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REVIEW ARTICLE
Regulation of natural genetic transformation and acquisition of
transforming DNA in Streptococcus pneumoniae
Ola Johnsborg & Leiv Sigve Håvarstein
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway
Correspondence: Leiv Sigve Håvarstein,
Department of Chemistry, Biotechnology, and
Food Science, Norwegian University of Life
Sciences, PO Box 5003, N-1432 Ås, Norway.
Tel.: 147 6496 5883; fax: 147 6496 5901;
e-mail: [email protected]
Received 13 November 2008; revised 1
February 2009; accepted 1 February 2009.
First published online 10 March 2009.
DOI:10.1111/j.1574-6976.2009.00167.x
Editor: Eduardo Rocha
Keywords
natural genetic transformation; Streptococcus
pneumoniae ; fratricide; acquisition of
transforming DNA.
Abstract
The ability of pneumococci to take up naked DNA from the environment and
permanently incorporate the DNA into their genome by recombination has been
exploited as a valuable research tool for 80 years. From being viewed as a marginal
phenomenon, it has become increasingly clear that horizontal gene transfer by
natural transformation is a powerful mechanism for generating genetic diversity,
and that it has the potential to cause severe problems for future treatment of
pneumococcal disease. This process constitutes a highly efficient mechanism for
spreading b-lactam resistance determinants between streptococcal strains and
species, and also threatens to undermine the effect of pneumococcal vaccines.
Fortunately, great progress has been made during recent decades to elucidate the
mechanism behind natural transformation at a molecular level. Increased insight
into these matters will be important for future development of therapeutic
strategies and countermeasures aimed at reducing the spread of hazardous traits.
In this review, we focus on recent developments in our understanding of
competence regulation, DNA acquisition and the role of natural transformation
in the dissemination of virulence and b-lactam resistance determinants.
Introduction
A growing body of evidence shows that horizontal gene
transfer is a dominant force in the evolution of bacteria. Of
the three known mechanisms mediating horizontal gene
transfer (natural genetic transformation, transduction and
conjugation), natural transformation appears to be the least
widespread. Still, 4 60 bacterial species have been reported
to be naturally transformable, a number that is probably
considerably underestimated (Johnsborg et al., 2007b). In
the broadest sense, sex can be defined as any natural process
that combines genes from more than one source in a single
cell (Margulis & Sagan, 1986). According to this definition,
all the gene transfer mechanisms mentioned above represent
different forms of bacterial sex. However, while transduction
and conjugation rely on extrachromosomal genetic elements
promoting their own maintenance and distribution, natural
transformation is an integral part of the physiology of
bacteria possessing this property. Natural transformation
can therefore be considered as the only genuine form of
bacterial sex.
Natural genetic transformation was originally discovered
in Streptococcus pneumoniae (Griffith, 1928), and ever since
FEMS Microbiol Rev 33 (2009) 627–642
the pneumococcus has served as a paradigm for this important phenomenon. After Dawson & Sia (1931) achieved
transformation in vitro in 1931, the ‘nuts and bolts’ of the
transformation apparatus and its regulation has gradually
been unraveled. However, much remains to be learned,
especially with respect to environmental cues that promote
competence development in situ, and the complex nature of
the DNA uptake machinery. Recently, it was discovered that
competent pneumococci kill and lyse noncompetent sister
cells and members of closely related streptococcal species
during cocultivation (for a review, see Claverys &
Håvarstein, 2007). Accumulating evidence indicates that this
competence regulated mechanism, which has been termed
fratricide, has evolved to facilitate acquisition of homologous DNA during transformation (Johnsborg et al., 2008).
Thus, under in vivo conditions, liberation and uptake of
transforming DNA appears to be a highly coordinated
process, and not independent events as believed previously.
Here, we review the current state of knowledge regarding
natural transformation in S. pneumoniae, with particular
emphasis on competence regulation. In the second part, we
focus on the source of the donor DNA, and how it is
acquired. Thirdly, the impact of natural transformation on
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628
the dissemination of virulence and b-lactam resistance
determinants is discussed.
Regulation of natural genetic
transformation
Identification of the elusive competencestimulating peptide (CSP) and its transporter
Expression of competence for natural genetic transformation in S. pneumoniae is a transient phenotype. Typically,
laboratory cultures growing in a competence-promoting
medium will spontaneously induce this phenotype at an
OD550 nm of 0.15–0.2. However, after c. 30 min of competence, the ability to take up DNA is rapidly lost (Tomasz,
1966). It was reported 35 years ago that cell-free supernatants from various competent streptococcal cultures contained a proteinaceous compound that could induce
competence in noncompetent cells (Pakula & Walczak,
1963; Tomasz & Hotchkiss, 1964). Subsequent work concluded that the competence-inducing compound was acting
as an extracellular hormone-like substance, used by the
bacteria to coordinate competence development between
cells in a species-specific manner (Tomasz, 1965; Tomasz &
Mosser, 1966). The exact nature of the secreted competence
factor and the mechanism by which it would induce
competence remained elusive until the mid-1990s, when
the inducing molecule was purified and identified as an
unmodified peptide termed competence-stimulating peptide (CSP) (Håvarstein et al., 1995a).
Before the identification of CSP, a pneumococcal chromosomal region, termed the com locus, was found to be
essential for production of the competence inducer, as
supernatants from a deletion mutant lacking this region
did not contain this molecule. The com locus was shown to
contain two genes predicted to encode an ATP-binding
cassette (ABC) transporter (comA) and its accessory gene
(comB) (Chandler & Morrison, 1988; Hui & Morrison, 1991;
Hui et al., 1995). Interestingly, although comA mutants did
not secrete biologically active CSP, comA mutants could still
be induced to competence by addition of cell-free supernatants from competent pneumococcal wild-type cultures.
This indicated that ComA could be directly responsible for
the secretion of the competence-inducing molecule. ABC
transporters are omnipresent in bacteria, where they function in the import and export of a wide range of different
substrates (Davidson et al., 2008). In most cases, it is very
difficult, if not impossible, to predict the substrate specificity of an ABC transporter from its primary sequence.
Subsequent to the identification of ComA, a new subfamily
of ABC transporters was characterized (Håvarstein et al.,
1995b). This family is dedicated to the export of small
peptides, often peptide bacteriocins, which are synthesized
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O. Johnsborg & L.S. Håvarstein
with an N-terminal leader sequence containing a characteristic Gly–Gly motif (Håvarstein et al., 1994). The leader
sequence is removed concomitant with export by a proteolytic domain located in the N-terminal end of the ABC
transporters. This N-terminal protease domain could be
identified in ComA, indicating that the competence inducer
was a bacteriocin-like peptide with a double-glycine type
leader (Håvarstein et al., 1995b). Using a strategy developed
for the purification of small peptides, CSP was successfully
isolated from strain CP1200 and identified as a 17-residue
peptide (N-EMRLSKFFRDFILQRKK-C). Sequencing of the
comC gene confirmed the prediction that CSP is synthesized
as a precursor peptide containing a double-glycine type
leader at its N-terminal end (Håvarstein et al., 1995a).
Double-glycine leaders contain a conserved sequence motif
that in addition to the conserved glycine residues at position
1 and 2 relative to the processing site also include
hydrophobic amino acids at positions 4, 7, 12 and
15 (Håvarstein et al., 1994). Recently, the N-terminal
peptidase domain of ComA was expressed in Escherichia coli
and purified. Subsequent biochemical studies strongly suggested that the peptidase domain functions as a cysteine
protease. The strict substrate specificity of the enzyme
appears to be dependent on specific interactions with the
conserved hydrophobic sequence motif of the ComC leader
peptide, demonstrating that ComA function is dedicated to
the secretion and maturation of CSP (Ishii et al., 2006;
Kotake et al., 2008).
Extracellular CSP is sensed by a two-component
regulatory system
The core protein machinery regulating competence induction in S. pneumoniae is encoded by the comCDE operon,
where comC encodes the secreted peptide pheromone CSP,
comD the CSP receptor and comE the cognate response
regulator (Håvarstein et al., 1996; Pestova et al., 1996).
Together, ComD and ComE form a classical two-component
signal transduction system (for a review on two-component
systems, see Stock et al., 2000; Laub & Goulian, 2007) that
monitors and responds to the extracellular concentration of
CSP (Fig. 1). Similar to most histidine kinases, ComD is
comprised of an N-terminal sensor domain coupled to a
C-terminal kinase domain (Håvarstein et al., 1996;
Håvarstein, 2003; Iannelli et al., 2005). Based on amino acid
sequence similarity in the kinase domain, histidine kinases
have been divided into 11 subfamilies. ComD fall into one
distinct group, the HK10 subfamily, which also include the
staphylococcal AgrC and PlnB from Lactobacillus plantarum
(Grebe & Stock, 1999). All histidine kinases belonging to
this subfamily are specifically activated by a cognate secreted
peptide. In contrast to most membrane-localized histidine
kinases, where the N-terminal sensor domain consists of two
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Acquisition of transforming DNA in S. pneumoniae
Fig. 1. Schematic representation of competence regulation in Streptococcus pneumoniae. The CSP precursor, which is encoded by the comC gene, is
processed and secreted by the dedicated ComAB transporter, resulting in extracellular accumulation of mature CSP. Basal transcription of the comCDE
operon is subjected to regulation by global regulators such as the serine/threonine protein kinase StkP and the CiaRH two-component system (see text
for details). Binding of CSP to its ComD receptor is believed to result in autophosphorylation of ComD and subsequent transfer of the phosphoryl group
to the ComE response regulator. ComE then binds to and activates transcription from the various early gene promoters. ComE binding sets off increased
transcription of the comCDE operon, leading to a boost in the production of CSP, ComD and phosphorylated ComE. This auto-induction loop ensures
rapid accumulation of the alternative s factor ComX, ComW and the ComM fratricide immunity protein. ComW protects ComX from proteolytic
cleavage and stimulates the latter protein to activate transcription of the late genes encoding the fratricide trigger factors CbpD and CibAB as well as the
protein apparatus for DNA uptake and recombination. While cibAB is cotranscribed with a cognate immunity gene (cibC), competent cells are protected
from the CbpD murein hydrolase by the product of the early gene comM.
transmembrane segments flanking an extracytoplasmic
loop, the HK10 subfamily members contain a large polytopic sensor domain. Structural analyses of AgrC, PlnB and
ComD have indicated that this domain is anchored to the
cytoplasmic membrane by five to seven transmembrane
helices (Håvarstein et al., 1996; Lina et al., 1998; Johnsborg
et al., 2003; O. Johnsborg, unpublished data). In the case of
ComD, little is known about the mechanism underlying CSP
activation and signal transduction.
Following the initial identification of the comC gene in
strain CP1200 (Håvarstein et al., 1995a), this gene has been
sequenced in a large number of strains from species such as
S. pneumoniae, Streptococcus mitis, Streptococcus oralis and
other members of the mitis phylogenetic group (Pozzi et al.,
1996; Håvarstein et al., 1997; Ramirez et al., 1997; Whatmore et al., 1999; Johnsborg et al., 2007a). This work has
revealed that there exists extensive diversity with respect to
the primary amino acid sequences of CSPs produced by
various strains and species. In line with this observation, the
ComD receptors also display high sequence diversity in their
ligand-binding domains (Håvarstein et al., 1996, 1997).
Most of the CSPs contain a conserved sequence fingerprint
composed of a negatively charged N-terminal residue, an
arginine in position 3 and a positively charged C-terminal
tail (Håvarstein et al., 1997; Johnsborg et al., 2007a). In
contrast, the central region of the peptides is highly variable.
FEMS Microbiol Rev 33 (2009) 627–642
Circular dichroism (CD) analysis of the CSP produced by
S. pneumoniae R6 (hereafter referred to as CSP-1) revealed
that the peptide becomes structured upon exposure to
membrane-mimicking environments such as trifluoroethanol and dodecyl phosphocholine (DPC) micelles. This could
indicate that initial structuring of CSP-1 is initiated upon
interaction with the membrane of target bacteria. Nuclear
magnetic resonance spectroscopy analysis of CSP-1 in the
presence of DPC demonstrated the formation of an a-helix
between residues 6 and 12 (Johnsborg et al., 2006). The
a-helical region of CSP-1 is amphiphilic, with the nonpolar
residues Phe-7, Phe-8, Phe-11 and Ile-12 facing one side of
the helix and Lys-6, Arg-9 and Asp-10 facing the opposite
side. This hydrophobic patch has been shown to be involved
in the binding of CSP-1 to its cognate receptor ComD-1
(Johnsborg et al., 2006). ComD-1 differs from the CSP-2
receptor (ComD-2) of S. pneumoniae A66 in only 12 amino
acid positions (Håvarstein, 2003; Johnsborg et al., 2004).
Because CSP-1 cannot cross-activate ComD-2 and vice
versa, some or all of these positions must be involved in
determining receptor specificity. Interestingly, six of the
substitutions involve replacement of hydrophobic amino
acids with other hydrophobic amino acids, an observation
that supports the hypothesis that hydrophobic interactions
might be important for correct alignment of the
CSP–ComD interface. However, ComD does not appear to
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control ligand specificity through sequence-specific hydrophobic contacts with the cognate CSP, and the exact
mechanism by which the hydrophobic patch of the CSPhelix facilitates receptor recognition remains elusive (Johnsborg et al., 2006).
CSP binding presumably brings about a conformational
change in the ComD transmembrane domain, resulting in
activation of the intracellular kinase domain. Once activated,
the kinase domain presumably phosphorylates the transcriptional regulator ComE. All response regulators activated by
kinases from the HK10 subfamily have DNA-binding effector domains of the LytTR type (Nikolskaya & Galperin,
2002). The LytTR domain is a DNA-binding motif that is
typically found in response regulators regulating virulence
factor and toxin production in pathogenic bacteria (Galperin, 2006). Using the staphylococcal response regulator AgrA
as a model, this domain was recently shown to be composed
of a 10-stranded b fold that upon DNA binding induces a
bend in the target DNA (Sidote et al., 2008). In the case of
ComE, phosphorylation of the regulatory domain is believed
to enable the effector domain to bind a 9-bp imperfect direct
repeat motif found in the promoter regions of a set of genes
termed the early competence genes (Ween et al., 1999).
Binding of ComE activates transcription from the early gene
promoters, which include the promoters of the comAB and
the comCDE operons. As a consequence, the level of extracellular CSP, and hence the level of phosphorylated ComE,
increases rapidly, driving the cell toward the competent state.
Microarray analyses have shown that ComE activates the
expression of about 20 genes, known as the early competence
genes, which include two copies of the alternative s factor
ComX (Lee & Morrison, 1999; Luo & Morrison, 2003;
Dagkessamanskaia et al., 2004; Peterson et al., 2004).
The alternative r factor ComX
ComX directs the transcription of around 60 late competence genes, some of which encode the DNA uptake and
recombination machinery (Fig. 1). In addition to the early
and late genes, two other groups of CSP-responsive genes
have been defined. They include the delayed competence
genes, which mainly consist of stress-response genes, and the
repressed genes (Dagkessamanskaia et al., 2004; Peterson
et al., 2004). Some of the genes from the latter two classes are
dependent on ComX, while expression of others appears to
be more indirectly influenced by competence induction. The
global influence of ComX activity on cell physiology is
reflected in the tight control of the synthesis and stability of
this protein. In addition to the transcriptional regulation of
comX by ComE, ComX protein stability is also subjected to
regulation by the ClpP protease. ClpP-deficient mutants
display a prolonged competence induction as compared
with wild-type cells, suggesting the involvement of ClpP as
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O. Johnsborg & L.S. Håvarstein
a negative regulator of competence induction (Chastanet
et al., 2001; Robertson et al., 2002). clpP mutants also display
increased levels of ComX, suggesting that the level of ComX
is negatively regulated by ClpP-mediated proteolytic degradation (Sung & Morrison, 2005). The protein encoded by
the early competence gene comW, which is essential for
efficient induction of competence, has the ability to transiently prevent degradation of ComX (Luo et al., 2004; Sung &
Morrison, 2005). ComW also appears to be involved in the
regulation of ComX activity, possibly by processing ComX
to an active form, or by influencing ComX-RNA polymerase
interactions (Sung & Morrison, 2005). Because ComW
expression is directly controlled by ComE, the regulatory
function of ComW must have evolved to prevent ComX
accumulation in the absence of high levels of comCDE
expression, thereby ensuring that the competent state does
not develop at the wrong time and place.
Complex regulation of the ComCDE
autocatalytic circuit
The discovery of CSP and the ComDEX signal transduction
pathway readily explained how competence is induced in a
pneumococcal culture once the extracellular level of CSP has
reach a critical concentration (Fig. 1). However, the molecular mechanisms regulating CSP production are still only
partially understood. It has long been known that environmental factors such as high phosphate concentration, bovine
serum albumin, CaCl2, as well as slightly alkaline pH,
stimulate spontaneous competence development in laboratory cultures (reviewed in Claverys & Håvarstein, 2002).
Recently, it has also been established that competence can be
induced in response to the DNA-damaging agent mitomycin
C and antibiotics such as norfloxacin, levofloxacin, moxifloxacin, kanamycin and streptomycin (Prudhomme et al.,
2006). At present, it is still unclear how any of these
environmental cues are recognized and processed by the cell,
but several regulatory proteins, apart from ComCDEX, have
recently been demonstrated to affect competence development. The autoregulatory CiaRH two-component system
was first identified in a screen for b-lactam resistance
determinants in laboratory mutants (Guenzi et al., 1994).
As for most of the histidine kinases encoded by the pneumococcal genome, the signal that regulates CiaH activity in
pneumococci remains unknown. There are, however, reasons to believe that this system is part of a pneumococcal
response to cell wall stress (Dagkessamanskaia et al., 2004;
Mascher et al., 2006). The fact that CiaRH is involved in
protecting the cells against b-lactam antibiotics could suggest that the CiaRH system is activated during cell wall
damage. This hypothesis is supported by the observation
that pneumococcal cells harboring a pbp2x mutation conferring penicillin resistance display increased susceptibility
FEMS Microbiol Rev 33 (2009) 627–642
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Acquisition of transforming DNA in S. pneumoniae
to autolysis in a ciaR background (Mascher et al., 2006).
Microarray analyses have demonstrated that the transcription of ciaRH is increased upon competence induction, and
the operon has been assigned to the group of delayed
competence genes (Dagkessamanskaia et al., 2004; Peterson
et al., 2004). Activation of this system appears to allow the
cells to exit normally from the competent state, as competence induction in ciaR mutants triggers growth arrest and
stationary phase autolysis (Dagkessamanskaia et al., 2004). A
T230P mutation in the CiaH histidine kinase has been
shown to result in the complete inhibition of competence
(Guenzi et al., 1994). This mutation is thought to activate
the CiaRH system because enhanced transcription of CiaR
could be demonstrated in this background (Giammarinaro
et al., 1999; Mascher et al., 2003). The fact that the comCDE
operon was shown to be strongly repressed in this mutant
suggested that the CiaRH system controls the early steps in
competence regulation. Recently, it was established that the
CiaR response regulator directly controls expression from 15
promoters, of which 14 are positively regulated (Halfmann
et al., 2007). CiaR does not bind directly to any of the
promoters that are known to direct the synthesis of early or
late competence genes. Hence, CiaR must regulate competence development by increasing or decreasing the expression of one or several factors that are able to interact with the
competence regulon. One CiaR-regulated gene that has been
implicated in the downregulation of competence is htrA. It
has been hypothesized that the extracellular HtrA protease
degrades a protein product that is essential for the early
regulation of competence (Sebert et al., 2005). Given the fact
that HtrA does not degrade CSP and ComD, and that several
authors have reported no stimulation of competence induction upon deletion of htrA (Dagkessamanskaia et al., 2004;
Ibrahim et al., 2004), further experiments are required to
clarify the putative role of HtrA in competence regulation.
The pneumococcal protein StkP is a eukaryotic-type
serine/threonine protein kinase that contributes to the
resistance of S. pneumoniae to environmental stresses (Echenique et al., 2004; Nováková et al., 2005). Deletion of this
global regulator results in increased basal expression of the
core competence regulators, including ComCDE, ComAB,
ComX and ComW. The mechanism underlying increased
expression of the competence regulators is not known
(Sasková et al., 2007). Curiously, although stkP mutants
display a low level of constitutive competence, they are not
able to respond to exogenously added CSP. Hence, mutation
of stkP blocks high frequency transformation. Interestingly,
Guiral et al. (2006) have reported that a point mutation in
the upstream region of comCDE results in increased basal
expression of comE. Similar to the observations with the stkP
mutants, this mutant also displayed reduced CSP-induced
transformability. From the above, it is clear that pneumococci have evolved complex molecular circuits involving
FEMS Microbiol Rev 33 (2009) 627–642
several regulatory networks to finely tune both induction
and downregulation of the competence phenotype.
Competence-induced cell lysis
Interestingly, the level of transcription of 4 180 genes is
changed following the induction of the competent state in
S. pneumoniae. However, only 23 of these genes have been
found to be directly required for natural transformation, i.e.
uptake of extracellular DNA that is incorporated into the
recipients genome by homologous recombination (Burghout et al., 2007; Guiral et al., 2007). For the most part, the
role of these additional CSP-regulated genes is unknown. As
described above, pneumococci have evolved a complex
system to control the timing of competence development.
However, none of the involved regulatory factors are known
to respond to the presence of free DNA in the environment
surrounding the cell. This is a paradox, because cells would
risk developing competence under circumstances where
transforming DNA is not available. One way to get around
this problem would be to postulate that naked DNA is
released from bacteria that die and fall apart, and that this
kind of DNA is practically always present in the natural
habitat of naturally transformable streptococci. Interestingly, recent studies of competent pneumococci have revealed that these cells express a small set of proteins that
apparently constitute a molecular mechanism directed at
active acquisition of DNA from sister cells and closely
related streptococcal species. Steinmoen et al. (2002) observed that induction of competence in a liquid monoculture of the pneumococcal strain CP 1415 resulted in leakage
of an intracellular b-galactosidase reporter protein. Using a
transformation assay, the authors were also able to demonstrate that the development of competence in this strain was
associated with increased leakage of chromosomal DNA into
the culture supernatants. The fact that both DNA uptake
and DNA release in monocultures was induced by the
ComCDE signal transduction pathway led to the possibility
that a subpopulation of the competent cells undergoes
autolysis, and thereby provides transforming DNA that can
be taken up by the survivors. However, subsequent work on
the DNA release mechanism revealed that the ability to act
as a DNA donor is not dependent on competence development in the donor cells. In liquid cultures containing a
mixture of competence-deficient (DcomE) and competenceproficient cells, addition of CSP was shown to result in
significant release of DNA from the competence-deficient
cells (Steinmoen et al., 2003). The ability of competent cells
to lyse noncompetent but otherwise isogenic pneumococci
was also observed by Guiral et al. (2005) in a different
experimental setup. They demonstrated that competent
pneumococci lyse and release intracellular pneumolysin
from noncompetent siblings when the cells are cocultivated
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O. Johnsborg & L.S. Håvarstein
on agar plates (see below for more details). The term
fratricide was adopted to describe this mechanism.
Target cells are killed by murein hydrolases
Early investigations on the fratricide mechanism revealed
that addition of 2% choline to the growth medium blocked
reporter enzyme release from competent monocultures of
strain CP1415 (Steinmoen et al., 2002). In other words, the
presence of choline inhibited competence-induced cell lysis.
Choline is well known for its ability to interfere with the cell
wall anchoring of pneumococcal choline-binding proteins
(CBPs), which normally bind noncovalently to the phosphorylcholine moiety of the S. pneumoniae cell wall through
a conserved choline-binding domain (Giudicelli & Tomasz,
1984; López & Garcı́a, 2004). Three such proteins, CbpD,
LytA, and LytC, were later demonstrated to be essential for
fratricide in liquid cultures (Steinmoen et al., 2002; Moscoso
& Claverys, 2004; Guiral et al., 2005; Kausmally et al., 2005;
Håvarstein et al., 2006; Claverys & Håvarstein, 2007) (see
Fig. 2). LytC is a lysozyme that is expressed from a housekeeping promoter throughout the exponential growth phase
(Garcı́a et al., 1999). LytA, the major autolysin of pneumococci, is activated during the late stationary phase and causes
extensive lysis of the bacterial culture (Garcı́a et al., 1986;
Sanchez-Puelles et al., 1986). Similar to LytC, the amidase
LytA is constitutively synthesized during growth. However,
upon competence induction, LytA expression is upregulated
from a late gene promoter upstream of cinA (MortierBarrière et al., 1998; Dagkessamanskaia et al., 2004; Peterson
et al., 2004). In contrast to LytA and LytC, CbpD is
expressed exclusively during competence from a late gene
promoter (Kausmally et al., 2005). The findings that CbpD
is synthesized only during competence, and that deletion of
CbpD in attacker cells abolishes lysis of competence-deficient target cells in liquid cultures (Kausmally et al., 2005;
Johnsborg et al., 2008), demonstrated that CbpD is a key
player and a triggering factor of the fratricide mechanism.
The subsequent discovery that the so-called clumping reaction, an indirect assay of competence-induced cell lysis, does
not take place when attacker cells lacking CbpD are used
further substantiates the central role of CbpD in pneumococcal fratricide (Håvarstein et al., 2006). The CbpD protein
consists of an N-terminal cysteine, histidine-dependent
amidohydrolase/peptidase (CHAP) domain, two Src
homology 3b (SH3b) domains in the middle and a
C-terminal choline-binding domain comprising four choline-binding repeats (Kausmally et al., 2005; Claverys et al.,
2007). While it has been firmly established that cholinebinding repeats mediate the cell wall anchoring of proteins
containing such domains (López & Garcı́a, 2004), the exact
functions of the CHAP and SH3b domains of CbpD remain
to be elucidated. Closely related structural homologs of the
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Fig. 2. Factors influencing cell lysis during fratricide in Streptococcus
pneumoniae. Concomitant with competence induction, pneumococci
attack the cell wall of noncompetent cells by secretion of the CbpD
murein hydrolase and the two-peptide bacteriocin CibAB. While CbpD is
essential to trigger fratricide in liquid cultures, CibAB appears to be the
main trigger factor on solid media. Efficient fratricide is dependent on
the presence of the amidase LytA and the LytC lysozyme. While both
hydrolases are expressed constitutively during growth, LytA synthesis is
upregulated during competence. LytA and LytC can be provided either
by the target cells, or in trans by the competent attacker cells. To protect
themselves against their own lysins, the competent cells produce the
immunity proteins ComM and CibC. In the case of ComM, it has not
been established whether this protein protects the cells specifically
against the action of CbpD, or whether the immunity is directed against
LytA and/or LytC. Fratricide triggers lysis of the noncompetent cells,
resulting in the release of intracellular material. CbpD-mediated fratricide has been shown to result in rapid release of transforming DNA that
can be taken up by the competent cells. CibAB-mediated fratricide has
been demonstrated to result in leakage of intracellular pneumolysin, but
the exact kinetics of this release has not been determined.
CHAP domain of CbpD have been demonstrated to act as
murein hydrolases, either by acting as endopeptidases that
cleave within murein stem peptides or by acting as amidases
that cleave the N-acetylmuramyl-L-ala bond (Bateman &
Rawlings, 2003; Rigden et al., 2003). It is therefore reasonable to assume that the CHAP domain of CbpD possesses
similar properties. Recently, it was found that substitution of
alanine for the predicted active-site cysteine 75 resulted in a
nonfunctional CbpD protein, indicating that the CHAP
domain functions as a cysteine protease as suggested by the
phylogenetic data. Interestingly, deletion of the murM gene
does not affect the activity of CbpD toward noncompetent
pneumococci (V. Eldholm, O. Johnsborg, K. Haugen, H.S.
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Acquisition of transforming DNA in S. pneumoniae
Ohnstad & L.S. Håvarstein, unpublished data). The murM
mutation removes the seryl-alanine/alanyl-alanine dipeptide
bridges that link the stem peptides in the pneumococcal cell
wall (Fiser et al., 2003). This result indicates that the CHAP
domain of CbpD cleaves within the murein stem peptides
rather than in the dipeptide bridges. Similar to the CHAP
domain, the SH3b domains are essential for CbpD function
during fratricide in liquid culture (O. Johnsborg, unpublished data). Prokaryotic SH3b domains are structurally
homologous to the classical SH3 domains of eukaryotes
(Lu et al., 2006). In contrast to their eukaryotic counterparts, which interact with proline-rich motifs in target
proteins (Mayer & Eck, 1995), prokaryotic SH3b domains
appear to interact with cell wall murein (Lu et al., 2006). It is
therefore possible that the SH3b domains of CbpD mediate
binding of the protein to the cell wall of target bacteria.
Autolysis or fratricide?
CbpD also contributes to fratricide when cells are grown on
agar plates. However, in this context, CbpD is not essential
for lysis to occur. Rather, fratricide under these conditions
relies on the synthesis of the two-peptide bacteriocin CibAB
(Fig. 2), which is expressed from a late competence gene
promoter (Guiral et al., 2005). Although inactivation of
cibAB does not have an impact on fratricide in liquid
cultures (Håvarstein et al., 2006), deletion of cibAB abolishes
fratricide on agar plates (Guiral et al., 2005). Hence, the
ability of CbpD and CibAB to act as fratricide trigger factors
appears to depend on the growth conditions. The cibAB
genes are cotranscribed with cibC, which encodes an immunity protein that protects the competent cells from their
own bacteriocins (Guiral et al., 2005). Similarly, the early
competence gene comM encodes an integral membrane
protein that by an unknown mechanism protects competent
cells from their own lysins in liquid cultures (Håvarstein
et al., 2006). Because the competent cells are protected,
fratricide always relies on a mixture of competent and
noncompetent cells (Fig. 2). Then how is it possible that
fratricide was first observed in pneumococcal monocultures
treated with CSP? This paradox can be explained by the fact
that the efficiency of competence induction in laboratory
cultures varies between pneumococcal strains, growth media
and even different batches of media used. Thus, under
suboptimal conditions, a subfraction of the cell population
will remain noncompetent even in the presence of CSP,
resulting in a mixture of competent and noncompetent cells.
Contributions and roles of the cell wall
hydrolases CbpD, LytA and LytC
Although CbpD produced by the competent cells is absolutely essential for fratricide in liquid cultures, CbpD does
not appear to be very active on its own under these
FEMS Microbiol Rev 33 (2009) 627–642
conditions (Steinmoen et al., 2002; Moscoso & Claverys,
2004; Guiral et al., 2005; Håvarstein et al., 2006). Rather,
CbpD triggers efficient cell lysis only in the presence of LytC
and/or LytA. Intriguingly, using an assay in which attacker
and target cells were cocultivated overnight in blood agar
plates, Guiral et al. (2005) discovered that LytC and LytA can
be provided in trans by the competent attacker cells as well
as in cis by the noncompetent target cells themselves.
Eldholm and colleagues investigated the relative contributions of LytA and LytC to fratricide by measuring the release
of an intracellular b-galactosidase reporter enzyme from
target cells after cocultivating them with competent attacker
cells in liquid medium for 30 min. The results for the most
part agree with those of Guiral and coworkers. However, in
liquid culture, lysis of target cells were found to be more
efficient when LytA and LytC were supplied in cis compared
with the situation where these autolysins were provided
in trans (V. Eldholm, O. Johnsborg, K. Haugen, H.S.
Ohnstad & L.S. Håvarstein, unpublished data).
LytC is present in relatively large amounts in the medium
of noncompetent pneumococcal cultures during the exponential growth phase, demonstrating that LytC does not
damage the cells during normal growth (unpublished data).
Likewise, exogenously added LytA is not able to degrade the
cell wall of growing cells (Lacks, 1970; Tomasz & Waks, 1975;
Dı́az et al., 1990). This shows that CbpD is able to activate
LytA and LytC, either through direct interaction or by a
more indirect mechanism. It is, for instance, possible that
CbpD triggers the action of the autolysins by introducing
damage to the cell walls of target cells. Recently, it was
discovered that competent S. mitis also use the fratricide
mechanism to kill and lyse closely related strains and species
(Johnsborg et al., 2008). In contrast to S. pneumoniae, most
strains of S. mitis do not contain the lytA gene (Kilian et al.,
2008). Given the high degree of identity between the CbpDs
from S. mitis and S. pneumoniae, it is unlikely that this
protein has evolved as a specific LytA activator. Therefore,
activation of LytA probably requires that CbpD alone or
together with LytC damages or kills the target cells through
limited hydrolysis of their cell walls. This might indirectly
activate LytA in the target cells, resulting in autolysis. We
have recently found that most of the secreted CbpD protein
remains attached to the cell wall of the producer cell,
whereas a smaller fraction is released to the growth medium
(V. Eldholm, O. Johnsborg, K. Haugen, H.S. Ohnstad & L.S.
Håvarstein, unpublished data). A possible explanation for
this result is that fratricide under natural conditions involves
cell–cell contact between attacker and target cells. As CbpD
probably attaches to protruding teichoic acid and lipoteichoic acid in the cell wall of the competent cells, it is
conceivable that contact between the cell wall surface of
competent and noncompetent cells could result in lysis of
the latter cell type.
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634
Fratricide and its impact on horizontal
gene transfer
A newly published study by Johnsborg et al. (2008) demonstrated that the fratricide mechanism dramatically increases
the efficiency of horizontal gene transfer between pneumococci in vitro. Transfer of an antibiotic marker from noncompetent target to competent attacker cells was shown to
be a thousand-fold more efficient with wild type than
CbpD-deficient attacker cells. Similarly, it was shown that
the fratricide mechanism has a large positive impact on the
efficiency of gene transfer from the commensals S. mitis and
S. oralis to S. pneumoniae. Furthermore, fratricide is not
unique to pneumococci, as this mechanism has been demonstrated in competent S. mitis as well (Johnsborg et al.,
2008). Fratricide is also likely to be used by competent
S. oralis, as both cbpD and comM are known to be present in
the genome of this species (R. Hakenbeck, pers. commun.).
In order to function, the fratricide mechanism requires a
mixture of competent and noncompetent pneumococci
and/or members of closely related species. How do such
populations arise in vivo? Studies indicate that due to the
large diversity of CSPs produced by different strains and
species of naturally transformable streptococci, mixed populations of competent and noncompetent cells arises
naturally (Johnsborg et al., 2008). The reason for this is that
in most cases cross-induction of competence between different pherogroups will not take place. Interestingly, competent pneumococci are not able to lyse more distantly
related species, such as Streptococcus gordonii (Johnsborg
et al., 2008). The mechanism behind this relatively narrow
target range is not known, but it is reasonable to assume that
the ability of CbpD to mediate cell lysis is restricted by the
structure of the cell wall in the target bacteria. For instance,
phosphorylcholine is part of the cell wall teichoic acid of
S. pneumoniae, S. mitis, S. oralis and some Streptococcus
infantis strains (Kilian et al., 2008), but is not present in
more distantly related species such as S. gordonii and
Streptococcus sanguinis (Gillespie et al., 1993). Thus, CbpD
might be unable to attach to the cell wall of S. gordonii and
S. sanguinis. CbpD activity could also be impaired by the
lack of a suitable binding site for the SH3b domains, or lack
of a suitable substrate for the CHAP domain. Hence, it
appears that the fratricide apparatus has evolved as a lysis
mechanism that specifically targets noncompetent cells that
are relatively closely related to the competent attacker cells.
If the purpose of natural genetic transformation is to
capture genetic material from other cells in order to
facilitate repair of damaged genes, generate genetic diversity
and acquire novel functions, this makes sense. A mechanism
that discriminates between DNA from related and unrelated
bacteria has also evolved in some naturally transformable
members of the families Neisseriaceae and Pasteurellaceae
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O. Johnsborg & L.S. Håvarstein
(Smith et al., 1999; Treangen et al., 2008). These bacteria
strongly prefer to take up DNA containing 10–12-bp
sequence motifs termed DNA uptake sequences (DUS). As
the genomes of naturally transformable members of these
families are enriched in their respective DUS sequences,
uptake of homologous DNA is favored.
Different terms have been used in the literature to
describe lysis of various target bacteria. Lysis of noncompetent isogenic bacteria was originally termed heterolysis
(Steinmoen et al., 2003). Later on, allolysis was suggested as
a better alternative (Guiral et al., 2005), whereas the term
sobrinicide was coined for lysis of nonisogenic strains of the
same species (Claverys et al., 2006). Because fratricide,
heterolysis, allolysis and sobrinocide are driven by the same
molecular mechanism, one might ask whether there is need
to differentiate the various types of killing by different terms.
Thus, for the sake of simplicity, we have only used fratricide
in the current review.
Dissemination of virulence and penicillin
resistance genes
Streptococcus pneumoniae continues to be a significant cause
of morbidity and mortality in humans. Consequently, the
emergence and spread of penicillin-resistant pneumococcal
clones has become a major concern worldwide. Despite the
increasing prevalence of resistance to penicillin and other blactams, these antibiotics remain the first-line drugs for
treatment of pneumococcal disease. Resistance is not due to
the expression of b-lactamases, but it arises from alterations
in the penicillin-binding proteins (PBPs) of S. pneumoniae.
Specific mutations within the transpeptidase domain of
these cell wall-synthesizing enzymes give rise to PBPs that
have decreased affinity for b-lactam antibiotics (Hakenbeck
et al., 1980). Streptococcus pneumoniae contains five highmolecular-weight PBPs (1a, 1b, 2a, 2b and 2x), plus a lowmolecular-weight (D,D) carboxypeptidase (PBP3). Among
these six PBPs, alterations in PBP2x, PBP2b and PBP1a are
most frequently encountered in penicillin-resistant clinical
isolates (Grebe & Hakenbeck, 1996; Hakenbeck et al., 1999).
Nucleotide sequence analysis of PBP genes from resistant
strains revealed the presence of highly divergent mosaic
blocks in the region encoding the transpeptidase domain.
These mosaics must have arisen by intra- and interspecies
recombinational events mediated by natural transformation
(Dowson et al., 1989, 1993; Coffey et al., 1991; Laible et al.,
1991; Sibold et al., 1994). The degree of sequence divergence
between the acquired mosaic blocks and the corresponding
region in the wild-type pneumococcal genome indicates that
they originate mainly from different but closely related
species. A large variety of mosaic blocks has been identified,
demonstrating that they have been captured from different
sources by independent recombination events (Chi et al.,
FEMS Microbiol Rev 33 (2009) 627–642
635
Acquisition of transforming DNA in S. pneumoniae
2007; Izdebski et al., 2008). Potential ancestor genes have
been identified in S. mitis and S. oralis, strongly indicating
that these commensals constitute a reservoir for penicillin
resistance determinants. How did such determinants arise in
the first place? Presumably, treatment of patients with blactams over an extended period of time resulted in the
emergence of S. mitis and S. oralis clones with mutated PBP
genes that conferred increased resistance to these antibiotics.
Once S. mitis and S. oralis clones harboring low-affinity
PBPs had evolved, functional resistance determinants were
transferred to related strains and species by natural
transformation. This scenario is based on considerable
experimental support, and provides a framework for understanding the sequence of events that have led to the observed
increase in b-lactam resistance in clinical isolates of
S. pneumoniae (Zapun et al., 2008).
Fratricide dramatically increases the efficiency of gene
exchange between and within the species S. pneumoniae,
S. mitis and S. oralis in vitro (Johnsborg et al., 2008), and it is
therefore reasonable to assume that the fratricide mechanism plays the same role in vivo. If fratricide turns out to be
the predominant mechanism for acquisition of donor DNA
under natural conditions, i.e. in multispecies biofilms in the
oral cavity and the nasopharynx, competent streptococci act
as DNA predators and not as DNA scavengers as believed
previously. It makes sense to capture DNA from living cells,
because these cells represent survivors that are well adapted
to the environment they live in. Prudhomme et al. (2006)
have shown that sublethal concentrations of the antibiotics
norfloxacin, kanamycin and streptomycin induce the competent state in S. pneumoniae. Evidently, these bacteria
respond to stress by seeking new genetic material that might
help them survive. Chances are that the best source of the
sought-after genetic information is thriving neighboring
cells, and not extracellular genetic material originating from
cells that have died and fallen apart.
Virulence genes, such as the cps genes involved in the
synthesis of the pneumococcal polysaccharide capsule, are
also transferred between pneumococci by natural transformation. Sometimes horizontal transfer of cps genes, which
are localized in a single cluster flanked by the dexB and aliA
genes, results in a change of serotype, a phenomenon termed
capsular switching (Coffey et al., 1991, 1999; Jefferies et al.,
2004; Porat et al., 2004; Gherardi et al., 2009). In the year
2000, a pneumococcal conjugate vaccine was introduced in
the United States that protects against seven (4, 6B, 9V, 14,
18C, 19F and 23F) out of 91 known serotypes. Different
serotypes vary considerably in their propensity to cause
invasive disease. Therefore, the seven serotypes included in
the conjugate vaccine represent those most commonly
recovered from patients with invasive disease. Since its
introduction, a significant decline in the incidence of
pneumococcal invasive disease has been reported. In addiFEMS Microbiol Rev 33 (2009) 627–642
tion, carriage of vaccine serotypes has been reduced in the
general population, leading to less disease in unvaccinated
individuals also (Whitney et al., 2003, 2006). However, by
targeting only seven serotypes, a vacant niche has been
created. As a result, the replacement of vaccine serotypes by
nonvaccine serotypes has taken place (Beall et al., 2006;
Hanage, 2008). This replacement was expected in advance,
but it was hoped that the replacing serotypes were of low
virulence and that they would not evolve into more virulent
strains. Similar to serotype replacement, capsular switching
has the potential to cause a serious threat to the long-term
efficacy of the conjugate vaccine. In principle, members of
the vaccine serotypes 4, 6B, 9V, 14, 18C, 19F and 23F can
transform into nonvaccine serotypes in a single recombination step. In fact, proof of this principle has already emerged.
Brueggemann et al. (2007) identified an isolate expressing
the nonvaccine serotype 19A capsule, which possessed a
genotype previously associated only with serotype 4. A
prerequisite for serotype switching is that more than one
serotype is carried by the same individual. Interestingly, this
appears to be quite common, as several studies have reported
that children may carry two or more serotypes at the same
time (Gratten et al., 1989; Rapola et al., 1997; Syrjänen et al.,
2001). As mentioned, the fratricide mechanism has been
shown to increase the efficiency of gene exchange between
different pneumococcal strains a thousand-fold in vitro
(Johnsborg et al., 2008). An important task for the future
will therefore be to determine whether this mechanism is the
driving force behind serotype switching under natural conditions. If this turns out to be the case, more detailed
information about the conditions that facilitate such gene
transfer in situ might help optimize the formulation of the
next generation of pneumococcal conjugate vaccines.
CSP diversity -- possible biological
significance
Sequencing of a large number of comC genes from different
strains and species belonging to the mitis phylogenetic
group revealed that different species always produce different CSPs and that the pheromone diversity is considerable
even within each species (Johnsborg et al., 2007a). This
finding could be interpreted in two different ways. One
alternative is that no selection pressure exists that conserves
the primary structure of the CSP peptide. This seems
unlikely, as mutations causing amino acid changes in the
signaling peptide in most cases would affect its functionality.
Because of the complementarity between CSP and its
cognate ComD receptor, changes in one of them must be
compensated for by the appropriate changes in the other.
Consequently, it must be assumed that most changes in the
amino acid sequence of CSP, caused by random mutations,
are selected against. Another, and in our opinion more
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636
likely, explanation for the observed diversity of CSP peptides
is that there exists a selection pressure that drives the
evolution of new functional CSP/ComD pairs. The need for
concealed communication could be the driving force behind
this development. To mount a successful attack on neighboring cells, it is crucial to avoid that these cells detect the
oncoming attack and protect themselves by producing the
ComM and CibC immunity proteins. In other words, the
competent cells must be able to cooperate efficiently in
order to predate on other cells, but this communication
must not be sensed by the noncompetent target cells.
Interestingly, we found that the pneumococcal R6 strain
in some cases can sense foreign CSPs (Johnsborg et al.,
2008). This strain, which makes and responds to a peptide
pheromone termed CSP-1, was cross induced by two out of
seven S. mitis pheromones tested. One of these, produced by
the S. mitis type strain (NCTC12261), shares very little
homology with CSP-1. Thus, it appears that ComD receptors have evolved to be promiscuous instead of developing
high specificity for their cognate ligands. What could be the
driving force behind this particular adaptation? Because
ComM is part of the competence regulon in S. pneumoniae,
S. mitis and S. oralis (Claverys et al., 2007; unpublished
data), potential target cells can protect themselves by sensing
an imminent attack and turn on the expression of the
ComM immunity protein (Johnsborg et al., 2008). Consequently, the best survival strategy for members of these
species presumably is to (1) produce a CSP type that escapes
detection by other streptococcal strains sharing the same
niche and (2) possess a ComD receptor that can detect the
presence of as many foreign CSP peptides as possible,
especially those CSP types that are produced by strains
adapted to the same habitat.
The line of reasoning outlined above can also be used to
explain a phenomenon called pherotype switching. Streptococci that have undergone pherotype switching have exchanged the comC gene, and the part of comD encoding the
CSP-binding domain, with the corresponding region from a
Streptococcus belonging to a different pherogroup. Several
examples of such switching have been discovered, demonstrating that it represents a relatively common event
(Håvarstein et al., 1997; Kilian et al., 2008). It therefore
appears likely that the acquisition of a new pherotype
through horizontal gene transfer is often beneficial to the
recipient. A possible reason for this is that the new pherotype
is not detected by members of other pherogroups sharing the
same niche, and/or that the new ComD receptor enables the
recipient to sense the pheromones produced by its neighbors.
Quorum, diffusion or efficiency sensing
In classical quorum sensing, as exemplified by the lux system
of Vibrio fischeri, populations of bacterial cells measure their
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O. Johnsborg & L.S. Håvarstein
own density by secreting and sensing low-molecular-weight
autoinducers. When these signaling molecules reach a
threshold concentration, reflecting the number of cells in a
particular environment, expression of the appropriate target
genes is switched on (Waters & Bassler, 2005). This cell–cell
signaling mechanism allows bacterial populations to carry
out cooperative activities that require a minimum cell
density to be efficient. How do competence development in
S. pneumoniae and other members of the mitis phylogenetic
group fit into this classical model? In vitro studies with
planktonic cells have demonstrated that, depending on the
pH of the growth medium, pneumococci develop the
competent state when subjected to 2–10 ng mL1 of synthetic CSP (Håvarstein et al., 1995a). Thus, in accordance
with classical quorum sensing, a threshold level of CSP is
required to trigger competence development in liquid
cultures. On the other hand, Claverys et al. (2006) discovered that regardless of their initial densities, pneumococcal
cultures begin to develop the competent state about 45 min
after a so-called alkaline shift (pH 6.9 ! pH 7.9). Hence, in
this particular case, CSP acts more like a timing device than
a quorum-sensing signal. Another example of quorumsensing-independent competence development in S. pneumoniae is the finding that sublethal concentrations of
some antibiotics induce the competent state in this species
(Prudhomme et al., 2006).
The natural habitat of S. pneumoniae and its commensal
relatives is complex multispecies biofilms in the oral cavity
and nasopharynx. In such biofilm communities, where
streptococcal cells often grow within confined spaces, the
concentration of CSP depends not only on the number of
CSP-producing cells but also on the rate at which CSP is lost
to the environment. Redfield (2002) has gone so far as to
argue that quorum sensing is just a side effect of diffusion
sensing. She has suggested that bacteria make and respond
to autoinducers solely to determine how rapidly these
secreted molecules move away from the producer cell. In
the case of competence regulation in S. pneumoniae and its
relatives this makes sense. If CSP is rapidly lost to the
environment by diffusion or advection, the same would be
true for the key effector of fratricide CbpD, and possibly
LytA, LytC and CibAB. Thus, one of the functions of CSP
may be to act as a probe that the producer cells use to
explore their immediate surroundings. In other words, the
concentration of CSP tells the cells whether production of
the fratricide effectors would be effective or not. However, as
mentioned above, the concentration of CSP does not only
depend on how rapidly this autoinducer is lost but is also a
function of how rapidly it is produced. Consequently, both
the number of cells that contribute to CSP production and
the physical barriers that limit diffusion of this signaling
molecule are crucial factors that together control competence development (Fig. 3). This model is in agreement with
FEMS Microbiol Rev 33 (2009) 627–642
637
Acquisition of transforming DNA in S. pneumoniae
the efficiency sensing hypothesis recently proposed by Hense
et al. (2007). According to this hypothesis, autoinducers are
used as ‘a proxy for testing the efficiency of producing
costlier diffusible extracellular effectors’. Whether or not
CSPs function as an efficiency sensor ultimately depends on
whether cell–cell contact is a prerequisite for fratricide.
There is no need for an efficiency sensor if the relevant
effector proteins are associated with the surface of the
attacker and target cells. We have detected CbpD as well as
LytC in the growth medium of competent cultures, but in
the case of CbpD the majority of extracellular protein
appears to be bound to teichoic acid in the cell wall of the
producer cells (V. Eldholm, O. Johnsborg, K. Haugen, H.S.
Ohnstad & L.S. Håvarstein, unpublished data). Consequently, it is still an open question whether competent
pneumococci kill their siblings through cell–cell contact or
at a distance by effectors that diffuse from the attacker to the
target cells. An attractive third alternative is that target cells
are lysed by a combination of these two mechanisms.
In sum, it is possible to envision three different but
mutually nonexclusive functions for the CSP peptide.
Firstly, CSP might act to coordinate competence development in a population of cells belonging to the same
pherogroup. Such coordination would presumably result in
higher extracellular concentrations of the fratricide effectors,
leading to increased lysis of target cells. Secondly, CSP might
serve as a warning signal that enables members of the same
pherogroup to synthesize the ComM immunity protein
Fig. 3. Pneumococci and related commensal streptococci such as Streptococcus mitis and Streptococcus oralis inhabit multispecies biofilms in
the upper respiratory tract. These bacteria (red dots) develop the
competent state when the extracellular concentration of their respective
CSP-pheromones reaches a critical concentration. A number of different
factors will influence the external concentration of CSP in situ. The most
important are (1) the rate of CSP synthesis per streptococcal cell, (2) the
number and density of cells in a particular location within the biofilm, (3)
advection and (4) physical barriers that limit diffusion of CSP. Consequently, competence development in these bacteria is not simply
regulated by a classical quorum-sensing mechanism, but by a combination of factors collectively referred to as efficiency sensing (Hense et al.,
2007).
FEMS Microbiol Rev 33 (2009) 627–642
before they are killed by the secreted effector proteins.
Thirdly, as discussed above, CSP might function as a probe
that the producer cells use to measure the rate at which
secreted molecules diffuse away from the producer cell. It
follows from all this that it is the predatory DNA acquisition
mechanism (fratricide), and not DNA uptake per se, that is
the reason why competence for natural transformation in
S. pneumoniae, S. mitis and S. oralis is regulated by a secreted
autoinducer. Furthermore, based on the reasoning above, it
is likely that a competence-induced lysis mechanism also
exist in other members of the mitis phylogenetic group such
as S. infantis, Streptococcus crista, S. gordonii and S. sanguinis. All of these species are known to produce and respond
to their own particular CSP peptides by a mechanism that
appears to be the same as that for S. pneumoniae, S. mitis
and S. oralis (Claverys et al., 2007).
Concluding remarks
Through studies that, for the most part, have been carried
out in vitro, a detailed picture of natural genetic transformation in S. pneumoniae is now beginning to develop. We have
no doubt, however, that this phenomenon still holds many
secrets that are waiting to be revealed. One important aspect
of natural transformation that is poorly understood is
where, and under what circumstances, DNA is exchanged
in vivo. As described above, sequence analysis of PBP genes
from a number of penicillin-resistant pneumococcal isolates
has revealed that their resistance-determinants are frequently acquired from related species such as S. mitis and
S. oralis. These species all colonize the naso- and oropharynx
of their human host, and it is therefore highly likely that
multispecies biofilms are the natural environment for gene
exchange between pneumococci and their commensal relatives. Future research should therefore aim at developing
techniques that can be used to investigate gene transfer
between naturally transformable streptococci in biofilms
in vitro. Equally important, efforts should be made to
improve our understanding of transformation in a living
host. In his original experiments, Griffith (1928) injected
mice with a mixture of live and dead pneumococcal cells. As
his approach involves the use of large amounts of heat-killed
donors, it relies on artificial conditions and is not useful as a
method for studying gene transfer in vivo. However, a few
pioneering studies from the 1960s have demonstrated the
occurrence of transformation during mixed pneumococcal
infection in mice and man (Ottolenghi & MacLeod, 1963;
Conant & Sawyer, 1967; Ottolenghi-Nightingale, 1969,
1972). In mice, transformation was shown to take place even
when living donor and recipient cells were injected at
different sites or 6 h apart. Furthermore, gene transfer
among living pneumococci has also been observed in mice
infected by intrabronchial inoculation. Considering the vast
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638
amount of knowledge gained from studying natural transformation over the last 40 years, the time is now ripe for
revisiting this issue.
Acknowledgements
This work was supported by the Research Council of Norway. A special acknowledgement goes to Hilde Johnsborg
for artwork.
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