Enterococcal Genetics
KEITH E. WEAVER
Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069
ABSTRACT The study of the genetics of enterococci has
focused heavily on mobile genetic elements present in these
organisms, the complex regulatory circuits used to control
their mobility, and the antibiotic resistance genes they frequently
carry. Recently, more focus has been placed on the regulation of
genes involved in the virulence of the opportunistic pathogenic
species Enterococcus faecalis and Enterococcus faecium. Little
information is available concerning fundamental aspects of DNA
replication, partition, and division; this article begins with a brief
overview of what little is known about these issues, primarily by
comparison with better-studied model organisms. A variety of
transcriptional and posttranscriptional mechanisms of regulation
of gene expression are then discussed, including a section on the
genetics and regulation of vancomycin resistance in enterococci.
The article then provides extensive coverage of the pheromone-
responsive conjugation plasmids, including sections on regulation
of the pheromone response, the conjugative apparatus, and
replication and stable inheritance. The article then focuses on
conjugative transposons, now referred to as integrated,
conjugative elements, or ICEs, and concludes with several
smaller sections covering emerging areas of interest concerning
the enterococcal mobilome, including nonpheromone plasmids
of particular interest, toxin-antitoxin systems, pathogenicity
islands, bacteriophages, and genome defense.
Interest in enterococcal genetics began with three land-
mark discoveries: (i) identification of the first conjugative
plasmids whose transfer systems are induced by an iden-
tifiable signal (1), (ii) identification of the first “transpo-
sons” capable of intercellular (conjugative) transposition
(2, 3), and (iii) the acquisition of vancomycin resistance
(4, 5). Since the most prevalent antibiotic resistance genes
are located on plasmids and transposons, early work on
enterococcal genetics focused heavily on mobile genetic
elements (MGEs). Examination of the complete sequence
of a vancomycin-resistant clinical isolate of Enterococ-
cus faecalis, V583, reaffirmed the importance of MGEs
in the evolution of this species, revealing that over a
quarter of the genome consists of mobile and/or exog-
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enously acquired DNA (6). Further genome sequencing
has revealed extensive variability in the proportion of
horizontally acquired DNA in E. faecalis strains, with the
presence of clustered regularly interspaced short palin-
dromic repeats (CRISPR) with CRISPR-associated (Cas)
protein (CRISPR-Cas) loci playing an important role in
regulating MGE acquisition (7, 8). More recent genomic
analysis has revealed that MGEs are important drivers of
genome diversity and evolution in Enterococcus faecium
as well (9).
While work on enterococcal MGEs continues apace,
other aspects of enterococcal genetics are beginning to
come into sharper focus. While the basic mechanisms of
chromosome replication, partition, and cell division re-
main poorly defined, progress on other chain-forming
ovococci provides a framework for further detailed anal-
ysis. Meanwhile, substantial progress has been made in
understanding transcription and posttranscriptional reg-
ulation of gene expression, intercellular communication,
and global response via second messengers, particularly
of genes involved in virulence of the two major pathogens
Received: 4 December 2018, Accepted: 8 January 2019,
Published: 8 March 2019
Editors: Vincent A. Fischetti, The Rockefeller University, New York,
NY; Richard P. Novick, Skirball Institute for Molecular Medicine, NYU
Medical Center, New York, NY; Joseph J. Ferretti, Department of
Microbiology & Immunology, University of Oklahoma Health
Science Center, Oklahoma City, OK; Daniel A. Portnoy, Department
of Molecular and Cellular Microbiology, University of California,
Berkeley, Berkeley, CA; Miriam Braunstein, Department of
Microbiology and Immunology, University of North Carolina-Chapel
Hill, Chapel Hill, NC, and Julian I. Rood, Infection and Immunity
Program, Monash Biomedicine Discovery Institute, Monash
University, Melbourne, Australia
Citation: Weaver K. 2019. Enterococcal Genetics. Microbiol
Spectrum 7(2):GPP3-0055-2018. doi:10.1128/microbiolspec.GPP3-
0055-2018.
Correspondence: Keith E. Weaver, kweaver@usd.edu
© 2019 American Society for Microbiology. All rights reserved.
1
Weaver
of the genus, E. faecalis and E. faecium (for a compre-
hensive review see 10). This article will focus primarily on
the most recent advances in this field, with references to
detailed reviews where available. Unless otherwise stated,
identification of E. faecalis genes orthologous to genes
in other bacteria utilized the V583 genome for searches
(6), and gene annotations where given will use the V583
designation.
CHROMOSOMAL REPLICATION, PARTITION,
AND CELL DIVISION
The fundamentals of chromosomal DNA replication in
enterococci are probably similar to those in the model
organism Bacillus subtilis (11). The E. faecalis genome
includes homologs of all known components of the typ-
ical Gram-positive DNA polymerase III (Pol III) holo-
enzyme, including a replicative DNA helicase (which is
named DnaB according to the Escherichia coli conven-
tion rather than DnaC as in B. subtilis), helicase loaders,
primase, clamp and clamp loaders, and DnaE- and PolC-
type catalytic alpha subunits. Most have also been iden-
tified in the E. faecium genome. It has been proposed that
PolC may be the primary leading strand DNA poly-
merase, while DnaE plays dual roles as the lagging strand
polymerase and as an error-prone polymerase during
DNA repair in B. subtilis and Streptococcus pyogenes
(12, 13). The E. faecalis PolC and DnaE proteins have
been purified and found to have characteristics similar to
the B. subtilis homologs, even cross-reacting with anti-
bodies generated against B. subtilis PolC and DnaE (14),
suggesting that E. faecalis and B. subtilis enzymes are
structurally and functionally similar. The putative en-
terococcal oriC has a bipartite organization with DnaA
binding sites flanking the dnaA gene as in B. subtilis and
distinct from E. coli.
In addition to DNA Pol III, the E. faecalis V583 chro-
mosome also encodes a DNA Pol I homolog, two putative
phage-encoded DNA polymerases, and one member of
the mutagenic Y-family polymerases (15) that have been
shown to be involved in lesion bypass in other organisms.
Y-family polymerases are commonly found on bacterial
plasmids (16), and all three of the plasmids in E. faecalis
V583 encode at least one member of the family. The pre-
viously described uvrA gene of E. faecalis plasmid pAD1
(17) also belongs to this group and was shown to impart
increased resistance to UV exposure.
Other genes associated with DNA repair present in
the E. faecalis genome include homologs required for
transcription-coupled DNA repair (mfd), nucleotide ex-
cision repair (uvrA, uvrB, and uvrC), mismatch repair
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(mutS and mutL homologs annotated as hexA and
hexB, respectively), and numerous other nucleotide re-
pair proteins, including seven potential mutT homo-
logs. It should be noted that the previously designated
uvrABC genes located on plasmid pAD1 (17) are not
homologs of the excision repair enzymes that usually
bear this name but are instead associated with a lesion
bypass polymerase. Multiple homologs of genes involved
in recombination repair were also identified, including
genes for a LexA-like regulator, RecA, a RexAB DNA
processing enzyme, a RuvAB Holliday junction heli-
case, a PriA replication restart primase, a RecFOR com-
plex, and a variety of other recombination proteins. A
recombination-deficient mutant of E. faecalis, UV202,
has a phenotype similar to recA mutants in other bacteria
(18). Sequencing and complementation analyses con-
firmed that this phenotype is due to a single point mu-
tation that results in a G265D change in the RecA protein
(19). Other putative recombinational repair proteins have
not been characterized.
The E. faecalis genome includes topoisomerases I to
IV, a Streptococcus pneumoniae condensin homolog, and
a homolog for the nucleoid associated protein HU, sug-
gesting that the chromosomal DNA is organized and
compacted as in other bacteria. No replication termina-
tor protein is annotated in the genome, and no identifi-
able homolog of the B. subtilis Rtp terminator could be
identified. In fact, Rtp appears to be limited to the Bacillus
genus, suggesting that proteins involved in termination of
replication are poorly conserved. A single XerD homo-
log, involved in chromosome dimer resolution in other
bacteria, is annotated in the E. faecalis genome, but
several other integrase/recombinases have been identified
that could play the role of the XerCD resolution recom-
binase. The C-terminus of the cell division protein FtsK has
been implicated in organizing both the XerCD dimer res-
olution complex and the topoisomerase IV decatenation
complex near the chromosomal replication terminus for
efficient separation of daughter chromosomes in E. coli
(20, 21). The C-terminal domain of E. faecalis FtsK is 42%
identical to the C-terminus of E. coli FtsK, but the N-
terminal domains of the two proteins are not similar,
suggesting that they may play similar roles at the end of
chromosomal replication but different roles in cell division.
The division/cell wall cluster of E. faecalis was iden-
tified in strain A24836 (22) and appears to be identical
in the V583 genome (note that divIB and ftsQ are or-
thologs). This cluster includes ftsZ, the gene for the bac-
terial tubulin homolog essential for contraction of the cell
membrane during cell division, two other cell division
genes, ftsA and ftsQ, and several genes involved in pep-

tidoglycan synthesis. There are also multiple ftsW- and
ftsK-like genes in the V583 genome that may be involved
in cell division. Details of the mechanism of FtsZ ring
closure have been examined in S. pneumoniae (23–25)
and may be applicable to enterococci.
In the model rod-shaped organisms E. coli and B. sub-
tilis the primary systems responsible for division site se-
lection and coordination with chromosome segregation
are the Min and nucleoid occlusion systems (26, 27).
In the Min system, the MinCD division inhibitor func-
tions with a topological specificity determinant (MinE in
E. coli and DivIVA in B. subtilis) to prevent division site
placement at the poles, while nucleoid occlusion prevents
septum closure over the bulk chromosomal DNA. Gram-
positive cocci lack recognizable genes for MinCD and
known nucleoid occlusion systems, and evidence suggests
that nucleoid occlusion does not function in these organ-
isms (28, 29). Recent work in S. pneumoniae suggests
that proper origin placement and chromosome compac-
tion drive division site selection, and a protein unique
to the Enterococcaceae and Streptococcaceae families,
MapZ (30), is required for proper perpendicular orien-
tation of the division plane (31). The mechanism of origin
placement remains unclear but may involve as yet un-
identified proteins, coupling of transcription to segrega-
tion (32), or entropic effects (31).
The S. pneumoniae system provides a useful model for
understanding enterococcal partition and cell division.
Enterococcal division is at least superficially similar to
streptococcal cell division, and the enterococcal chro-
mosome includes orthologs for many of the important
players, including genes for partition, chromosome com-
paction, and MapZ. But there are also several potentially
significant differences. First, the S. pneumoniae parB
gene, whose product is required for proper partition and
recruitment of the nucleoid organizing protein Smc (33),
is located immediately adjacent to the origin region and is
not genetically linked to a gene for the ParA protein,
which is normally essential for ParB-mediated partition
function. The closest S. pneumoniae parB homolog on
the E. faecalis genome (EF3298) is not closely linked to
the putative origin region and has a clear parA partner
(EF3299). Gene order at the E. faecalis and E. faecium
origins conform more closely to that found in B. subtilis
than in S. pneumoniae (34). Further, the homology be-
tween S. pneumoniae and enterococcal MapZ is pri-
marily in the C-terminal peptidoglycan binding domain
and does not extend to the transmembrane and cyto-
plasmic domains potentially involved in midcell divi-
some placement. While these observations may simply
represent variations on a general theme, further research
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Enterococcal Genetics
is required to determine how similar coordination of
chromosome partition and cell division are in the en-
terococci and streptococci.
REGULATION OF GENE EXPRESSION
Like many other aspects of enterococcal genetics,
regulation of gene expression was initially investigated
in the MGEs of E. faecalis. These include the two-
component signal transduction system (TCSTS) regula-
tion of transposon-borne vancomycin resistance genes,
signal peptide-dependent and antisense RNA-mediated
regulation of conjugation, and antisense RNA regula-
tion of plasmid-encoded toxin-antitoxin (TA) genes. The
mechanisms of regulation of these systems will be exam-
ined in detail in later sections of the article. This section
will focus on more recent advances in the understanding
of the regulation of enterococcal chromosomal genes.
The E. faecalis genome encodes genes for RNA poly-
merase α, β, β′, ω, and δ subnits and four recognizable
σ factors. The σ factor genes include a putative house-
keeping σ factor (rpoD or sigA), two members of the
extracytoplasmic family (ECF), and a σ54-like sigma fac-
tor (rpoN). σ54 is unique among bacterial sigma fac-
tors in that σ54 RNA polymerase is unable to form open
complexes and initiate transcription without the help of
ATP-dependent activator proteins (35). Such “enhancer
binding proteins” are highly conserved, and genes en-
coding four such proteins have been identified in the
E. faecalis genome (36). All belong to the LevR subfam-
ily of σ54 activators, which includes a conserved DEAH
helicase motif (37). Immediately downstream of each of
these genes is a putative σ54 promoter and operons en-
coding four distinct sugar phosphotransferase systems
(PTSs) of the mannose family (36). Mutation of σ54, en-
hancer binding protein MptR, or PTS component MptD
resulted in decreased sensitivity to class IIa bacteriocins,
implicating MptD as the bacteriocin receptor and σ54
and MptR as its regulators (36, 38). V583 σ54 mutants
showed increased biofilm formation while decreasing au-
tolysis, cell death, and extracellular DNA release (39).
This phenotype was independent of the four known en-
hancer binding proteins. Because biofilm development is
believed to be dependent on extracellular DNA, it was
proposed that σ54 mutants adapt an alternative matrix
for biofilm establishment. The E. faecium genome also
encodes an rpoN and five enhancer binding proteins of
the LevR family, but the functions of the genes are so far
undefined.
ECF σ factors generally facilitate transcription of genes
involved in bacterial stress responses. They are seques-
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tered by membrane-localized anti-σ factors and released
by stress-induced anti-σ factor degradation by regulated
intramembrane proteolysis (40, 41). The SigV ECF of
E. faecalis was shown to contribute to survival following
heat, acid, and ethanol stress (42) and to play a key role in
lysozyme resistance and virulence (43). The gene adjacent
to sigV, rsiV, encodes the anti-σ factor of the system (42)
and is targeted by the Eep membrane-embedded zinc
metalloprotease and at least one other unidentified pro-
tease for degradation in response to lysozyme and per-
haps other stresses (44). The function of the second E.
faecalis ECF σ factor is unknown.
TCSTSs have emerged as ubiquitous bacterial signal-
ing systems for regulating gene expression in response to
environmental conditions (45). The canonical TCSTSs
are composed of a sensor histidine kinase (HK) that
modulates a response regulator (RR) via phosphotrans-
fer. The RR is frequently a transcriptional regulator.
Analysis of the E. faecalis genome revealed the pres-
ence of 17 HK/RR pairs and 1 orphan RR (46). The best-
characterized enterococcal TCSTSs are the Van system,
required for regulation of vancomycin resistance genes
(see below), and the Fsr system. The Fsr system regulates
production of two proteases, gelatinase and serine pro-
tease, that are associated with virulence and biofilm for-
mation (47–51). Fsr is a quorum sensing system that
responds to the accumulation of a peptide lactone, des-
ignated GBAP (52). GBAP is the product of a small open
reading frame, fsrD, that is processed and secreted by
FsrB (53). GBAP is sensed at high cell density by the FsrC
HK, which then activates the FsrB RR by phosphoryla-
tion. Phosphorylated FsrB then activates transcription
from the gelE-sprE target promoter and the fsrBDC pro-
moter, completing the autoinduction loop. The fsr system
is analogous to the well-studied agr system of Staphylo-
coccus aureus (54) except for the absence of the global
small RNA (sRNA) regulator RNA III; FsrA appears to be
the terminal regulator of the Fsr system.
The CroRS TCSTS is essential for PBP5-mediated
intrinsic cephalosporin resistance in both E. faecalis and
E. faecium (55–57). The CroS HK is a sensor of cell
wall homeostasis and is stimulated to phosphorylate the
CroR RR in response to a number of cell wall-active
antibiotics. The CroR-regulated effector genes respon-
sible for cephalosporin resistance have not been identi-
fied, but neither PBP5 itself nor its substrates appear to
be the primary target. In some E. faecalis strains, cross-
regulation is observed with a second TCSTS CisRS (56).
The situation is further complicated by the involvement of
a transmembrane serine/threonine kinase, IreK, its cog-
nate phosphatase, IreP, and their substrate, IreB, which
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also modulate intrinsic cephalosporin resistance by an
unknown mechanism (58–61).
The GrvRS TCSTS system, also known as EtaRS, is
orthologous to the important S. pyogenes virulence reg-
ulatory TCSTS CovRS (62). In E. faecalis it has been
demonstrated to be involved in virulence as well as re-
sistance to low pH and high temperature (63). GrvRS has
also been implicated in the regulation of the important
extracellular matrix adhesin Ace (64), catalase (65), and
the heat shock proteins DnaK and GroEL (66). It seems
likely that the GrvRS regulon will continue to grow with
further investigation.
Disruption of RR 04, 08, and 18 (as defined in 46)
each resulted in sensitivity to heat, and transcription of
HK-RR 07, 13, and 14 was induced by heat, suggesting
that these TCSTSs may also play a role in stress resis-
tance (66).
In E. faecium the EntKR TCSTS regulates expression
of the bacteriocin enterocin A (67). EntKR is a quorum
sensing system that responds to a small peptide produced
from the same locus, designated EntF. There appear to
be no close homologs to the EntKR system in E. fae-
calis. The ChtRS TCSTS contributes to resistance to both
bacitracin and the disinfectant chlorhexidine (68), and
CroRS is associated with intrinsic cephalosporin resis-
tance (57). Clear homologs of E. faecalis HK-RR 01, 03,
04, 05, 07, 09, and 10 (as defined in 46) are present in the
E. faecium genome sequence. In addition, a system with
linked homologs of the S. aureus agrA, agrB, and agrC
genes is present in E. faecium, but this system is not
closely related to the E. faecalis fsr system. The E. fae-
cium genome contains 10 more annotated RRs and 9
more HKs that either have no strong homology to coun-
terparts in the E. faecalis genome or are no more closely
related to E. faecalis genes than to genes from any other
Firmicutes. Therefore, E. faecalis and E. faecium have
about the same number of TCSTSs but not necessarily the
same complement of homologs.
The E. faecalis cyl cytolysin-producing operon is in-
duced by a quorum sensing mechanism operating through
a two-component system, but in this case the sensor
and regulator show no homology to HK/RR of typical
TCSTSs (69). Active cytolysin is composed of two post-
translationally modified peptides, CylLL and CylLS, the
latter acting as the autoinducer of the system. CylR2 is
a DNA binding protein that represses expression of the
cyl operon encoding the structural genes for the toxin,
an enzyme for modification of the toxin peptides, and
an ABC transporter for secretion. The structure of the
CylR2-promoter interaction has been determined (70).
The CylLS inducing signal is transmitted to CylR2 via

the membrane receptor CylR1. The system is sensitive
to both bacterial cell density and the presence of target
cells. Target cells are detected by preferential binding of
CylLL from the dual toxin complex, releasing CylLS to
induce the operon (71).
The E. faecalis genome includes numerous other pu-
tative regulatory proteins in a variety of other classes,
but few of these regulators have been characterized. The
E. faecalis version of the catabolite control protein gene,
ccpA, has been mutationally inactivated and shown to
regulate a variety of glucose starvation proteins and was
capable of complementing a B. subtilis ccpA mutant (72).
CcpA/P-Ser-Hpr binding to catabolite responsive ele-
ments (cre) in E. faecalis has been shown to participate in
(i) the repression of citrate utilization operons through
transcriptional inhibition of both the structural genes
and the gene for the GntR-type citrate-responsive posi-
tive regulator CitO (73), (ii) the partial repression of
genes for malate utilization, including maeKR encoding
a malate response TCSTS (74), and (iii) repression of
the agmatine deiminase pathway in cooperation with the
mannose PTS and in opposition to the AguR-positive
regulator (75). CcpA has also been shown to be impor-
tant for the growth and virulence of E. faecium (76).
Metal homeostasis is frequently important in bacterial
pathogens, and regulatory responses to copper and man-
ganese have been investigated in the enterococci. Copper
uptake in Enterococcus hirae is encoded in the four-gene
cop operon consisting of the copA and copB genes,
which encode proteins for copper uptake and efflux, re-
spectively, copY, which encodes a copper-responsive re-
pressor, and copZ, which encodes a copper chaperone
that delivers copper from CopA to CopY (77). At low
copper levels, CopY in complex with Zn(II) binds to
DNA sequences overlapping the promoter as a dimer and
inhibits transcription. At high copper levels, CopZ de-
livers Cu(I) to CopY, displacing Zn(II) and causing dis-
sociation from the promoter. CopZ complexed with Cu
(I) is subject to proteolysis, preventing excessive accu-
mulation of intracellular copper, which can damage the
cell. The Efa adhesin, encoded by the E. faecalis efaCBA
operon, is regulated in response to manganese by the
EfaR protein, a member of the DtsR/MntR repressor
family (78). The EfaR binding motif was later identified
in the promoter regions of 30 genes. efaR mutation im-
paired biofilm formation, survival in macrophages, and
oxidative stress tolerance, suggesting that EfaR is an
important modulator of virulence (79).
Four regulators, OxyR (80), PerR (81), Spx (82), and
HypR (83), have been implicated in the response of E.
faecalis to oxidative stress. HypR is the best studied of
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Enterococcal Genetics
these regulators, acting as a transcriptional activator of
at least six genes encoding catalase, glutathione reduc-
tase, two subunits of alkyl hydroperoxide reductase,
thiol peroxidase, and HypR itself through binding to a
“HypR box” (84, 85). HypR mutants are deficient in
macrophage survival and virulence.
Transcriptomic analyses have identified a number of
regulatory networks and putative regulatory proteins
involved in the virulence of E. faecalis. These have been
recently reviewed (10). Information relevant to stress re-
sponses in E. faecalis may also be found in a recent re-
view on the stress physiology of lactic acid bacteria (86).
Posttranscriptional regulation by various RNA-
mediated mechanisms has recently increased in promi-
nence in the regulation of gene expression in bacterial
cells. As in most other bacteria, the first examples of
RNA-mediated regulation in enterococci were identified
in MGEs (see below). More recently, a number of ex-
amples of RNA-mediated regulation were identified on
the enterococcal chromosome. The best studied of these
is regulation of the eut regulon, which encodes genes
essential for ethanolamine (EA) utilization (87, 88) (Fig. 1).
Regulation of the eut regulon requires a response to
two regulatory inputs: the presence of EA substrate and
the availability of adenosyl cobalamine (AdoCbl), a co-
factor in the first enzymatic step of EA catabolism. The
presence of EA is sensed by the EutVW TCSTS. Bind-
ing of EA by the EutW HK leads to phosphotransfer
to and dimerization of the EutV RR. Unlike most other
RRs, EutV is an RNA binding protein in the ANTAR
family of antiterminator proteins (89). EutV∼P dimers
bind to and stabilize a dual hairpin structure in nas-
cent RNA that precludes formation of multiple intrinsic
terminators in four eut polycistronic RNAs, allowing
transcriptional read-through into downstream eut genes.
AdoCbl is sensed via a unique riboswitch located up-
stream of the eutG structural gene, designated EutX. In
the absence of AdoCbl, EutX adopts a structure that
allows read-through to a EutV∼P dimer binding site.
The relative abundance of EutX allows sequestration of
EutV∼P dimers, preventing mRNA binding and anti-
termination (+EA) (Fig. 1). AdoCbl binding to nascent
EutX causes a conformational change that allows for-
mation of an intrinsic terminator that stops transcrip-
tion before the EutV∼P binding site (+AdoCbl) (Fig. 1),
allowing EutV∼P dimers to perform their antitermina-
tion function (+AdoCbl,+EA) (Fig. 1).
Several other examples of posttranscriptional regula-
tion have been identified in the enterococcal core genome.
The pyr operon of E. faecalis appears to be regulated
by PyrR via an attenuation mechanism similar to that
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FIGURE 1 Model for EutV- and EutX-mediated regulation of the eut regulon. In the absence
of either EA or AdoCbl (–EA), EutV∼P dimers are not available to disrupt terminators up-
stream of eut genes. In the presence of EA but the absence of AdoCbl (+EA), the EutX
riboswitch adopts a conformation that binds EutV∼P dimers, titrating them away from the
eut gene terminators. In the presence of AdoCbl but the absence of EA (+AdoCbl), EutX
adopts an alternative conformation that is not competent for binding EutV∼P dimers, but
such dimers are absent due to the lack of EA. In the presence of both EA and AdoCbl
(+AdoCbl, +EA), EutX is unable to bind EutV∼P dimers, which then interfere with termination
in eut transcripts, allowing the genes to be transcribed. (Adapted from reference 87.)

described for B. subtilis (90). Bioinformatic analysis sug-
gests that genes involved in amino acid biosynthesis and
transport are subjected to T-box regulation in E. faecalis
and E. faecium (91). A transcription attenuation mecha-
nism for regulation of the tetM gene of Tn916 was pro-
posed to be based on the observation of (i) a short leader
peptide encoded between the promoter and the TetM-
encoding open reading frame, (ii) the existence of mul-
tiple alternative RNA secondary structures surrounding
the leader peptide coding sequence, and (iii) the apparent
extension of a short transcript upon induction with tet-
racycline (92). RNase J2 has been implicated in the reg-
ulation of the ebp pilus operon in E. faecalis independent
of an effect on the level of the mRNA for EbpR, the
transcriptional activator of the operon (93). Mutation of
the E. faecalis rnjB gene encoding RNase J2 was further
shown to affect the expression of 62 genes and biofilm
formation, bile salt resistance, and virulence in a model
system (94).
Recently, a comprehensive map of all 5′ RNA ends
in E. faecalis V583, discriminating processed from pri-
mary ends, was obtained by a combination of differen-
tial tagging and RNA-seq (95). This study identified 85
novel putative regulatory RNAs, which when added to
those identified in previous studies (96, 97), brings the
total number to >120. This includes putative trans-acting
sRNAs and antisense RNAs as well as such functional
RNAs as transfer-messenger and 4.5S RNA. The specific
functions and targets of these putative sRNAs and anti-
sense RNAs remains a mystery, although four have been
implicated in modulating virulence and stress responses
(98). E. faecium has also been subjected to experiments
designed to detect putative regulatory RNAs with the
identification of 61 sRNA candidates (99). Several of
these RNAs were demonstrated to be induced or re-
pressed during exposure to daptomycin or development
of resistance to it. One putative sRNA, sRNA_0160, was
demonstrated to be downregulated in the presence of
daptomycin and repressed in a daptomycin-resistant
mutant. Further work will be required to determine the
target of this sRNA and its functional mechanism.
VANCOMYCIN RESISTANCE
Enterococci are intrinsically resistant to a variety of
commonly used antibiotics and have acquired resistance
to many others either by chromosomal mutation or ac-
quisition of MGEs (100). As such, the enterococci provide
a collection and distribution center for the dissemination
of antibiotic resistance genes to other, potentially more
pathogenic, Gram-positive bacteria. Dramatic evidence
of this was provided by the characterization of the first
high-level vancomycin-resistant S. aureus (VRSA) iso-
late (101, 102). Examination of the plasmid content of

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the VRSA isolate and coisolated vancomycin-resistant
E. faecalis (VRE) and vancomycin-sensitive S. aureus
(VSSA) strains suggested that a vanA-carrying transpo-
son, Tn1546, was delivered from the VRE to the VSSA
on a conjugative plasmid. The Tn1546 then transposed to
a plasmid resident in the VSSA, and the donating plasmid
was subsequently lost. Further studies have implicated
Inc18 plasmids, in particular, as the driving force for dis-
semination of vancomycin resistance to S. aureus (103).
However, even though the Tn1546-bearing plasmids are
conjugative in S. aureus, vancomycin resistance has not
become widespread in S. aureus infections. A total of only
14 VRSA were isolated between 2002 and 2015 (104).
Whether this is due to some fundamental incompatibil-
ity between vancomycin resistance and pathogenesis in
this species or some bottleneck in vanA transmission is
unknown.
Vancomycin is a cell wall-active antibiotic that rep-
resents the last line of defense against many multiply
resistant Gram-positive pathogens. Vancomycin and re-
lated glycopeptide antibiotics inhibit cell wall synthe-
sis by binding to the terminal D-Ala-D-Ala dipeptide of
peptidoglycan precursors, preventing their transfer from
the lipid carrier to the cell wall by transglycosidases (105).
Transpeptidases and D,D-carboxypeptidases involved in
cell wall synthesis are also inhibited. VRE circumvent
vancomycin action by substituting D-Ala-D-Lac or D-Ala-
D-Ser for the D-Ala-D-Ala dipeptide, decreasing the affinity
of peptidoglycan precursors for glycopeptides. Hydrolysis
of pre-existing D-Ala-D-Ala dipeptides is also required for
full resistance (106).
Unlike resistance to most other antibiotics, resis-
tance to vancomycin involves the acquisition of a suite
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Enterococcal Genetics
of enzymes required to reprogram peptidoglycan syn-
thesis. Nine genotypically and phenotypically distinct
systems designated VanA to VanE and VanG (reviewed
in 107), VanL (108), VanM (109), and VanN (110) have
been identified in VRE. Van gene clusters generally in-
clude three components: (i) a TCSTS, VanRS, required
for sensing the presence of the antibiotic and inducing
the resistance operon; (ii) an essential three-gene core
encoding enzymes responsible for producing the D-Lac
(VanH) or D-Ser (VanT) precursors, ligating those pre-
cursors to D-Ala (VanA to VanN), and eliminating D-Ala-
D-Ala dipeptides (VanX, VanY, and/or a VanXY fusion);
and (iii) accessory genes performing various and some-
times unknown roles. While the core and accessory genes
are generally transcribed as an operon, the TCSTS is
transcribed from a separate promoter (Fig. 2).
The VanC, VanE, VanG, VanL, and VanN systems all
encode low-level vancomycin resistance by producing pep-
tidoglycan peptides terminating in D-Ala-D-Ser. All encode
a VanXY bifunctional dipeptidase/carboxypeptidase for
degrading D-Ala-D-Ala, a VanT serine racemase for pro-
ducing D-Ser, and their respective ligases. The VanC sys-
tems are responsible for the intrinsic resistance of motile
enterococci, are chromosomally encoded, and do not ap-
pear to be on mobile elements. Although VanC systems
appear to encode a TCSTS, resistance is either expressed
constitutively or only sluggishly induced (111, 112). The
VanE system is very similar to VanC in sequence and gene
organization but is present in an E. faecalis strain (113).
Although likely acquired from one of the intrinsically re-
sistant enterococci, it is chromosomally located and could
not be transferred to a vancomycin-susceptible strain.
VanE is inducible in spite of a nonsense mutation in the
FIGURE 2 Organization of the VanA and VanB van-
comycin resistance systems. Gene functions are color
coded: green, sensory and regulatory; red, essential
resistance genes; blue, auxiliary resistance genes. De-
tailed functions of each gene are described in the text.
Green arrows show the position of the VanR inducible
promoters. The percentage identity of the VanB genes
to their VanA homologs is given below the individual
VanB genes. Homologs have identical names in both
systems except for the vanA and vanB ligases.
7
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gene for the VanS HK, suggesting some level of cross-talk
with other cellular TCSTSs. The VanG system is also
chromosomally located in an E. faecalis strain but resides
on a large (∼240 kb) mobile element that also encodes
an ermB erythromycin resistance gene (114). The VanG
system is inducible, probably due to the presence of a
functional VanRS TCSTS. VanL and VanN were re-
cently described, with VanN being the first D-Ala-D-Ser-
producing system in E. faecium (108, 110).
The VanA, VanB, VanD, and VanM systems produce
peptidoglycan precursors terminating in D-Ala-D-Lac. The
VanA and VanB systems are widespread in both E. fae-
calis and E. faecium. In general, vancomycin resistance is
more prevalent in E. faecium than in E. faecalis (115).
Phenotypically, VanA-type resistance is characterized by
high-level resistance to both vancomycin and teicoplanin,
while VanB-type resistance provides variable levels of re-
sistance to vancomycin only. Susceptibility to teicoplanin
in the VanB system is due to a failure to induce the re-
sistance genes in response to this antibiotic; VanB cells
induced with vancomycin are resistant to both antibi-
otics. Genotypically, the VanA and VanB systems are
quite similar (Fig. 2). Each encodes a VanRS TCSTS, a
VanH pyruvate dehydrogenase for production of D-Lac,
their respective ligase VanA or VanB, and separate VanX
dipeptidase and VanY carboxypeptidase enzymes. Only
the vanH, vanA/B, and vanX genes are essential for re-
sistance. In addition, the VanA system encodes an addi-
tional gene, vanZ, which confers low-level resistance to
teicoplanin by an unknown mechanism, and the VanB
system encodes a gene, vanW (also present in VanG), of
unknown function.
The VanRS TCSTS of the VanA and VanB systems
have been extensively investigated, and reference 116
provides an excellent review. In vitro the VanRS sys-
tem functions much like other TCSTSs. The HK, VanS,
autophosphorylates its conserved histidine residue and
then transfers the phosphate to the VanR RR (117).
Phosphorylated VanR (VanR∼P) produces a larger foot-
print and binds with higher affinity to its target pro-
moters than unphosphorylated VanR, consistent with
its in vivo role as a transcriptional activator (118).
However, in the absence of VanS, dephosphorylation of
VanR∼P is unusually slow compared to related RR, and
VanS stimulates dephosphorylation (117). In addition,
VanR is capable of stimulating its own phosphoryla-
tion in the absence of VanS, using acetyl phosphate.
These in vitro results are consistent with the observation
in vivo that inactivation of VanS results in constitutive
activation of vancomycin resistance rather than failure
to induce expression. Apparently, VanR∼P is the default
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condition in vivo, with phosphorylation occurring either
by acetyl phosphate or by cross-talk with other cellular
HK. The primary role of VanS, then, is to dephosphor-
ylate VanR in the absence of the inducing signal (119,
120). The nature of the inducing signal is beginning to
emerge. Given the differences in induction patterns and
the poor conservation of VanS, the signal may be dif-
ferent for the VanA and VanB systems. Interestingly,
single amino acid substitutions in the signaling domain
of VanSB allow induction by teicoplanin (121). A re-
cent report examining structural and activity effects on
VanSA of a range of potential ligands including vanco-
mycin, teicoplanin, and a variety of peptidoglycan pre-
cursers identified the likely inducers as the glycopeptides
themselves (122).
Both the VanA and VanB gene clusters are frequently,
if not always, associated with MGE. VanA is located on a
Tn3-type transposon, Tn1546, and is frequently found
on conjugative plasmids. In one E. faecium outbreak, a
unique mechanism of facilitating horizontal transfer of
VanA was observed (123). In this case, a nonconjugative
Tn1546-bearing plasmid was observed to integrate into
a pheromone-responsive plasmid via a mechanism in-
volving a series of insertion sequences located outside of
Tn1546. Conjugative transfer was enhanced by the fact
that plasmid fusion occurred within a homolog of prgX,
the central negative regulator of the pheromone response
in pCF10-like plasmids (see below). The resulting in-
sertional inactivation of prgX derepressed conjugation
functions, and transfer occurred in the absence of pher-
omone. Tn1546-like elements have also been identified
on plasmids related to the conjugative non-pheromone-
responsive plasmid pMG1 (124) (see below). VanB has
been localized on a variety of MGEs. In some strains,
conjugative transfer of VanB has been associated with
the movement of large (90 to 250 kb), chromosomally
located elements that may be complex conjugative trans-
posons (125). VanB was further localized on one of these
elements to a 64-kb composite transposon, Tn1547, ca-
pable of transposition to plasmids under laboratory con-
ditions. In other cases, VanB resistance was associated
with conjugative plasmids (126), but the identification of
similar genes on a variety of plasmids also suggested a
transposon location. A VanB gene cluster was localized
on a Tn916-like element designated Tn5382 (127). Al-
though independent mobility of this element was not
demonstrated, it appears to be associated with a larger,
130- to 160-kb chromosomally located conjugative ele-
ment. A similar element, designated Tn1549, was iden-
tified on a pAD1-like pheromone-responsive plasmid
(128). Mobility of chromosomally located vancomycin

resistance genes may also be accomplished by Hfr-like
transfer mechanisms as described below and in references
129 and 130.
Unlike VanA and VanB, VanD systems provide con-
stitutive vancomycin resistance due to various lesions in
the vanS gene (131–133). In spite of the presence of an
apparently intact vanX gene, VanD systems produce very
little VanX D-Ala-D-Ala dipeptidase activity. Neverthe-
less, peptidoglycan precursors in VanD strains contain
very little or no D-Ala-D-Ala terminal peptides due to
mutations in the chromosomally encoded ddl gene re-
sponsible for producing the D-Ala-D-Ala precursors (131,
133, 134). Thus, a combination of constitutive expres-
sion of VanD and mutation of the chromosomal ddl
ensure vancomycin resistance. VanD systems have been
found only in E. faecium and do not appear to be mobile.
The complex nature of the vancomycin resistance
systems argues against their acquisition by accumulation
of mutations or piecemeal acquisition of individual genes.
More than likely, they were acquired en masse from
a single source. In this context it is interesting to note
that the gene organization and sequence of the VanA
and VanB systems are highly similar to self-protection
systems present in glycopeptide-producing species (135,
136).
Resistance to Other Antibiotics
In addition to vancomycin, enterococci express intrinsic
and/or acquired resistance to a wide variety of other anti-
biotics, including daptomycin, aminoglycosides, rifampi-
cin, quinolones, macrolide/lincosamide/streptogramin B,
β-lactams, and linezolid. For information on the genetics
of these resistance mechanisms and the MGEs with which
they are associated the reader is referred to a recent ex-
tensive review (100).
PHEROMONE-RESPONSIVE PLASMIDS
The enterococci host a multitude of plasmids, including
both theta and rolling circle replicating replicons (RCRs)
(137). The majority of these plasmids fall into six clas-
ses based on conserved domains in their putative ini-
tiator proteins: three RCR classes (Rep_trans, Rep_2,
and Rep_1) and three theta classes (Rep_3, inc18, and
RepA_N). These plasmids have been further subdivided
into 10 families (of 19 present in the Firmicutes) based
on initiator sequence homology (138). RCR and inc18
plasmids generally have a broad host range and are
widely disseminated in Gram-positive and even some
Gram-negative bacteria. They have been extensively
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Enterococcal Genetics
studied in bacteria other than the enterococci and have
been the subject of several excellent reviews (137, 139,
140). Rep_3 replicons are ubiquitous in bacteria and
include some of the best-studied bacterial plasmids, in-
cluding F, P1, and pSC101 (141, 142). The RepA_N
plasmids are a class of replicons broadly distributed
among the Firmicutes but whose individual members
have a narrow host range (143). They include both
conjugative and nonconjugative plasmids ranging in size
from 3.3 to 281 kb. RepA_N proteins have also been
detected on streptococcal bacteriophages and integrated
conjugative elements (ICEs). Of particular relevance to
enterococcal genetics are the RepA_N replicons that
encode conjugation systems that are induced by small
peptide pheromones secreted by potential recipients, the
pheromone-responsive plasmids. These plasmids repli-
cate primarily in E. faecalis and remain one of only two
plasmid systems in which a specific signal for induction
of conjugation has been identified (the quorum sensing
system of Agrobacterium Ti plasmids is the other [144]).
These plasmids will be discussed below, but the reader is
referred to detailed reviews that discuss their replication,
stable inheritance, and conjugation (137, 145–150).
Pheromone-responsive plasmids range in size from 37
to 92 kb and are maintained at a low copy number in
their native host. In general, the pheromone-responsive
conjugation system works as follows. Conjugation in cells
containing pheromone-responsive plasmids is normally
repressed in the absence of an appropriate recipient.
When cocultivated with or exposed to culture filtrates
of plasmid-free cells, plasmid-containing cells respond by
producing a plasmid-encoded surface adhesin called ag-
gregation substance (AS). AS binds to a ligand called
enterococcal binding substance that is present on the
surface of both donor and recipient cells, leading to the
formation of macroscopic cellular aggregates that pro-
vide the contact required for plasmid transfer. Once
transfer is complete, production of the pheromone spe-
cific for the transferred plasmid is shut down in recipient
cells by the combined action of two plasmid-encoded
components: a peptide that competitively inhibits pher-
omone activity and a membrane protein that reduces
pheromone secretion. These inhibitor mechanisms are
specific for the cognate pheromone of the particular
plasmid acquired; production of pheromone(s) specific
for other plasmids continues, allowing the recipient to
induce a mating response in other potential donors and
continue to collect plasmids. A cartoon showing the
relevant players involved in the pheromone response,
how they interact, and the organization of the plasmid-
encoded regulatory genes is shown in Fig. 3.
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FIGURE 3 Regulatory circuitry of the pheromone response. (A) Events occurring at the cell
surface. Important chromosomally encoded determinants are colored yellow with the
exception of the pheromone itself, which is shown as blue circles. Plasmid-encoded de-
terminants are color coordinated with their genes shown in panel B. The inhibitor peptide
(ip) is shown as red circles and competitively inhibits pheromone binding to TraC. TraB
inhibition of pheromone secretion is depicted here as sequestration, but may also involve
peptide degradation. (B) Events occurring at the DNA level. Binding of pheromone by TraA
links events at the cell surface with events at the DNA level. A conformational change in
TraA due to pheromone binding is indicated by the change in shape of the molecule and
the change in dimerization state, although more subtle conformational changes are likely.
Pheromone-free TraA binds Po and inhibits transcription. Direction of transcription from
Po and Pa is indicated by the arrows. The antisense RNA, generalized to aR in this figure,
stimulates termination at t1, indicated by the green arrow. Positive regulatory elements vary
between different pheromone-responsive plasmids, and their mechanisms of inducing
downstream transcription of the conjugation structural genes may also vary. For simplicity,
the more common gene names and order are used. It should be noted that the gene order
of the traC and traB genes are reversed in pAD1. The RepA gene is shown to orient the
reader relative to Fig. 4. (C) Events at the RNA level. Relative levels of RNA produced from
the Po and Pa promoters under uninduced (red) and induced (green) conditions are
depicted by the sizes of the arrows.

Regulation of the Pheromone Signal
Transduction Circuit
All sex pheromones and inhibitors characterized to date
are linear, hydrophobic peptides of seven or eight amino
acids. The genetic determinants responsible for the pro-
duction of five pheromones were identified in the E.
faecalis V583 genome and found to be encoded within
signal peptide coding sequences of different lipopro-
tein genes (151). The involvement of these genes in the
production of pheromones specific for pAD1 (cAD1),
pCF10 (cCF10), and pAM373 (cAM373) was confirmed
by cloning and mutagenesis experiments (152–154). Mu-
tations of the mature lipoprotein-encoding portion of the
genes for cAD1 and cAM373, cad and camE, respec-
tively, had no effect on pheromone production or mat-
ing potential, suggesting that the lipoproteins themselves
are not involved in conjugation. Pheromone sequences
are generally found at the C-terminus of the lipoprotein

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signal peptide, indicating that signal peptidase II is re-
quired for the initial processing step. Maturation of the
pheromone is then completed by the site 2 protease Eep in
most cases (155). As described above, Eep belongs to a
family of intramembrane processing proteins involved
in regulated intramembrane proteolysis in organisms
as diverse as bacteria and humans (156). The plasmid-
encoded genes for the inhibitor peptides (iAD1, iCF10,
iAM373, etc.) encode prepeptides resembling unattached
lipoprotein signal peptides (157) and therefore require
only Eep for processing. Some variability is observed in
the processing of specific pheromones, suggesting further
complications and potential alternative pathways. For
example, Eep-processed cCF10 is further processed by
removal of three C-terminal amino acids by an unknown
mechanism (153, 158), and Eep is not required for cOB1,
cAM373, or iAM373 processing (155, 158). Interest-
ingly, both cAD1 and cAM373 activities can be detected
in some S. aureus strains. In both cases the responsible
peptides are produced from the signal sequences of lipo-
proteins unrelated to those that produce the same pep-
tides in E. faecalis (154, 159). Linear synthetic peptides of
the appropriate sequence function as effective inducing
signals for the cognate plasmids, indicating that no fur-
ther processing is necessary. Hydrophobic septa- and oc-
tapeptide pheromones are pumped out of the membrane
by a membrane ABC pump (160). Release of lipoprotein
signal sequence fragments appears common among the
Firmicutes, and perhaps pheromone-responsive plasmids
evolved to take advantage of this process. It is interesting
and perhaps surprising that so far, this efficient cell-cell
communication system is mainly found in the species E.
faecalis and is not more widespread.
Pheromones are imported into plasmid-containing cells
by the chromosomally encoded oligopeptide permease
(Opp) system in combination with a plasmid-encoded
OppA homolog, TraC/PrgZ, that provides increased sen-
sitivity and specificity for the cognate pheromone (161–
164). The chromosomal Opp system transports sufficient
pheromone to induce conjugation at high peptide con-
centrations, but TraC/PrgZ allows responses to phero-
mone levels of 10–10 to 10–11 M (161). Evidence indicates
that inhibitor peptides compete with their cognate pher-
omones for TraC/PrgZ binding and that this at least par-
tially accounts for their function (but see below) (102,
165).
Once inside the cell, pheromone is bound by a key
negative regulatory protein, TraA/PrgX (161, 166). These
proteins belong to the RRNPP (Rap, Rgg, NprG, PlcR,
and PrgX) family of linear peptide pheromone-responsive
regulators that are widespread in Firmicutes (167).
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Results with three TraA/PrgX proteins, those from pAD1,
pPD1, and pCF10, show that the repressors bind both the
stimulatory pheromone peptides and their cognate in-
hibitor peptides (166, 168, 169). In pPD1 and pCF10,
both inhibitor- and pheromone-bound repressors bind
the promoter required for expression of the conjugative
apparatus (Po in Fig. 3) but with subtly different con-
figurations that make the difference between prevent-
ing or allowing RNA polymerase binding (168, 170).
In pPD1 this involves the binding of TraA to alternate
sites within the promoter region (168). In pCF10 it in-
volves subtle conformational changes in the peptide-
bound PrgX tetramers that allow greater competition
with RNA polymerase promoter binding in the inhibitor-
bound than in the pheromone-bound state (170). Thus,
competition between pheromone and inhibitor peptides
occurs at two levels, import via TraC/PrgZ and inter-
action with repressor protein TraA/PrgX. The complex
dual peptide regulation of TraA/PrgX systems is unique
among RRNPP family members, giving rise to several
interesting evolutionary questions that have been ad-
dressed elsewhere (148).
As shown in Fig. 3, even in the absence of pheromone,
transcription from Po is not completely repressed. In fact,
basal-level expression is required to maintain the unin-
duced state since the gene for the inhibitor peptide, ip in
Fig. 3, is under Po control. In fact, results in pCF10 sug-
gest that pheromone induction results in only a 5-fold or
less increase in transcription from Po (147, 171). Thus,
the pheromone response must rely on regulatory mech-
anisms beyond simple derepression of transcription from
Po. Multiple lines of evidence suggest that this added
layer of regulation occurs at the t1 terminator (Fig. 3),
which separates the Po promoter from the rest of the
conjugation genes. First, pPD1 TraA has been shown to
bind to both t1 and t2 either alone or when iPD1-bound
but not when cPD1-bound (168). It is proposed that this
facilitates termination in the uninduced state, but pre-
cisely how this occurs is not known. Second, transcrip-
tion from the Pa promoter results in the production of
an antisense regulatory RNA (aR in Fig. 3) that binds
to the nascent Po transcript, stabilizing a conformation
including an intrinsic terminator. mD and Qa antisense
RNAs have been identified and described in pAD1 and
pCF10, respectively (172, 173), and their sequences and
location are highly conserved in other pheromone plas-
mids. Details of this countertranscript-driven attenuation
mechanism have been described for the pCF10 system
(174, 175). In pCF10, PrgX and Qa are transcribed from
Pa, and PrgX is required both for positive autoregulation
of Pa and proper processing of Qa (171). In both the pAD1
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Weaver
and pCF10 systems, pheromone induction results in a
significant decrease in antisense RNA production (176–
178). Based on these observations, it was proposed that,
under noninducing conditions, the level of antisense RNA
is precisely poised to ensure that all Po transcripts adopt a
termination-competent structure. Even a moderate in-
crease in transcription from Po tips the balance in favor of
antitermination by three amplification mechanisms: (i)
titration of the Pa transcript by the excess Po transcript,
(ii) transcriptional interference favoring the Po transcript
and further reducing Pa transcript levels (179), and (iii)
RNase III-mediated degradation of the complex between
the Po and Pa transcripts, which reduces translation of
TraA/PrgX (180). These overlapping regulatory mecha-
nisms allow the cell to precisely set the pheromone:
inhibitor ratio high enough to prevent fruitless induction
of the costly conjugation apparatus but low enough to
allow efficient transfer of the plasmid DNA when suffi-
cient numbers of recipients are present. Tight regulation
at t1 is most likely required to prevent transcription of
strong downstream positive regulators. In the case of
pAD1, this regulator is TraE1, which positively auto-
regulates its own transcription and is essential for ex-
pression of the downstream structural genes required for
conjugation (177, 181). The pCF10 plasmid lacks a
TraE1 homolog but produces several RNAs in the re-
gion immediately downstream of t1 and t2 that posi-
tively regulate downstream structural genes (182–184).
The mechanisms of action of these regulators are un-
known but may involve both stimulation of transcrip-
tional read-through and translation.
The inhibitor peptide is essential for modulating the
plasmid-containing cell’s response to exogenous phero-
mone, but it is not sufficient to prevent self-induction by
endogenously produced pheromone. A second plasmid-
encoded protein, TraB/PrgY, is required to specifically
reduce secretion of the cognate pheromone (185–187).
The pCF10-encoded PrgY protein has been studied in
detail (158, 188). It consists of four transmembrane do-
mains with the N-terminal two-thirds of the protein lo-
cated on the outer surface of the cytoplasmic membrane.
The cCF10 pheromone binding region has been mapped
to an approximately 120-amino acid region in the extra-
cytoplasmic domain adjacent to the first transmembrane
segment. PrgY has been proposed to function by speci-
fically binding to cognate pheromone as it emerges from
the membrane, either following or concomitant with
cleavage by Eep. The protein then either degrades the
peptide directly or chaperones it to a cell wall bound
protease for degradation (147). Recent data favor direct
degradation (G. Dunny, personal communication). TraB/
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PrgY proteins appear to be part of an orthologous family
of proteins found in all domains of life but only spottily
distributed (188). For instance, there are representatives
in humans, plants, and Gram-negative bacteria but not in
any other Firmicutes species outside of the enterococci.
This phylogenetic distribution suggests that horizontal
gene transfer has been critical for their distribution. No
function has been assigned for any of the TraB/PrgY
orthologs, but homology suggests that they are involved
in some form of peptide signaling or processing. Inter-
estingly, pAM373 appears to control extracellular pher-
omone levels without a clear TraB/PrgY homolog (189).
Altogether then, pheromone plasmids encode three
proteins, TraC/PrgZ, TraA/PrgX, and TraB/PrgY, that
bind their cognate pheromone yet are divergent enough
to be able to selectively respond only to the pheromone
specific for their encoding plasmid. Structural analyses
of these proteins suggest that the pheromone binding
pockets did not diverge from a single ancestral domain
but rather evolved independently and convergently (148).
Assuming that all pheromone plasmids descended from
a single ancestor, divergence to respond to new phero-
mone and inhibitor peptides requires coordinated evolu-
tion of the binding pockets of all three proteins. How
these specificity switches occur is an intriguing evolu-
tionary question.
The Conjugation Apparatus
Downstream of the positive regulatory elements of pAD1,
transcription of a contiguous region of ∼20 kb is induc-
ible by pheromone (190). In most pheromone plasmids,
the first two genes of this region encode a surface exclu-
sion protein and AS (191). Both proteins encode signal
sequences and LPXTG cell wall-anchoring motifs and
have been demonstrated to be present on the surface
of only pheromone-induced plasmid-bearing cells (192).
The surface exclusion proteins limit conjugation between
strains harboring the same pheromone plasmid (193,
194). AS is essential for the cellular aggregation response
to pheromone and efficient mating in broth but is not
required for plasmid transfer on solid surfaces, suggesting
that it is not an integral part of the mating pore (195). AS
is a cell wall-anchored protein and is highly conserved in
all pheromone plasmids except pAM373. pAM373 does
not encode an entry exclusion function (196) and encodes
an AS that is half the size of other AS proteins and shows
only localized homology (189). AS facilitates intercellu-
lar aggregation by binding to an enterococcal binding
substance which is present on the surface of both donor
and recipient cells. Lipoteichoic acid (LTA) appears to
be at least one component of enterococcal binding sub-
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stance, since free LTA inhibits aggregation (197) and cells
containing mutations affecting LTA are poor recipients
(198). Mutagenesis and in vitro binding analysis of the
AS from pCF10 has identified two distinct aggregation
domains, one of which binds tightly to LTA in vitro (199,
200). However, pCF10 AS also binds LTA from E. hirae
which does not aggregate with AS-expressing E. faecalis
cells, suggesting that LTA binding may not be sufficient
for aggregation. AS has also been implicated as a viru-
lence factor in several pathogenicity models (201–205)
and is induced by plasma by means of the pheromone-
sensing machinery (206). A recent study demonstrated
that the pCF10 surface exclusion protein, AS, and the
product of the prgC gene immediately downstream all
play roles in conjugation, biofilm formation, and viru-
lence (207). Another gene located between the AS gene
and prgC, designated prgU, encodes a small protein with
an RNA binding fold that mitigates the toxicity associated
with overproduction of AS and other related adhesins in
multiple Gram-positive bacteria (208). Its mechanism of
action remains to be determined.
Comparison of the sequences downstream of the AS
gene of five completely sequenced pheromone-responsive
plasmids, pAD1 (209), pAM373 (189), pCF10 (210),
pTEF1, and pTEF2 (6), show a high degree of conser-
vation in the presumed structural genes required for
conjugation. Comparison of these sequences to those of
conjugative systems of both Gram-negative and Gram-
positive bacteria suggests that they are orthologous to the
family of protein and DNA secretion machineries col-
lectively referred to as type 4 secretion systems, or T4SSs
(211). Functional studies of the products of these genes
from pheromone-responsive plasmids are limited, but
work on pCF10 has identified PcfC as the coupling pro-
tein responsible for bringing the nicked DNA substrate
to the secretion apparatus (212, 213), PrgJ as a VirB4-
like ATPase in the T4SS apparatus (214), and PrgK as a
peptidoglycan hydrolase required to generate localized
lesions in the cell wall for assembly of the T4SS (215).
Within the conserved region encoding the T4SS is a
short segment of localized reduced homology. In pAD1,
this region has been shown to encode a functional origin
of transfer (oriT2) located between a relaxase gene, traX,
and traW, the pAD1 coupling protein homolog (209). A
similarly located oriT was identified in pAM373, and the
two oriTs were found to be mobilized only by their
cognate plasmids. Plasmid-specific single-strand nicking
by the putative relaxase was demonstrated within a large,
conserved inverted repeat in oriT. Specificity was im-
parted by nonconserved direct repeats, presumed to be
the relaxase binding site, adjacent to the conserved
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inverted repeat (216). The relaxase of these plasmids is
unique in that it lacks the “three-histidine motif” com-
mon to most other plasmid relaxases (217) and instead
relies on a catalytic domain related to the PD-(D/E)XK
family of restriction endonucleases for nicking activ-
ity. Thus, the pAD1 TraX relaxase is the prototype for
a distinct family of relaxases designated MOBC (218).
pAD1 also contains a second oriT located within the repA
initiator protein gene that is similar to IncP-type oriTs
(209), but this oriT is utilized with 1,000-fold lower ef-
ficiency than oriT2 and its biological relevance is un-
clear. The pCF10 relaxase, PcfG, is unrelated to pAD1
TraX and instead is closely related to the pheromone-
independent Lactococcus lactis plasmid pRS01 relaxase
(210, 219, 220). A comparative study of the pCF10 and
pRS01 systems identified related accessory proteins im-
portant for assembly of the relaxosome. In spite of clear
sequence homology between the pCF10 and pRS01 re-
laxases and their target oriTs, each system effectively
mobilized only its cognate target (221).
Replication and Stable Inheritance
On the opposite side of the pheromone-response regu-
latory module from the conjugation structural genes
are the genes required for replication and stable inher-
itance (189, 222–224). The genes of the basic replicon
include a replication initiator protein RepA/PrgW, a
partition system RepBC/PrgPO, and an antisense RNA-
regulated type I toxin-antitoxin (TA-1) module desig-
nated par (Fig. 4). The repA/prgW genes belong to the
RepA_N family of replication initiator proteins, which
along with their associated partition modules, have been
recently reviewed (143). RepA_N initiator proteins are
not restricted to pheromone plasmids and are wide-
spread among conjugative and nonconjugative plasmids
in the Firmicutes. The genes encoding RepA_N initiators
encode a centrally located series of repeats whose or-
ganization and sequence vary between plasmids and, in
some cases, have been demonstrated to function as an
origin of replication (oriV) (225–228). Work on pAD1
has shown that (i) oriV is sufficient to support plasmid
replication if its RepA_N initiator, RepA, is supplied
in trans, (ii) the repA gene itself is sufficient to support
plasmid replication if RepA is overexpressed from an
artificial promoter, and (iii) RepA binds specifically to
oriV in double-stranded DNA and nonspecifically to
single-stranded DNA (225). The pCF10 initiator pro-
tein, PrgW, binds cCF10 pheromone, and provision of
cCF10 in a host not normally permissive for plasmid
replication, L. lactis, allows plasmid establishment (229).
The role of cCF10 in plasmid replication and whether
13
Weaver

FIGURE 4 Genetic organization of a general pheromone-responsive plasmid replicon.

Gene names are given as pAD1/pCF10. The stipled box within repA/prgW represents the
origin of replication (oriV) to which the repA/prgW product binds. The lined boxes at each
end of the repB/prgP-repC/prgO operon represent the likely centromere-like sites to
which the repC/prgO products bind and, with the repB/prgP product, direct plasmid
partition. The organization of the par region is blown up below the replicon. The arrow-
heads at each end of the locus marked P represent the promoters for the RNAI and RNAII
transcripts. The extent and direction of transcription of the transcripts are shown above and
below the genes for RNAII and RNAI, respectively. The fst gene is designated by the di-
agonally lined box, and the sequence of the peptide is shown at the bottom. DRa and DRb
are direct repeats in the DNA sequence that provide complementarity between RNAI and
RNAII when transcribed in opposite directions. Overlapping transcription at the bidirec-
tional transcriptional terminator also provides complementarity.

this observation can be extended to other pheromone-
responsive plasmids has not been determined. The mech-
anisms of strand opening, initiation of replication, and
regulation of initiation frequency have yet to be deter-
mined for the pheromone-responsive plasmids. How-
ever, work with the RepA_N proteins of staphylococcal
plasmids pSK41 and pTZ2162 may be instructive (230).
Both domains flanking the central repeat region of these
proteins adopt a structural fold similar to the DnaD pri-
mosome. However, the N-terminal domain has evolved
to bind the oriV repeats bending the DNA and presum-
ably facilitating separation of the DNA strands. The C-
terminal domain retains primosome function and recruits
DnaG primase to initiate replication. Furthermore, the
N-terminal domain was also demonstrated to be capable
of bridging two plasmid origins, suggesting that it could
function in replication control by handcuffing (231). It
had been previously proposed that the pattern of con-
servation of RepA_N proteins, with a highly conserved
N-terminal domain and genus-specific conservation of
the C-terminal domain, suggested a universally conserved
function (e.g., origin binding) for the former and a genus-
specific function (e.g., interaction with host replication
proteins) for the latter (143). These results support that
prediction. A second method of copy number control in-
volving an antisense RNA that regulates expression of the
initiator protein is superimposed on handcuffing in the
staphylococcal plasmids (232). No analogous antisense
RNA has yet been detected, either physically or bioin-
formatically, on the pheromone plasmids.
The pheromone-responsive plasmid partition systems
are variants of the previously described type Ib class
(233). Type I systems encode a well-conserved Walker-
type ATPase designated ParA, a poorly conserved DNA
binding protein designated ParB, and a centromere-
like site to which ParB binds. In type Ib systems, the
ParA component lacks a DNA-binding domain, and the
centromere-like site is located upstream of the parA-
parB operon where binding of the ParB component can
autoregulate transcription as well as direct partition.
The RepB/PrgP and RepC/PrgO proteins correspond to
type Ib ParA and ParB proteins, respectively. In all four
pheromone-responsive plasmids examined, including
pAD1, pCF10, pPD1, and pAM373, a series of repeats
is present upstream of the repB/prgP gene (Fig. 4), with
at least one repeat overlapping a putative promoter se-
quence, suggesting that protein binding to the repeats
could suppress transcription. A series of similar repeats
is also located downstream of the repC/prgO gene (143,
150). Each plasmid has repeats with a distinct DNA
sequence and organization. For example, pAD1 contains
26 TAGTARRR (R = purine) repeats upstream of repB

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in two clusters of 13 and 12 tandem repeats with a single
isolated repeat overlapping the promoter. Three more
tandem repeats are located downstream of repC. pCF10
contains six tandem ATATATNNN (N = any nucleo-
tide) repeats upstream of prgP with a single isolated re-
peat overlapping the promoter and two sets of four
tandem repeats downstream of prgO. Work with the
pAD1 RepBC system indicates that it functions similarly
to other partition systems (234). Thus, RepBC, with the
flanking repeats, is capable of stabilizing heterologous
plasmids, the upstream repeats act as a strong incom-
patibility determinant, RepC binds specifically and with
high affinity to the repeats, RepB binds the repeats only
in the presence of RepC and ATP, and RepC down-
regulates its own expression. Plasmids bearing either the
full complement of 25 upstream repeats or the half site
containing 12 repeats but not the downstream 3 repeats
can be stabilized by repBC supplied in trans. Sponta-
neous expansion of the pAD1 repB upstream repeats
has also been associated with loss of regulation of the
conjugative response and constitutive aggregation (235).
The molecular basis of this phenomenon is unknown but
may not be directly related to the normal function of
the repeats. RepB proteins are broadly distributed in the
Firmicutes but are not phylogenetically congruent with
the associated replication initiator proteins, suggesting
mosaicism of partition and replication modules (143).
TA modules are ubiquitous on bacterial plasmids
and function as stability determinants by programming
plasmid-free segregants for death (236). To accomplish
this, TA modules encode a stable toxin and an unstable
antitoxin; as long as the plasmid is retained, antitoxin is
continuously replenished and the cell is protected, but if
the plasmid is lost, the antitoxin is degraded and the
toxin kills the cell. There are now five classes of TA
modules (237), but only two are currently relevant to
enterococci. In TA-1 the antitoxin is an unstable small
regulatory RNA that binds to the toxin message and
suppresses its translation. In TA-2 the antitoxin is an
unstable protein that binds to the toxin protein and in-
hibits its activity. A general review of these systems is
available (238), and enterococcal TA systems have been
reviewed (137). Only one TA system has been charac-
terized on pheromone-responsive plasmids, the TA-1 par
locus (see 137, 239–241 for extensive reviews). par en-
codes two convergently transcribed RNAs, RNAI and
RNAII, initiating from each end of par and terminat-
ing at a common bidirectional intrinsic terminator (242,
243) (Fig. 4). RNAI encodes the toxic component of the
system, a 33-amino-acid peptide designated Fst. RNAII
is a 65-nucleotide small regulatory RNA that is the par
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antitoxin. Although the RNAI and RNAII genes overlap
only at the terminator, each reads in opposite directions
across a pair of direct repeats (DRa and DRb in Fig. 4).
Therefore, RNAII contains regions of complementarity
with both the 3′ and 5′ ends of RNAI. Furthermore, the
RNAI repeats are positioned such that interaction with
RNAII sequesters the translation initiation region of fst,
preventing ribosome binding and translation (244, 245).
Formation of the RNAI-RNAII complex proceeds in
at least two steps, with the interaction initiating at a
U-turn motif in the RNAI terminator loop which stim-
ulates interaction with the complementary sequences in
the RNAII terminator loop. Interaction then extends to
the repeats at the 5′ end, suppressing translation (246)
(Fig. 5). An intramolecular stem-loop within RNAI that
partially sequesters the fst ribosome binding site is re-
quired to inhibit translation until RNAII binding can be
completed (244, 247). A long distance interaction be-
tween sequences near the 3′ end of RNAI and its 5′
nucleotides is responsible for the stability of RNAI rela-
tive to RNAII (248). Unlike most antisense RNA sys-
tems, binding of RNAI by RNAII results in formation of
a highly stable complex rather than specifically targeting
the complex for degradation (248, 249). Formation of
such a stable complex allows the toxin-encoding RNAI
to persist in plasmid-containing cells. Over time, how-
ever, RNAII is slowly removed from the complex to al-
low translation of fst. The mechanism of this preferential
degradation of RNAII from the complex is unknown.
The Fst toxin sequence is shown in Fig. 4. It consists of
a central hydrophobic region flanked by charged N- and
C-terminal tails. Overexpression of Fst has been shown
to be toxic in E. faecalis, S. aureus, B. subtilis, and E. coli
(247, 250–252). In all four species overexpression re-
sults in condensation and mislocalization of the nucleoid
and interference with proper partition and cell division.
Membrane permeabilization is only a secondary effect.
Internal expression of Fst and external addition of the
lantibiotic nisin synergistically effect cell killing, suggest-
ing that the two peptides have different but comple-
mentary effects on the cell envelope (252). Saturation
mutagenesis experiments revealed that the central hy-
drophobic region and charged residues in the N-terminus
were important for overexpression-mediated toxicity but
that the C-terminal charged tail was nonessential (251).
An atomic resolution structure determined in a mem-
brane mimetic by nuclear magnetic resonance spectros-
copy revealed that the essential hydrophobic region
forms an α-helix that spans the membrane with the
charged N- and C-termini protruding (253). The charged
C-terminal seven amino acids were disordered and pre-
15
Weaver

FIGURE 5 Model of RNAI-RNAII interaction. RNAI is the larger, black structure and RNAII is
the smaller, blue structure. Stems, loops, and bulges are depicted in their approximate
locations and sizes as determined by experimentation. The red stem-loop structure within
RNAI sequesters the ribosome binding site and prevents translation until a complex is
formed. The initial interaction occurs at a U-turn motif in the terminator loop of RNAI (A),
followed by interaction between the complementary repeats in the 5′ end of each RNA
(B and C). Since RNAII-mediated protection from the RNAI-encoded toxin occurs in vivo even
when one of the repeats is mutated, the structure shown in panel C is apparently sufficient
to prevent translation in vivo. Once complex formation is complete (D), the structure is
extremely stable in vivo and in vitro perhaps due to the gap between the interacting repeats.

dicted to extend from the cytoplasmic side of the mem-
brane. The authors suggested that the primary function
of membrane insertion was to facilitate interactions with
a specific target, rather than being directed against the
membrane itself. They also predicted that the disordered
C-terminus might become structured upon recognition of
the target. This would appear to contradict results indi-
cating that the C-terminal amino acids are nonessential
for toxicity, but it should be noted that the expression
system used in reference 251 could not distinguish levels
of toxicity. Therefore, it is possible that the toxicity is
substantially decreased in the truncated mutants and
could even be an artifact of overexpression. This possi-
bility is under further investigation.
TA-1 type toxins in general are small proteins, less
than 60 amino acids, that have hydrophobic regions ty-
pical of transmembrane domains (254). These toxins
have been divided into eight superfamilies, and Fst is
the founding member of the Fst/Ldr family (255). The
Fst members of this family are widespread among the
Firmicutes and appear to be regulated and organized
similarly to the pAD1 par determinant (251, 255, 256).
The Ldr members are present in the γ-proteobacteria and
are regulated in a manner more similar to hok-sok fam-
ily members. In spite of their phylogenetic distance, Ldr
overexpression has effects on nucleoid condensation and
cell division similar to those Fst has in E. coli (247, 257).
Like most other TA systems, the par-like Fst-encoding
systems have both MGE and chromosomal members,
and like other TA systems, the function of the chromo-
somal members is obscure. One of these chromosomal
members, the toxin of which is annotated as EF0409, is
conserved in E. faecalis genomes (251). For purposes of
nomenclature, we have established a convention of using
the locus or MGE name as a subscript for the relevant
element. While the parEF0409 and parpAD1 systems show
clear genetic similarity, the predicted RNAI-RNAII in-
teraction sites are divergent, and it has been demon-
strated that the antitoxins do not cross-protect (258).
ParEF0409 is located between two paralogous mannitol-
type phosphotransferase sugar transport systems, sug-
gesting that it may be involved in regulating carbon flux.
Transcriptomic analysis of cells overexpressing either
FstpAD1 or FstEF0409 revealed that effects on the expres-

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sion of a variety of transporters were predominant (258,
259), including effects on several genes for PTS compo-
nents other than those to which parEF0409 is genetically
linked. It was also observed that interference with the
function of ABC transporters rescued cells from FstpAD1
overexpression (259). Utilizing a novel expression vector,
pCIE, that allows tight regulation and a large dynamic
range of induction, the effects of low-level expression of
FstpAD1 and FstEF0409 were compared. It was found that,
while there was some overlap, overexpression of the two
toxins had distinct transcriptomic effects (258). This sug-
gests that the toxins have different targets or have dis-
tinct effects on the same target. Finally, overexpression of
the Fst proteins is approximately 10-fold more toxic to
mannitol-grown than to glucose-grown cells (K. Weaver,
unpublished observation), strengthening the circumstan-
tial link between par function and carbon flux. Somewhat
surprisingly a mutant deleted for the parEF0409 antitoxin
was isolated in E. faecalis strain EryS (98). The mutant
strain showed increased virulence and improved survival
in macrophages under several stress conditions. Prote-
omic analysis performed on this strain showed signifi-
cant differences from the wild type, but these differences
did not correlate with the transcriptome differences ob-
served in strain OG1RF (258). These differences could
be strain-related, due to differences between transcription
and translation effects, or due to the accumulation of
compensatory mutations in the deletion strain. Further
investigation of the enterococcal par systems should elu-
cidate the function of the chromosomal parEF0409 locus,
determine the mechanism of action of the Fst toxins, and
provide insight into the evolution of the par-like systems.
CONJUGATIVE TRANSPOSONS (ICES)
The enterococci carry the full range of transposon vari-
eties found in other bacteria. Two Tn3-type transposons,
Tn917 encoding erythromycin resistance and Tn1546
encoding vancomycin resistance, have been extensively
studied (260, 261), and Tn917 and its derivatives have
been used for transposon mutagenesis in a number of
Gram-positive organisms. The genome sequence of E.
faecalis strain V583 revealed the presence of 38 insertion
sequence elements, with ISEf1, IS256, and IS1216 being
the most frequent (6). Both IS256 and IS1216 have been
associated with simple composite transposons (123, 262)
and large, complex, mosaic elements (263, 264). Many
of these elements contain antibiotic resistance genes, and
some are conjugative by means that have yet to be fully
explained (see below). For a more detailed description of
these elements see references 137 and 150.
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The E. faecalis transposon Tn916 was the first well-
characterized self-conjugative transposon (2, 3) although
large, conjugative chromosomal elements had been pre-
viously identified in other streptococci (265). Conjuga-
tive transposons were later found to be widespread in
the lactic acid bacteria, and mechanistically similar but
unrelated elements were identified in Bacteroides spe-
cies (266). Characterization of new elements from both
Gram-positive and Gram-negative organisms and the
reinterpretation of data on older elements formerly clas-
sified as integrative plasmids have revealed that MGEs
with characteristics similar to conjugative transposons
(i.e., that excise from their chromosomal locus, circular-
ize, transfer to a new host cell by conjugation, and in-
sert in the recipient cell’s chromosome without extensive
replication) are probably as ubiquitous in bacteria as
plasmids. Since the mobility of most of these elements
is dependent on a tyrosine or serine recombinase rather
than a DDE-type transposase and since many of them
have relatively site-specific integration preferences, the
appropriateness of the term “conjugative transposons”
has been questioned and alternative names have been
suggested, the most inclusive of which is ICE, for inte-
grative and conjugative element (267). The full spectrum
of ICEs has been described in detail in numerous reviews
(268–271). This section will focus primarily on Tn916 and
related elements and other ICE-related issues specific to
enterococci. A map of Tn916 is shown in Fig. 6. For more
detail on the Tn916-family of ICEs see references 272 and
273. For a comprehensive list of Tn916-like elements and
other ICEs in enterococci see reference 137.
Unlike transposons using a DDE-type transposase,
Tn916-like transposons are not flanked by direct repeats
of target sequence. Preferred insertion sites tend to be AT-
rich and possess intrinsic curvature, but the DNA se-
quence is not conserved and flanking sequences on each
end are heterologous (274, 275). Unlike many other ICEs,
Tn916 has relatively little insertion site preference. Two
transposon-encoded proteins, Int and Xis, are required
for excision (276, 277). Int is a tyrosine recombinase
(278) with N-terminal and C-terminal DNA binding do-
mains (279). The N-terminal domain (Int-N) binds via a
three-stranded β sheet to direct repeat sequences near
each end of Tn916 (280, 281). The C-terminal domain
(Int-C) binds to the transposon termini as well as to target
sequences. Int-C contains the conserved active site resi-
dues involved in nicking-closing. Like other tyrosine re-
combinases, Int produces staggered cuts at each end of the
integrated transposon. One cut is made flush with the end
of the transposon, producing a recessed 3′ end covalently
jointed to Int via a phosphotyrosine linkage. The second
17
Weaver

FIGURE 6 Genetic organization of Tn916 and the relative positions of Int and Xis binding
sites. (A) Functions of Tn916 genes are color coded: blue, recombination; green and red,
positive and negative regulation, respectively; yellow, tetracycline resistance; magenta, con-
jugation. Gene names and open reading frame numbers are shown above the individual
genes. The origin of transfer is designated by the line between orfs 20 and 21, labelled oriT.
Promoters and the direction of transcription are designated by labeled arrows below the gene
line. (B) Binding sites for the Int-C and Int-N DNA binding domains and Xis are designated by
marked circles and triangles. Int and Xis binding sites are color coordinated with the genes
shown in panel A.

cut is made 6 nucleotides into the host sequence, leaving
a free 5′ OH that attacks the phosphotyrosine linkage at
the other end of the transposon, resulting in circulariza-
tion (282). Since the joint is derived from chromosomal
DNA at each end that is not complementary, a 6-bp
heteroduplex is formed in the circular intermediate, called
the coupling sequence (283). The fact that Int must bind
and cleave at the coupling sequences, which vary from
insert to insert, may explain the wide range of conju-
gation frequencies (from <10–9 to >10–4 per donor cell)
observed using different donor strains (284). How the
coupling sequences affect conjugation frequency is not
known, but it does not appear to be due to effects on
integrase affinity (285) or on excision frequency (286).
Xis binds adjacent to the Int-N binding sites on both
ends of the transposon (287) and stimulates Int-mediated
excision of Tn916 in E. coli (276, 288), L. lactis (289),
B. subtilis (277), and in vitro (290). In E. coli, Xis bind-
ing to the left end is implicated in playing an architec-
tural role similar to λ-Xis in excision of bacteriophage
λ (291). HU protein plays a significant role in excision in
E. coli, but no host factors affecting excision have been
identified in Gram-positive hosts. Interestingly, Xis bind-
ing to the right end of Tn916 inhibits excision, perhaps
by competing with Int-N for binding, suggesting that Xis
has dual roles in excision, both stimulating and inhib-
iting the process (292).
Once the circular intermediate is formed, the trans-
poson is transferred to the recipient cell by conjugation.
DNA strand transfer is initiated by nicking at oriT di-
rected by the Orf20 relaxase and the Int protein (293,
294). While the Orf20 protein is responsible for strand
nicking, Int functions as a specificity factor, conferring
both strand and sequence specificity on its endonucleo-
lytic activity (294). Although Tn916 oriT bears simi-
larities to IncP-type origins of transfer (293), Orf20 does
not show similarity to any of the identified families of
plasmid-encoded relaxase proteins (217). Instead, it ap-
pears to have similarity to the Rep_trans family of RCR
initiator proteins and may represent a novel solution to
the problem of conjugative nicking (217, 294). On the
opposite side of the oriT from orf20 is orf21, which en-
codes a product in the FtsK/SpoIIIE/coupling protein
superfamily of DNA translocases. It is likely that Orf21
connects the oriT/Orf20 complex to the T4SS proteins
encoded in orfs14 to 16 (137). A combination of genetic
analyses and DNA sequence determination have identi-
fied a number of genes involved in Tn916 conjugation
(295, 296), but the precise roles of none of these genes
have been experimentally determined.

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Tn916 does not mobilize plasmids into which it has
integrated, nor does it mobilize chromosomal genes ad-
jacent to its integration site during intercellular trans-
position. This suggests that some mechanism suppresses
utilization of the oriT site until after the transposon has
excised from its host genome and circularized. Support-
ing this suggestion, induced expression of the transfer
genes requires a functional int gene, and transcription is
initiated from the orf7 promoter, which since it is lo-
cated at the opposite end of the transposon, is linked to
the transfer genes only in the circular intermediate (297).
Activation of the orf7 promoter requires orf7 and orf8
and is repressed by orf9, but the mechanisms of regu-
lation are unknown. Conjugation is also induced by the
presence of tetracycline, which induces transcription of
the tetM gene via ribosomal override of a transcriptional
attenuator at its 5′ end (92). Transcription then proceeds
through tetM, increasing transcription of the positive
regulators orf7 and orf8 and blocking the orf9 regu-
lator presumably by antisense RNA interactions and/or
transcriptional interference. This in turn increases tran-
scription through int and xis, stimulating excision and
circularization, leading to read through into the structural
conjugation genes (272). Recent evidence suggests that
subinhibitory concentrations of other ribosome-targeting
antibiotics also stimulate Tn916 conjugation and may
contribute to its spread (298).
Once a copy of the transposon is transferred, integra-
tion into the recipient chromosome requires Int but not
Xis (277). Since the known tyrosine recombinases func-
tion only on double-stranded DNA, replacement syn-
thesis of the transferred strand presumably takes place
prior to integration. Experiments with int- versions of
Tn916 where Int was provided in trans from a plasmid in
the donor revealed that int gene expression was not re-
quired in the recipient for integration, suggesting that the
Int protein might be transported with the transposon
DNA (299). Recipients frequently acquire multiple cop-
ies of the transposon, and there is no evidence that Tn916
expresses either an entry exclusion function or transpo-
son immunity (145, 300). Interestingly, in cells harboring
more than one copy of the transposon, excision of one
element transactivates excision and mobility of other
elements located elsewhere in the genome (301).
Sequence analysis of known and putative ICEs in se-
quenced genomes suggests that these mobile elements are
modular and that the modules may have different evo-
lutionary histories (267, 302, 303). For example, Tn5397
from Clostridium difficile is greater than 84% identical at
the DNA sequence level to Tn916 across all of the con-
jugation-related genes and the tetM gene, but Tn916 Int
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Enterococcal Genetics
is a tyrosine recombinase, while the recombinase protein
of Tn5397 is a serine recombinase. Conversely, the re-
combination module of the VanB transposon Tn1549 is
closely related to the Tn916 int/xis genes, but most of
the conjugation genes are unrelated (128). Putative ICEs
identified in the E. faecalis V583 genome sequence, EfaC2
and EfaD2, include conjugation-related genes apparently
acquired from pAD1 and pAM373 (267). A more global
analysis including plasmids, bacteriophages, and genomic
islands as well as ICEs indicates that the degree of ex-
change between these elements is very high, suggesting
that, rather than representing distinct groups, they rep-
resent a continuum of structures that maximize mobility
under various circumstances (304).
OTHER FEATURES OF THE ENTEROCOCCAL
MOBILOME
The above discussion focused on MGEs that are unique
to the enterococci and/or were originally described in
the enterococci, but the enterococcal “mobilome” is ex-
traordinarily diverse and includes plasmids, insertion
sequences, and transposons in a variety of families, group
II introns, integrons, pathogenicity islands (PAIs), and
bacteriophages. A recent review provides tabular lists
of many of these elements (137). For a comprehensive
overview of MGEs associated with the mobilome of clin-
ically important E. faecium and E. faecalis lineages, the
reader is referred to a recent report (305). Enterococcal
phages have also been recently reviewed (306). Addressed
below are some issues of particular interest related to the
enterococcal mobilome.
Important Non-Pheromone-Responding
Plasmids
pAMα1 is an ∼10-kb multicopy tetracycline resistance
plasmid that is a composite of two replicons (307, 308),
an RCR replicon similar to Bacillus cereus plasmid pBC16
that carries the tetracycline resistance gene but does not
function in E. faecalis and a putative theta-replicon simi-
lar to pS86 that supports replication in E. faecalis. Each
component plasmid encodes a relaxase gene, and the
junction sites contain oriT sequences recognized by them.
The oriT/relaxase complex allows mobilization of the
plasmid in the presence of another conjugative plasmid.
The presence of the oriT sites at the plasmid junctions
suggests that the fusion giving rise to pAMα1 occurred via
a site-specific recombination event catalyzed by the re-
laxases as was previously observed in other plasmids
(309, 310). Under tetracycline selection, cells accumulate
pAMα1 derivatives containing tandem repeats of the
19
Weaver
pBC16 replicon, amplifying the tetracycline resistance
gene. Amplification is initiated by a RecA-independent
recombination event requiring at least one of the relaxase
genes and both oriT sequences (311) followed by more
extensive RecA-dependent amplification between redun-
dant sequences in the duplicated region (312). Thus, the
relaxases of pBC16 and pS86 perform three functions:
(i) stimulate cointegration to form pAMα1, (ii) facilitate
mobilization of the cointegrate, and (iii) trigger amplifi-
cation of the tetracycline resistance gene in the presence of
the antibiotic.
pMG1 is an ∼65-kb conjugative plasmid originally
isolated from E. faecium and encoding high-level gen-
tamicin resistance (313). pMG1-like plasmids bearing
Tn1546 encoding the vanA determinant have been as-
sociated with the spread of vancomycin resistance in
enterococci (124, 314). Like the pheromone-responsive
plasmids, pMG1 is capable of high-frequency transfer
in broth. Unlike pheromone plasmids, pMG1 transfer is
not induced by exposure to culture filtrates from po-
tential recipient cells, conjugating broth cultures do not
form macroscopic cellular aggregates, and transfer occurs
freely between E. faecium and E. faecalis cells. Further-
more, no homology between pMG1 and the conjuga-
tive systems of pheromone-responsive plasmids or Inc18
plasmids was observed (313). Rather, sequencing results
indicated significant homology of suspected conjugation
genes to those from pXO2, the capsule-encoding plasmid
of Bacillus anthracis (315, 316). Microscopic aggregates
were observed during mating, and this phenomenon is
related to a region of the plasmid encoding five open
reading frames. One open reading frame encodes a large
protein with similarity to a pXO2-encoded surface protein
that could represent an “aggregation substance” unrelated
to the pheromone plasmid AS (316). Genes potentially
encoding the coupling protein, a VirB11-family protein, a
relaxase, and the oriT were also identified. A gene desig-
nated traA appears to encode a key positive regulatory
protein (317).
TA Systems
In addition to parpAD1 and parEF0409, several other TA
systems have been identified on enterococcal plasmids
and chromosomes. Par homologs are present on sev-
eral other enterococcal plasmids, although no chromo-
somal loci analogous to parEF0409 have been described
outside of E. faecalis (215, 256). The E. faecium multi-
resistance plasmid pRUM (318) and the related van-
comycin resistance plasmid pS177 (319) encode a TA-II
locus designated Axe-Txe. Axe-Txe stabilizes heterolo-
gous plasmids in Bacillus thuringiensis, E. coli, and its
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native host. The Txe toxin belongs to the RelE super-
family of RNA interferases and was shown to cleave
mRNA one base downstream of the AUG initiation co-
don (319, 320). Interestingly, Axe is more closely related
to Doc, the antitoxin for the Phd toxin, than it is to RelB,
the antitoxin most commonly associated with RelE tox-
ins, supporting the hypothesis that TA-II systems have
undergone extensive recombinatorial mixing and match-
ing during their evolution (321). The ωεζ TA-II system is
commonly present on the broad-host-range Inc18 plas-
mids (139), many representatives of which are present in
enterococci (322), including the VanA-encoding E. fae-
cium plasmid pVEF3 (323), pAMβ1 (308), and the pher-
omone plasmid pSL1 (324). Unlike most TA-II systems,
the ε antitoxin does not autoregulate the locus. Instead,
it is regulated by an independent regulatory component,
ω, which also regulates the plasmid active partition lo-
cus and the copy control factor CopS (325). ζ-family
toxins have been proposed to cause cell growth arrest
by depleting ATP and GTP pools in the cell and may
be associated with persistence to antibiotics (326). The
ubiquitous higBA and mazEF chromosomally encoded
TA-II systems are prevalent in E. faecium and E. faecalis
clinical isolates, and mazEF has been suggested as a
target for novel antibacterials (327).
In E. faecalis V583 a mazEF TA-II homolog is closely
linked to a TA-I locus distantly related to B. subtilis txpA-
ratA (328). Upstream of the mazEF locus is a direct repeat
motif that binds MazE and MazEF, suggesting that the
locus is negatively autoregulated as is observed in other
TA-II modules (329). It was also observed that ectopic
expression of MazE or MazEF stimulated transcription
of the ratA antitoxin RNA for the txp-ratA locus, and a
sequence with similarity to the direct repeat motif was
found upstream of the ratA gene. Thus, the MazE toxin
suppresses the toxicity of both mazEF, by reducing over-
all transcription, and the txpA-ratA locus, by increasing
synthesis of the ratA antitoxin, which then targets the
txpA mRNA for degradation. Conversely, one would
expect triggered degradation of MazE, by an as yet un-
known mechanism, to activate toxicity of both systems.
Thus far, this is the only example of coordinated regula-
tion of TA-I and TA-II loci, and the complete system has
been dubbed a TAI-II locus.
PAIs and Intercellular Transfer
by Hfr Formation
PAIs are chromosomal regions containing large clusters of
genes associated with bacterial virulence (330). Frequently,
PAIs include genes associated with known MGEs, such as
phage- and ICE-related integration/excision modules and
ASMscience.org/MicrobiolSpectrum

plasmid-related conjugation genes, and some have been
shown to be either independently mobile (331) or mo-
bilizable by other MGEs (332). The first enterococcal PAI
was identified in E. faecalis strain MMH594, and related
islands were described in strains V586 and V583 (333).
This PAI is ∼150 kb long, encodes several pathogenicity
factors, including the enterococcal surface protein (esp),
cytolysin, AS, a bile acid hydrolase, various surface pro-
teins, and general stress proteins, and contains numer-
ous transposons. The PAI also contains phage-related
integration/excision proteins, homologs of plasmid con-
jugation functions, and is flanked by 10-bp terminal di-
rect repeats, suggesting that it could be independently
mobile. Genomic analysis of clinical E. faecalis isolates
revealed that PAI genetic composition is highly variable,
suggesting the involvement of modular accretion during
its evolution and dissemination (334, 335). Intercellular
transfer of either portions of the PAI (336, 337) or the
complete PAI (130, 338) has been demonstrated be-
tween E. faecalis strains and between E. faecalis and E.
faecium. Complete transfer from V583 was shown in
vitro to be independent of PAI-encoded mobility genes
and to frequently include chromosomal genes outside of
the element. This in vitro transfer was dependent on the
conjugation functions of pTEF1 and pTEF2, two pher-
omone-responsive conjugative elements present in the
same strain, suggesting that the plasmids mobilized the
PAI via chromosomal integration, perhaps at IS256 ele-
ments, followed by mobilization either by an Hfr- or
F′-like mechanism (130). In contrast, precise excision,
circularization, and recipient insertion of the PAI was
observed in E. faecalis strain UW3114, suggesting that
the PAI is capable of independent mobility (338), per-
haps at a rate substantially lower than that mediated
by mobilization. Interestingly, the PAI circular interme-
diate was not detected in V583, suggesting that an im-
portant component for self-mobilization may be absent
in that strain or not expressed under the conditions tested.
However, even in UW3114, a coresident pheromone-
responsive plasmid, pLG2, was transferred in parallel
with the PAI and may have facilitated its transfer, and the
authors did not determine whether or which PAI genes
might be required for mobility. So whether the E. faecalis
PAI is fully self-mobile is still an open question, although
genes associated with excision, integration, and transfer
appear to be intact.
Similar esp-associated PAIs have also been identified
in E. faecium that may be subject to conjugative transfer
(337, 339, 340). Plasmid-mediated transfer of chromo-
somal determinants, including determinants involved
in β-lactam and vancomycin resistance, has been dem-
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Enterococcal Genetics
onstrated in E. faecium strain C68. Transfer in this case
was mediated by pLRM23 and a second plasmid and
appeared to proceed via cointegration of the plasmid
with the donor chromosome, excision of the plasmid
with neighboring chromosomal DNA, circularization in
the donor prior to transfer, integration of the chromo-
somal segment into the recipient chromosome by homo-
logous recombination, and subsequent loss of the unstable
plasmid remnant (129). Whether this mechanism can also
facilitate PAI transfer has not been determined.
Enterococcal Bacteriophage and Genome
Defense Systems
Bacteriophages provide a sizable fraction of the mobilome
of both E. faecalis and E. faecium, amounting to close to
10% of the genome of E. faecalis strain V583 (6) and 3 to
5% of seven sequenced E. faecium strains (340). V583
contains seven putative prophages, five of which have
sufficient gene content to generate functional virions. The
phage02 region was subsequently determined to be part of
the core genome of E. faecalis and is likely no longer
capable of mobilization (335). The phage07 region is ca-
pable of producing infectious virions but requires phage01
as a helper phage to provide packaging and structural
elements (341, 242). Sequencing of multiple E. faecalis
genomes has confirmed the variable presence of these
prophage elements except for phage02 (335, 343, 344),
and the OG1RF genome contains only phage02 (345).
Comparative genomic analysis of E. faecalis phages
suggests that they have a modular structure much like
bacteriophages infecting other bacteria (306). The mod-
ules consist of three transcriptional units, one encoding
the lysogeny control functions, one encoding genes re-
quired for the lytic pathway, and a variable “cargo” re-
gion encoding potential lysogenic conversion genes, some
of which could encode important virulence factors. Such
cargo genes include a set of putative membrane pro-
teins in the φEF11 phage (346) and a ferrochelatase gene
and cold shock protein gene in phage04 (6). However,
no systematic analyses have been performed to unequiv-
ocally link phage determinants to enterococcal patho-
genicity (306). Laboratory studies have demonstrated a
capacity for transduction by enterococcal phages, in-
cluding interspecies transfer of antibiotic resistance, so the
potential for phage effects on virulence does exist (347,
348).
CRISPR-Cas function as adaptive immune systems for
bacteria, generally targeting the DNA of invading MGEs
for degradation (see 349 for a recent review). Three dis-
tinct type II-A CRISPR loci have been identified in
E. faecalis: CRISPR1 and 3 are complete functional loci
21
Weaver
with associated Cas genes, while the CRISPR2 locus is
an “orphan” locus harboring repeats and spacers of the
same family as CRISPR1 but lacking Cas genes (8, 345).
CRISPR1-Cas and CRISPR3-Cas are variably present in
E. faecalis genomes and never cooccur in the same strain,
while CRISPR2 is ubiquitous and can thus be considered
part of the core genome of this species. CRISPR1-Cas-
related systems have also been detected in several envi-
ronmental and nonclinical enterococcal species (350).
Spacer sequences from all three CRISPR loci show iden-
tities to phage and plasmid sequences, suggesting that all
have acquired MGE segments for genome defense (8).
However, CRISPR2 only functions when the Cas genes
of CRISPR1 are provided in trans (351), suggesting that
strains containing only CRISPR2 should be more sen-
sitive to MGE invasion. Indeed, the absence of com-
plete CRISPR-Cas loci is associated with larger genomes,
higher percentages of horizontally acquired genes, greater
antibiotic resistance, the presence of certain virulence fac-
tors, and high-risk multilocus sequence type (7, 8). There-
fore, E. faecalis CRISPR-Cas loci, in cooperation with
restriction-modification systems (351), are effective in lim-
iting invasion by MGEs, but loss of CRISPR-Cas facilitates
the evolution of pathogenicity by allowing greater hori-
zontal gene transfer. Recently, a method for activating
CRISPR2 and using it to modify the E. faecalis population
structure was proposed (352).
A type II-A CRISPR-Cas locus has also been identified
in E. faecium, related to the E. faecalis CRISPR1-Cas
locus (8). Like in E. faecalis, the locus is not present in
all E. faecium strains and is absent in most multiple
antibiotic-resistant strains and in the major hospital-
adapted clade of this species (353).
CONCLUDING REMARKS
From the beginning, enterococcal genetics has been fo-
cused on MGEs. Pioneering studies of the enterococci
gave us one of the first examples of intercellular com-
munication in the bacterial world in the pheromone-
inducible conjugative plasmids and the first chromo-
somally located conjugative elements in the conjugative
transposons, later to be named ICEs after their distribu-
tion was found to be ubiquitous in bacterial cells. Stud-
ies of the complex regulatory circuits controlling the
pheromone response and the conjugative mechanisms of
ICEs continue to inform those interested in the molec-
ular mechanisms of gene regulation in prokaryotes in
general. The importance of MGEs in enterococci became
even more apparent with the emergence of vancomycin
resistance and the multitude of ways the van gene clus-
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ters could be shared among enterococci and with other
genera. Further studies identified the enterococci as a
promiscuous hub for the dissemination of a wide variety
of antibiotic resistance genes. The accumulation of ge-
nomic information on the enterococci has further revealed
just how important MGEs are to enterococcal patho-
genesis and evolution, particularly in the way that the
CRISPR MGE defense system impacts the structure of
E. faecalis and E. faecium populations.
Complicated regulatory schemes and important anti-
biotic resistance phenotypes are not limited to enterococ-
cal MGEs, and progress is now being made on important
systems in the core genomes, particularly those of of
E. faecalis and E. faecium. Work is proceeding on the
intrinsic mechanisms of antibiotic resistance in these or-
ganisms, and the complex transcriptional and posttran-
scriptional mechanisms of regulation of the EA regulon
presage more interesting regulatory phenomena to be
discovered. While roles for enterococcal TCSTSs are be-
ing defined, genomic analyses have revealed a plethora
of potential sRNA regulators about which we know very
little. To this point, most detailed genetic analyses have
been performed in vitro or have focused on the effects
of the various systems during pathogenic processes, but
the enterococci are common intestinal bacteria, and it is
likely that events occurring within the gut in the presence
of the microbiota are more relevant to normal entero-
coccal physiology. Here again, studies with MGEs are
likely to lead the way. Clearly, much remains to be dis-
covered about the genetics of these interesting and clin-
ically important Gram-positive bacteria.
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