 purQ/purL (Gu et al. 1992) genes of L. casei are par-
L. plantarum 11 GGAGG 6--12 tially overlapped, presumably ensuring the equi-
The nucleotide indicated is the one occurring most frequently.
molar synthesis of the two subunits of the enzymes
Bold capital: > 75%; capital; 50%-75%. T h e spacer is the (Oppenheim & Yanofsky 1980). In three genes
region between the RBS and the translation initiation codon. translation termination is effected by two consec-
utive stopcodons.
ever, deviates from that of the overall composition
of the genes. From Table 6 it is clear that the average Codon usage
A/T content of the first five codons after the startco- Codon usage of Lactobacillus genes shows a clear
don is significantly higher than that of the total bias for specific codons. All possible codons are
genes. This holds both for species with high A/T used by lactobacilli, but it appears not by all species.
content (e.g.L. helveticus) as well as for species with This also applies to stopcodons. U A A is most fre-
low A/T content (L. bulgaricus). When the data are quently used and is found in genes of all sPecies ex-
analysed for individual positions in the codons, sim- amined. The stopcodon UAG however is much
ilar conclusions can be inferred. At each position of more rare and is, except for an unidentified open
the codon the A/T content is significantly higher in reading frame in the insertion element IS1 (Shimi-
codons near the 5' end of the genes than in other zu-Kadota et al. 1985), not present in 17 genes be-
codons. These data may be interpreted to mean that longing to the L. casei group. Whether all these
a high G/C content of the gene 3' of the startcodon genes are truly L. casei genes remains to be estab-
might favour intra- or intermolecular RNA-RNA lished (Collins et al. 1989). The codon A G A for ar-
formation, which would interfere with translation ginine is frequently used in L. helveticus and L.
initiation. plantarum but is rarely or not at all used in most
Translational coupling of contiguous genes can other species. The same applies to the isoleucine co-
increase the efficiency of translation of the down- don AUA which is frequently used in L. acidophilus
stream gene. Overlapping start/stop codons, or but rarely used in all other species. Table 7 shows
partly overlapping reading frames, were observed that codon usage in two-codon sets, and in isoleu-
for thefgs (folylpoly-gamma-glutamate synthetase) cine codons, that use GNN as anticodon, is clearly

Table 6. A / T content of 1st, 2nd and 3rd nucleotide of codons in 5' region of Lactobacillus genes.

Species A / T content (%) Genes*

5'-end of gene** Total gene

1st 2nd 3rd Average 1st 2nd 3rd Average

L. acidophilus 83 79 80 81 56 60 70 62 5
L. bulgaricus 69 66 67 67 46 61 39 49 6
L. casei 56 73 ' 67 65 43 63 53 53 18
L. helveticus 63 89 88 80 54 69 71 65 7
L. pentosus 64 64 66 65 49 64 60 58 8
L. plantarum 58 65 85 69 56 69 73 66 7

* : n u m b e r of genes; ** : first five codons after the startcodon.

100

different for L. bulgaricus and L. plantarum. C is 1991). The genes coding for an a-amylase from L.
preferentially used over U, and G over A in L. bul- amylovorus, a proteinase from L. paracasei and
garicus, whereas the reverse it true for L. planta- bacteriocins, lactacin F and acidocin B from L. aci-
rum. These results may - in part - be explained by dophilus have recently been cloned and sequenced
differences in G + C content of the species (L. bul- (Jore et al., pers. comm.; Muriana & Klaenhammer
garicus 50%, L. plantarum 45%, Osawa et al. 1990; 1991; Holck & Naes 1992; Van der Vossen et al.,
Pouwels & Leunissen, in preparation). It seems pers. comm.). The a-amylase from L. amylovorus
likely, however, that also the constraint imposed by and proteinase from L. paracasei contain signal se-
tRNA affects codon usage. Efficiently expressed quences that are typical for secreted proteins. The
genes show a marked preference for C over T, and signal sequence of a-amylase from L. amylovorus is
G over A residues (except for Glu and Gln) in the 49 amino acids long, somewhat larger than the B.
third position of the codon in two-codon sets, and in subtilis enzyme (Jore et al., in preparation). The sig-
isoleucine codons (Table 8). These results indicate nal sequences of the two bacteriocins conform only
that in highly expressed genes in Lactobacillus loosely to those associated with secreted proteins,
more NNC codons are chosen than NNU as a result but show resemblance to signal sequences of bacte-
of selection pressure by GNN anticodons. In weak- riocins from other species (Leer et al., in prepara-
ly expressed genes codon choice apparently is much tion; Klaenhammer 1993). The genes of two sur-
less affected by tRNA. face-layer proteins from L. brevis and L. acidophi-
lus have recently been cloned and their sequence
determined (Vidgr6n et al. 1992; Boot et al. 1993).
Protein secretion The proteins contain signal sequences that are typ-
ical for exported proteins and are 30 and 24 amino
Excretion of proteins into the culture fluid is of con- acids long, respectively (Pugsley 1989).
siderable importance from the biotechnological
point of view, as the purification of secreted pro-
teins will in general be easier than that of intra-cel- Heterologous gene expression
lular proteins. Although lactobacilli do not appear
to be prodigious protein secretors, several species A number of studies have appeared describing the
are known to produce extra-cellular enzymes like expression of heterologous genes in Lactobacillus
a.amylase, insulinase and proteinase (Nakamura & spp. The successful transfer of conjugal plasmids
Crowel11979; Nakamura 1981; Szil~igi, pers. comm.; from heterologous hosts into Lactobacillus, and the
Holck & Naes 1992) and bacteriocins (Joerger & propagation of various plasmid vectors in a variety
Klaenhammer 1990; Muriana & Klaenhammer
Table 8. Codon usage in two-codon sets, and Ile of highly and
Table 7. Codon usage in two-eodon sets, and Ile of different Lac- weakly expressed Lactobacillus genes.
tobacillus species.
E* M-E N-E
L. bulgaricus L. plantarum
C/U + C (%) Asp (GAG/U) 65 37 25
C/U + C (%) Asp (GAG/U) 77 24 Asn (AAC/U) 86 51 25
Ash (AAC/U) 71 24 Tyr (UAC/U) 77 52 30
Tyr (UAC/U) 75 22 Phe (UUC/U) 85 48 33
Phe (UUC/U) 58 7 His (GAG/U) 76 47 29
His (CAC/U) 80 28 Ile (AUC/U) 67 27 22
Ile (AUG/U) 69 13 G/G + A (%) Glu (GAG/A) 1 18 28
G/G + A (%) Glu (GAG/A) 11 26 Lys (AAG/A) 73 62 40
Lys (AAG/A) 75 22 Gln (GAG/A) 12 22 42
Gln (GAG/A) 58 21 Total number codons 1315 1464 442
Total number codons 2919 1621
* E, efficiently; M-E, moderately-efficiently, N-E, not efficiently.
 101

of Lactobacillus spp., were the first demonstrations
that heterologous genes involved in plasmid repli-
cation and antibiotic-resistance are expressed in
lactobacilli. Besides these genes, which are ex-
pressed under the control of their own regulatory
elements, also a number of genes from other bacte-
rial sources encoding intra- or extracellular en-
zymes have been expressed in lactobacilli from
their own promoter. Amongst the genes expressed
in lactobacilli are a-amylases and glucanases from
Clostridium and Bacillus (Jones & Warner 1990;
Leer, unpublished observations; Chassy, pets.
comm.; Bates et al. 1989; Scheirlinck et al. 1989;
Baik & Pack 1990; Thompson & Collins 1991), the
lux gene from Vibrio fischeri (Ahmad et al. 1988),
and the lipase and lysostaphin genes from Staphylo-
coccus (Vogel et al. 1990; Gaier et al. 1992).
Expression of heterologous proteins under the
transcriptional control of Lactobacillus promoters
was recently demonstrated for a number of en-
zymes and fusion proteins (Table 9). For example,
the E. coli lacZ gene as well as fusion genes coding
for a foot-and-mouth disease epitope, or an epitope
of rotavirus capsid protein VP7 fused to lacZ, were
expressed under the control of the promoter of the
xylose repressor gene, xylR. The expression levels
are relatively low (up to 1.0%), but might be consid-
erably increased by making use of stronger promo-
ters.
Expression and secretion of extra-cellular pro-
teins into the culture fluid was reported for c~-amy-
lase from L. amylovorus in L. casei under control of
its own promoter (Jore et al., in preparation). The
regulation of expression was the same in the two or-
ganisms. Also genes from more distantly related or-
ganisms like S. aureus and E. coli are expressed in
Lactobacillus (Table 9).
Conclusions
At the time of the first international congress on
Lactic Acid Bacteria in 1984, our knowledge of ge-
netics of lactobacilli was non-existent while the de-
velopment of gene-transfer and gene-expression
systems for lactoccoci was well under way and po-
tential applications of that research were already
within reach. During the last few years, however,
considerable progress has been made in developing
the tools for genetic manipulation of lactobacilli.
Knowledge of the structure and mode of replication
of plasmids from Lactobacillus spp., and the use of
this knowledge for gene-transfer experiments in
Lactobacillus has come to a stage where applica-
tions in strain improvement programmes can be
foreseen. Also our insight into the question of how
to stably introduce and express foreign genes in lac-
tobacilli has reached a level similar to that previous-
ly demonstrated for other microorganisms.
A major limitation for a number of applications
is, however, the lack of detailed knowledge on con-
trol of gene expression. In order to exploit the me-
tabolic capacity of lactobacilli, it is of paramount
importance to understand the mechanisms under-

Table 9. Heterologous gene expression in Lactobacillus.

Protein Origin Species Promoter References

acidocin B L. acidophilus L. plantarum acdB Leer, unpublished

levanase B. subtilis L. plantarum/L, casei tac Wanker, pets. comm.
c~-Amylase L. amylovorus L. plantarum/L, casei amyA Jore, pets. comm.
Xylose isomerase L. pentosus L. casei xylA Posno et al. 1991b
13-Galactosidase E. coli L. plantarum/L, casei xylR, cbh Posno, pers. comm.
~-Glucuronidase E. coli L. plantarum cbh Posno, pers. comm.
FMDV-[3-gal FMDV-E. coli L. casei xylR Posno, per~. comm.
VP7-[~-gal Rotavirus-E. coli L. plantarum cbh Posno, pers. comm.
FMDV-c~-amylase FMDV-L. amylovorus L. casei arnyA Jore, pers. comm.
Chloramphenicol pC194 L. casei amyA, L-ldh This paper
acetyltransferase L. pentosus xylA, xylR Lokman, pets. comm.
102
lying control of gene expression under conditions
prevailing in situ. For example, the use of lactobacil-
li in the gastro-intestinal tract will require knowl-
edge about control of expression of LactobaciUus
genes in vivo. A second major handicap is our lack
of information regarding transfer of genes to and
from Lactobacillus by mechanisms other than
transformation. Improving knowledge of conven-
tional methods of gene-transfer such as conjugation
will facilitate strain improvement. This technology
can be used to make engineered Lactobacillus
strains available for application in food and feed in-
dustry. Such knowledge will also provide informa-
tion concerning the question whether or not inad-
vertent horizontal transfer of cloned genes takes
place. This issue is an essential element in discus-
sions about the acceptability of genetically engi-
neered microorganisms in human and/or animal
food. Despite these uncertainties, the dawning per-
spectives for using lactobacilli with improved prop-
erties also in areas other than the food/feed sector
such as in oral vaccination programmes or in con-
trolling bile acid metabolism warrant further inves-
tigations into this scientifically and biotechnologi-
cally highly rewarding field.
Acknowledgement
We thank Dr Bruce Chassy for critical reading of
the manuscript and Jack Leunissen for help with
computer analysis of nucleotide sequences.
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