RESEARCH ARTICLE

crossm

Genome-Wide Screens Reveal New Gene Products That

Influence Genetic Competence in Streptococcus mutans
Robert C. Shields,a Greg O’Brien,b Natalie Maricic,a Alexandria Kesterson,a Megan Grace,a Stephen J. Hagen,b
Robert A. Burnea

a
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA
b Department of Physics, University of Florida, Gainesville, Florida, USA

ABSTRACT A network of genes and at least two peptide signaling molecules

tightly control when Streptococcus mutans becomes competent to take up DNA from
its environment. Widespread changes in the expression of genes occur when S. mu-
tans is presented with competence signal peptides in vitro, including the increased
production of the alternative sigma factor, ComX, which activates late competence
genes. Still, the way that gene products that are regulated by competence peptides
influence DNA uptake and cellular physiology are not well understood. Here, we de-
veloped and employed comprehensive transposon mutagenesis of the S. mutans ge-
nome, with a screen to identify mutants that aberrantly expressed comX, coupled
with transposon sequencing (Tn-seq) to gain a more thorough understanding of the
factors modulating comX expression and progression to the competent state. The
screens effectively identified genes known to affect competence, e.g., comR, comS,
comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that
were not previously competence associated. The competence phenotypes of mu-
tants were characterized, including by fluorescence microscopy to determine at
which stage the mutants were impaired for comX activation. Among the novel
genes studied were those implicated in cell division, the sensing of cell envelope

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stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for
determining the specific chemical and physical cues that are required for genetic
competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S.
mutans to discover and study novel biological processes.
IMPORTANCE Streptococcus mutans acquires DNA from its environment by becom-
ing genetically competent, a physiologic state triggered by cell-cell communication
using secreted peptides. Competence is important for acquiring novel genetic traits
and has a strong influence on the expression of virulence-associated traits of S. mu-
tans. Here, we used transposon mutagenesis and genomic technologies to identify
novel genes involved in competence development. In addition to identifying genes
Received 21 August 2017 Accepted 26
previously known to be required for comX expression, 20 additional genes were October 2017
identified and characterized. The findings create opportunities to diminish the Accepted manuscript posted online 6
pathogenic potential of S. mutans, while validating technologies that can rapidly ad- November 2017
vance our understanding of the physiology, biology, and genetics of S. mutans and Citation Shields RC, O'Brien G, Maricic N,
Kesterson A, Grace M, Hagen SJ, Burne RA.
related pathogens. 2018. Genome-wide screens reveal new gene
products that influence genetic competence in
KEYWORDS ComX/SigX, transposon mutagenesis, single-cell behaviors, peptide Streptococcus mutans. J Bacteriol 200:e00508-
17. https://doi.org/10.1128/JB.00508-17.
signals, stress tolerance
Editor Victor J. DiRita, Michigan State
University
Copyright © 2017 American Society for

G enetic competence, the process by which bacteria are able to internalize DNA, is
a trait shared across bacterial phyla (1). The acquisition of the state of natural
genetic competence is transient, and under most conditions, the genes encoding
Microbiology. All Rights Reserved.
Address correspondence to Robert A. Burne,
rburne@dental.ufl.edu.
products required for DNA uptake are not expressed. The activation of the transcription

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Shields et al. Journal of Bacteriology

of these genes occurs in response to specific signals and, even then, only when
environmental conditions are permissive. In the Gram-positive streptococci, the com-
petence machinery is typically regulated by externalized peptides, which when present,
enhance the production of ComX (also known as SigX), an alternative sigma factor that
associates with RNA polymerase and enables the recognition of promoters of late
competence genes (2). ComX is essential for competence development and is highly
conserved in competent streptococci. However, multiple other networks influence the
expression of comX or the levels of ComX or turn off the competence regulon, and
these can differ substantially between species (2, 3). Genetic competence regulation in
Streptococcus mutans, an etiologic agent of dental caries, is particularly interesting
because it relies on two genetic circuits, including one that is integrated with the
production of bacteriocins, which appear to be critical for the establishment and
persistence of S. mutans in human oral biofilms (2).
S. mutans produces at least two signal peptides that can activate cells to develop
competence, namely, competence-stimulating peptide (CSP) and comX-inducing pep-
tide (XIP). The addition of synthetic CSP or XIP to early-exponential-phase cultures of S.
mutans can lead to enhanced expression of comX (4). The activation of comX expression
can occur directly when XIP, an extracellular 7-amino-acid (aa) peptide derived from its
17-aa precursor, ComS, is internalized and forms a complex with the transcriptional
regulator ComR (5). The ComR-XIP complex binds directly upstream of the promoters
for comX and comS. In the case of CSP, this 18-aa peptide is derived from ComC by
secretion and processing. Externalized CSP interacts with the histidine kinase ComD,
which leads to the phosphorylation of the response regulator ComE (ComE⬃P).
ComE⬃P directly activates the expression of a variety of genes encoding bacteriocins
and products involved in bacteriocin biogenesis and immunity (6). Although ComE⬃P
does not directly activate the transcription of comX, the provision of CSP to S. mutans
at levels as low as 30 nM can increase the transformation efficiency by as much as
1,000-fold, and higher concentrations of CSP trigger increased comX mRNA and ComX
protein levels (4, 7). The mechanism by which CSP activates the competence cascade
is poorly understood, but it requires ComRS and may involve the bacteriocin CipB;
transcription of cipB is activated by the binding of ComE⬃P to the cipB promoter region
(6). In addition to the ComRS and CSP-ComDE circuits, many other gene products and
environmental inputs can influence comX expression and ComX protein levels, and the

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effects of these factors differ depending on the signal peptide used and the medium
composition. Among the factors that have the greatest influence on S. mutans com-
petence gene expression and the transition to, or exit from, the competent state are
(p)ppGpp levels modulated by RelA (8), the products of the rcrRPQ operon (9, 10), the
pH of the local environment (11), the carbohydrate source (12), the exposure to oxygen
(13), the cell density (14), and the growth in biofilms versus planktonic culture (15). It
is also relevant that CSP induces comX expression and competence only in rich
(peptide-containing) media, whereas XIP is effective only in chemically defined media
lacking peptides (4).
The regulons of ComE⬃P, ComR-XIP, and ComX have been explored using microar-
rays and transcriptome sequencing (RNA-Seq) (16–19). In S. mutans, ComE⬃P controls
the expression of 4 operons, ComR-XIP controls 2 operons, and ComX affects the
expression of 21 operons (16). Collectively, these studies constitute a valuable founda-
tion for understanding how CSP and XIP influence the transcriptome. Still, there are
some significant gaps in our understanding of the regulation of genetic competence in
S. mutans, including how XIP is processed and externalized, the way in which the
ComCDE pathway stimulates the ComRS circuit to activate competence, the mecha-
nisms by which (p)ppGpp exerts its influence on many aspects of competence, and how
the ABC transporters and peptides encoded in the rcrRPQ operon so profoundly
influence comX-xrpA gene expression and the ability of S. mutans to be transformed.
Other significant deficiencies in our knowledge include how tolerance of environmental
stress and biofilm formation are integrated with regulators of the competence network
and how lytic behaviors are coordinated by inputs from stress tolerance and compe-

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tence systems. While functional genomics and approaches employing next-generation

sequencing (e.g., transcriptomics) are helping to understand the complexities of com-
petence and its integration with the biology of S. mutans, complementary approaches
are still needed to close the gaps in knowledge noted above. Interestingly, there have
been no classical genetic screens performed with the aim of identifying genes that
affect genetic competence in S. mutans or that may integrate competence with other
networks that are germane to the establishment, persistence, and virulence of the
organism. One reason for this may be that transposon mutagenesis techniques that
achieve unbiased saturation of the genome of S. mutans have not been available, in
contrast to the systems available for many Gram-negative organisms and for certain
Gram-positive bacteria, such as Streptococcus pneumoniae and Bacillus subtilis (20, 21).
Here, we employed classical transposon mutagenesis and screening, coupled with
transposon sequencing (Tn-seq), to examine the competence regulon and how it may
be integrated with pathways that govern physiologic homeostasis. Virtually all previ-
ously known regulators of genetic competence in S. mutans were identified in the
screen, as were an additional 20 strains with insertions in coding or noncoding regions
of the genome that had not previously been associated with competence. This study
also validates Tn-seq as a highly effective addition to the technologies available for
genetic studies with S. mutans.

RESULTS
Two screens for genes affecting competence in S. mutans. To develop a more
comprehensive overview of the gene products that may influence comX promoter
activity, a transposon insertion library was constructed in an otherwise wild-type
genetic background in S. mutans UA159 carrying a fusion of a Streptococcus salivarius
lacZ gene to the comX promoter (PcomX-lacZ) (12). Using in vitro transposon mutagen-
esis (22), a mariner-based transposon library was generated that contained 2.5 ⫻ 104
apparently random insertions, which is roughly equivalent to a collection of mu-
tants that carried transposon insertions every 80 bp in the 2 megabase genome of
S. mutans UA159. A Southern blot analysis of eight mutants using a probe specific
for the aad9 spectinomycin resistance gene in the transposon showed that the
mutants had single magellan6 insertions (see Fig. S1 in the supplemental material).
Sanger sequencing of PCR products, generated using a transposon-specific primer

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in conjunction with random primers (arbitrary PCR), from 168 individual PcomX-lacZ
defective mutants always gave unique results, providing further evidence that
multiple insertions rarely occurred in each mutant (data not shown). The library was
screened on tryptone-yeast extract-glucose (TYG) agar containing X-Gal (5-bromo-4-
chloro-3-indolyl-␤-D-galactopyranoside), on which the PcomX-lacZ strain formed blue
colonies after 40 h of incubation. Importantly, the deletion of the comR gene (ΔcomR
mutant), with ComR being required for transcriptional activation of comX, in the
PcomX-lacZ strain yielded gray colonies on TYG–X-Gal plates (see Fig. S2). Additionally,
cipB expression in colony biofilms was observed using green fluorescent protein (GFP)
reporters and confocal laser scanning microscopy, indicating that the CSP-ComDE
circuit was active under the conditions tested (Fig. 1A). A total of 233 PcomX-lacZ
magellan6 insertion mutants (which we designated mutants aberrant in competence
[mac] strains) were isolated on the basis of the formation of colonies that appeared
white on TYG–X-Gal plates (see Fig. 1B for an example of the appearance of a colony
with reduced lacZ expression). All mac strains were assayed to determine if they
displayed phenotypes with reduced transformation efficiencies (Fig. 1C) and decreased
sensitivity to an inhibition of growth when CSP was included in the medium. Of the
original 233, 168 mac strains met these initial criteria. By the use of PCR and sequenc-
ing, the insertion of the transposons in these 168 strains was determined to have
occurred in 55 distinct coding and noncoding regions of the genome. Table S1 shows
that our screen identified most of the genes that have been shown to influence comX
expression previously: comR, comS, rcrR, clpX, ciaH, cipB, comD, and comE. These strains
were also resistant to growth inhibition by 2 ␮M CSP and either displayed very low

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FIG 1 PcomX-lacZ and Tn-seq screens for novel genes required for optimal comX expression. (A) Photograph of otherwise wild-type S. mutans organisms carrying
PcipB-gfp or PcomX-gfp on the surface of TYG agar. Fluorescent strains were spotted (10 ␮l) on TYG agar and incubated for 24 h before the images were obtained
on a fluorescence microscope using a 20⫻ objective. (B) Example of the appearance of wild-type PcomX-lacZ activity (blue colonies) and a transposon mutant
(mac-1) with reduced PcomX-lacZ activity (light-blue colonies) on the surface of TYG–X-Gal agar. (C) Screening for transformation defects in transposon strains
(mac-1 shown) compared to wild-type S. mutans. Wild-type S. mutans and mac strains were incubated in BHI broth with 200 nM CSP and plasmid pIB184 (Ermr)
to monitor DNA uptake after plating 100 ␮l of the transformation sample on BHI-erythromycin agar. (D) Tn-seq results summarized using a scatterplot that

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shows changes in the numbers of transposon insertions in S. mutans genes before and after growth in 2 ␮M CSP. Reads were normalized to 1 million reads
per sample. Inset chart displays insertions in genes from SMu.481 to pnpB. The complete list of insertions that were overrepresented following passage in
BHI-CSP can be found in Table S2 in the supplemental material.

transformation efficiencies or could not be transformed, except for clpX and ciaH
mutants that were resistant to CSP but displayed only modestly diminished transfor-
mation efficiencies. That we could discover previously identified genetic competence
regulators validated our methodology; thus, we characterized the remaining mutants
carrying transposons inserted in the 40 coding and noncoding regions that represented
novel regulators of comX expression.
In addition to the high degree of randomness of insertion of the magellan6
transposon and the ability to rapidly determine insertion sites, another benefit of this
transposon library was the ability to perform deep sequencing (Tn-seq) on populations
prior to and after growth in selected in vitro or in vivo environments, thereby enabling
the enumeration of mutants before and after an imposed challenge to the population.
Tn-seq was used here to identify genes that influenced the fitness when cells were
exposed to 2 ␮M CSP. The sequencing of the starting library prior to selection showed
that the insertions were randomly distributed across the genome, except for genes that
were essential for growth under the conditions used (Fig. 1D) (R. C. Shields, L. Zeng, D. J.
Culp, and R. A. Burne, in preparation). The library was inoculated in brain heart infusion
(BHI) broth and BHI broth containing 2 ␮M CSP and passaged 5 times. More specifically,
fresh medium was inoculated with 107 cells from the initial library and grown to an
optical density at 600 nm (OD600) of 1.0 (⬃2 ⫻ 109 cells), and then the culture was

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diluted 1:100 in fresh medium and cultured to an OD600 of 1.0 four more times,
corresponding to approximately 30 generations. The hypothesis was that mutants
carrying transposon insertions that caused diminished comX expression or that dis-
rupted the connection between the CSP-ComDE circuit and those pathways that elicit
CSP-dependent growth inhibition and/or lysis would be present in greater proportions
in the library after passage with CSP (6, 16) than in the library grown in BHI broth alone.
As with the X-Gal screen, the strains carrying Tn insertions in genes known to affect
comX expression were identified as overrepresented after passage with CSP (Fig. 1D;
see also Table S2). The strongest positively selected transposon mutants (log2 fold
change greater than 0.5) were those with Tn insertions in the cipB (bacteriocin) and
comDE operons. Enhanced fitness was also observed for strains with Tn insertions in
irvR, comR, comX, rcrR, and ciaH, which are all known to impact competence, as well as
in 28 coding regions not previously reported to influence comX expression, compe-
tence, or sensitivity to CSP.
Validation of mutations affecting CSP-induced genetic competence. Across the
two screens, 37 coding and noncoding regions were identified that were deemed to
warrant further investigation. Three genes, pknB, liaS, and irvR, were previously linked
to genetic competence (23–26) but have not been extensively characterized, and so
these mutants were also examined. In total, 38 genes and 2 intergenic regions were
deleted and replaced with nonpolar kanamycin resistance cassettes. Phenotypic char-
acterization of these defined allelic exchange mutants began with the monitoring of
their growth characteristics in the presence of CSP or XIP. At high concentrations
of exogenously supplied peptides, both CSP and XIP caused growth arrest and/or lysis
of S. mutans UA159, phenotypes that have been shown to be dependent on ComX (25,
27). Mutant strains displayed a wide range of growth behaviors in response to CSP
during growth (Fig. S3A). For example, in the presence of CSP, the final OD600 of all
mutants was significantly higher than for the wild type (P ⫽ 0.0004). There was also
variability in the durations of the lag phase and exponential growth rates of the
mutants. To begin to interpret the basis for these various behaviors, a phenotypic
clustering model was developed that could be used to predict potential functional
interrelationships between genes (Fig. 2A; see also Text S1), with the rationale that if
growth phenotypes were similar between mutants in an environment, then the gene

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products that clustered together in the model may participate in a common pathway
or function. The model took into consideration the lag phase duration, exponential
growth rate, final yield (maximal OD600), and other parameters (Text S1). Using this
approach, we discovered putative gene-gene interactions between known genetic
competence regulators and the newly discovered genes (Fig. 2A), and previously
established interactions were also predicted. For instance, comD and comDE mutants
shared growth characteristics with the cipB mutant, consistent with the fact that cipB is
directly regulated by phosphorylated ComE (16). In addition, there was a strong
connection between liaS and pspC mutant strains. LiaS is a sensor kinase of a two-
component system (TCS), and its cognate response regulator, LiaR, binds to the
promoter region of the pspC gene in vitro (28). As another example, our clustering
model indicated that a ΔcomS mutant shared phenotypic behaviors with ΔciaH, ΔpspC,
ΔliaS, and ΔrgpI mutants. In Fig. S3B, we plotted the ratios of the relative growth rates
of mutants in BHI broth versus those in BHI broth with 2 ␮M CSP. The strongest
phenotypes were observed for strains with mutations in genes involved in the early
stages of the competence cascade, whereas the behaviors of strains carrying mutations
in genes for the latter phases of competence showed less profound phenotypes.
The primary function of the CSP-ComDE circuit of S. mutans appears to be to control
the production of bacteriocins. However, the expression and activity of bacteriocins,
particularly CipB (6), have also been linked to comX expression via ComRS, although the
mechanism is undefined. Therefore, we chose to examine cipB expression and bacte-
riocin production to begin to probe why certain mutants exhibited competence
defects. To evaluate bacteriocin production by the panel of mutants identified above,

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FIG 2 Discovery of mutant strains with aberrant responses to CSP. (A) Phenotypic clustering of growth characteristics in the presence of 2 ␮M CSP (see Text
S1 in supplemental material for criteria and analytical methods). Links between genes (lines) are based on correlations between phenotypes in the presence

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of 2 ␮M CSP. Genes with increased numbers of connections are highlighted by red circles. Additionally, values of linkages (denoted by the scale bar) were
determined from the normalized distance matrix discussed in Text S1. (B) Deferred antagonism assay to monitor bacteriocin production by wild-type S. mutans,
S. mutans ΔcomDE, and various mutant strains using Streptococcus gordonii DL1 as the indicator strain. Clear zones produced around the stabbed S. mutans
colonies indicate lysis of the indicator strain (S. gordonii). (C) PcipB-gfp activity was measured in defined medium (FMC) after the addition of 200 nM CSP.
Fluorescence of the PcipB-gfp reporter was measured every 30 min for 16 h using a Synergy microplate reader. Natural PcipB-gfp expression occurs in the wild-type
strain during the exponential growth phase. Raw fluorescence readings are plotted without any subtraction for background fluorescence. (D) Transformation
efficiencies of wild-type and mutant strains. Cells were grown to an OD600 of 0.1, and 200 nM CSP along with 100 ng pIB184 (Ermr) were added; cells were
incubated for 2.5 h and then plated on BHI-erythromycin agar and BHI agar. Bars represent the mean values derived from 3 or 4 biological replicates, with error
bars denoting the standard errors. Two-sample t tests were performed to determine statistical significance. *, P ⬍ 0.05; **, P ⬍ 0.01, ***, P ⬍ 0.001; ns, not
significant.

S. mutans UA159 and the deletion replacement mutants were stabbed into BHI agar,
and then a soft agar overlay containing either of two sensitive oral commensal
microorganisms, Streptococcus gordonii or Streptococcus sanguinis, was poured evenly
onto the plates. As deduced from the sizes of the zones of inhibition of growth of the
commensals by S. mutans, 11 mutants displayed statistically significant reductions in
inhibitory effects against S. gordonii and nine against S. sanguinis (Fig. 2B; see also Fig.
S4). In general, the mutant strains exhibited the most significant reductions in inhibition
of S. gordonii, as opposed to S. sanguinis. Additional studies will be needed to know if
this is related to the unequal impacts on expression of different bacteriocins and/or to
the sensitivities of the mutants to antagonistic factors produced by the commensal
streptococci. To investigate whether these mutants had reduced bacteriocin gene
expression, a PcipB-gfp reporter was introduced into each null strain, the strains were
incubated in the presence of CSP (200 nM), and GFP fluorescence measurements were
obtained in a microplate reader (Fig. 2C). PcipB-gfp activity was greatly reduced in the

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ΔpknB, ΔltaS, ΔrgpI, and ΔdltA mutants and moderately reduced in strains carrying
deletions of S. mutans 835 (SMu.835) and liaS.
To assess the transformation efficiency for the deletion mutants, we incubated
early-exponential-phase cells growing in BHI broth with CSP (200 nM) and 100 ng of
plasmid pIB184. For cells treated with CSP, 19 mutants exhibited reduced transforma-
tion efficiencies, ranging from 6-fold to over 10,000-fold, compared to those of S.
mutans UA159 (Fig. 2D). The deletion mutants with the lowest transformation efficien-
cies under the conditions tested were ΔpknB, ΔdivIB, ΔirvR, and ΔSMu.1913 to SMu.1904
(Δ1913-1904) mutants.
Identifying genes that are involved in XIP-mediated genetic competence. A
major question that remains unresolved is how the treatment of cells with CSP results
in enhanced transformability and induction of comX expression. CSP will not activate
competence in a comX or comR mutant, and CipB may play some role in connecting the
pathways, but details of how the CSP-ComDE circuit activates the ComRS pathway are
lacking. We hypothesized that, among the mutants, there may be isolates that dis-
played diminished competence because they could no longer couple CSP signaling to
the ComRS pathway. Thus, we investigated whether any of the mutants had alterations
in XIP-induced genetic competence, which requires the ComR circuit and is most
efficient when the comS positive feedback loop is intact (4). We began by monitoring
the growth of the mutant strains in the presence of 2 ␮M XIP in chemically defined
medium (FMC). The most substantial changes in the sensitivity to inhibition of growth
by XIP were seen with the ΔirvR, ΔltaS, ΔrgpI, and Δ835 deletion mutants, but another
9 strains exhibited an increased final OD600 compared with that of the wild-type strain
(Fig. 3A). Certain mutants, namely, ΔsufC, Δ462, ΔpknB, and ΔdltA mutants, grew more
poorly than the wild-type strain in FMC alone (see Fig. S5). When cultured in CDM, a
chemically defined medium with a composition different from that of FMC, only the
Δ462 mutant displayed a noticeable growth defect, although not as substantial as the
defect seen in FMC (Fig. S5). The principal albeit not the only difference between
the media is that CDM has substantially more phosphate and, as a result, a better buffer
capacity than FMC.
When XIP was used to induce competence in S. mutans growing in FMC, 14 of the
deletion mutants exhibited reduced transformation efficiencies, ranging from 5-fold to

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8,500-fold, compared to that of S. mutans UA159 (Fig. 3B). The mutants that displayed
the greatest decreases in transformation efficiencies carried deletions of sufC, SMu.462,
divIB, ltaS, rgpI, irvR, and dltA. Of these, only the ΔdivIB and ΔirvR mutants exhibited
similar reductions in transformation efficiency when treated with CSP in BHI broth. The
total numbers of null mutants with transformation defects in CSP only, XIP only, and in
both were 6, 1, and 13, respectively. Thus, mutants were identified that appear to have
lost the ability to couple the CSP signal to the induction of competence.
As shown in Fig. 1, comX is naturally expressed when cells are grown as colonies on
the surfaces of BHI agar plates, without the addition of CSP or XIP. Interestingly, comX
is also induced in CDM without the addition of exogenous XIP; this does not occur in
BHI broth or FMC, likely due to the suppressive effects of lower pH (11). The ability of
cells to “autoactivate” comX in CDM enabled us to quickly and quantitatively screen
reporter strains in a microtiter-based assay, measuring GFP fluorescence and cell
growth simultaneously for 16 h. The loss of ltaS, rgpI, irvR, and dltA significantly reduced
PcomX-gfp activity with or without the addition of synthetic XIP (Fig. 3). Strong reduc-
tions of PcomX-gfp activity in the absence of exogenously supplied XIP were evident in
strains carrying deletions of pknB, liaS, ppiB (also named SMu.488 or SMu.1631), pnpB,
pspC, SMu.835, or SMu.1781, but the induction of comX could be fully, or at least
partially, restored in these mutants by the addition of synthetic XIP (Fig. 3). Interest-
ingly, aberrant expression from PcomX was observed in the Δ462 and Δ695 mutants only
when exogenous XIP was added to the CDM, but these two mutants behaved like the
wild type in the absence of supplemental XIP (Fig. 3D). The Δ1193, Δ1388, and
Δ1913-1904 mutant strains did not behave differently than the wild type in terms of

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FIG 3 Newly identified competence mutants also exhibit comRS-dependent phenotypes. (A) Growth of S. mutans UA159, S. mutans ΔcomR, and competence

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mutant strains in the presence of 2 ␮M XIP monitored at an OD600. Data points are averages from triplicate samples with the standard errors shown for UA159
and ΔcomR strains. The maximal OD600 achieved by the competence mutants showed considerable variability (inset), with the wild type and a comR mutant
as reference strains. (B) Comparison of transformation efficiencies between wild-type S. mutans, S. mutans ΔcomR, and newly identified competence mutants.
Strains were grown in FMC using 200 nM XIP to activate transformation and 100 ng of purified pBGS plasmid (Spr) to identify mutants no longer able to take
up DNA at the same efficiency as the wild type. (C and D) Strains harboring a PcomX-gfp reporter plasmid were grown in CDM (C) and CDM containing 200 nM
exogenous XIP (D). Maximal green fluorescence relative to that of the wild-type strain is shown. Bars indicate means from three biological replicates, with error
bars denoting standard errors from the means. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001; ns, not significant.

PcomX-gfp activity in CDM. Importantly, these results demonstrate that certain mutations
may influence both XIP production and the sensing of exogenous XIP, whereas others
could be identified that could not “autoactivate” but were capable of sensing exoge-
nously supplied XIP. Since it is presently not known how XIP arises from ComS, further
analysis of these mutants could provide crucial new insights into competence regula-
tion.
Analysis of mutant behaviors at the single-cell level. The 20 mutants described
above displayed growth defects in the presence of CSP and XIP, as well as diminished
transformation efficiencies compared with that of the wild-type strain. Of relevance
here, in planktonic cultures growing batchwise, the induction of comX expression in S.
mutans UA159 by CSP in complex medium is bimodal (i.e., only a subpopulation of cells
activates comX transcription), whereas comX transcription is activated unimodally when
cells are exposed to XIP in a chemically defined medium lacking peptides (4). Also of
note, XIP-dependent activation of comX becomes bimodal in mature biofilms cultivated
in vitro (15). Therefore, we sought to determine whether the changes in the growth and
competence behaviors of the mutants were due to uniform changes across the

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FIG 4 Diverse comX expression phenotypes of competence-defective mutants revealed by fluorescence microscopy. A
selection of mutant strains that displayed CSP and/or XIP growth or transformation phenotypes were explored in greater detail
using a PcomX-gfp reporter system and fluorescence microscopy. Strains were grown in rich medium (RM) with 200 nM CSP or
defined medium (DM; FMC) with 200 nM XIP and incubated for 2.5 h before images were collected by fluorescence microscopy.
Overlays of the green and bright-field channels are shown. Deletion of pknB leads to a significant growth defect in FMC, and
so this condition was omitted. Images are representative of three independent experiments and were taken at ⫻63
magnification.

population or to changes in the behaviors of a subpopulation of cells. We examined the

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activation of comX expression at the single-cell level in various mutants in response to
CSP or XIP. In the ΔpknB, ΔliaS, ΔppiB, and ΔpnpB mutants, substantial defects in
transformation efficiency were observed in BHI broth with CSP (BHI-CSP) and FMC with
XIP (FMC-XIP) (Fig. 2D and 3B). Additionally, Tn insertions in these mutants were
overrepresented after passage in BHI-CSP (Table S2). The pknB gene encodes a serine/
threonine protein kinase, liaS encodes a histidine kinase of a TCS, ppiB encodes a
predicted protein with an N-terminal haloacid dehydrogenase (HAD)-like hydrolase
domain and a C-terminal peptidyl-prolyl cis-trans isomerase domain, and pnpB is
predicted to encode a polynucleotide phosphorylase involved in mRNA turnover and
processing. The pspC gene, encoding a putative phage shock domain and a stress-
responsive transcriptional regulator domain, was also included in the analysis as it is
regulated by LiaR (29). With respect to PcomX-gfp activity, the ΔpknB mutant was
noteworthy in that a substantially decreased proportion of the cells induced comX
expression when exposed to 200 nM CSP (Fig. 4A) compared with that observed in the
wild-type genetic background, and lower proportions of cells in the ΔliaS, ΔppiB, ΔpnpB,
and ΔpspC mutant strains were GFP positive than in the wild type (Fig. 4A). Although
ΔliaS, ΔppiB, ΔpnpB, and ΔpspC cells expressed comX unimodally when supplied with
exogenous XIP, the average fluorescence intensity of these mutants was lower than
that in the S. mutans UA159 genetic background (Fig. 4A).
We also focused on the ltaS, rgpI, and SMu.835 genes. The ltaS gene is predicted to
encode a phosphoglycerol transferase involved in envelope biogenesis. The rgpI gene
product shares homology with glycosyltransferase 2-like proteins, which are often
involved in cell wall biogenesis, and has been shown to affect rhamnose-glucose

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Shields et al. Journal of Bacteriology

polysaccharide synthesis by S. mutans (30). Lastly, SMu.835 is annotated as a possible

membrane protein that contains low-complexity repetitive sequences. Although the
deletion of these genes reduced the transformation efficiency and decreased the
sensitivity to growth inhibition relative to those in the wild type in BHI broth containing
CSP, more profound phenotypes were noted in cells growing in FMC and treated with
XIP (Fig. 2 and 3). In particular, the ΔltaS and ΔrgpI strains were essentially nontrans-
formable in FMC containing XIP. The deletion of these genes led to no visible induction
of PcomX-gfp activity when cells were treated with XIP in FMC compared with that in the
wild type (Fig. 4B). The mutant lacking SMu.835 still activated PcomX-gfp in response to
XIP in FMC, but the level of expression was significantly reduced compared with that
of the UA159 genetic background, and, importantly, this strain displayed evidence of
bimodality when treated with XIP in FMC (Fig. 4B).
Four additional mutants (ΔdivIB, ΔirvR, Δ1781, and Δ1913-1904 mutants) displayed
interesting PcomX-gfp single-cell phenotypes. The irvR gene encodes a LexA-like protein
that is required for CSP-induced transformation of S. mutans (24). In accordance with
what was previously reported (24), comX expression in the irvR mutant was not
adversely affected in cells treated with CSP. However, the loss of IrvR led to decreased
PcomX-gfp activity in cells growing in FMC and treated with XIP (Fig. 4C). The function
of the SMu.1781 product is unknown, although it shares homology with other unchar-
acterized proteins associated with RNase G and RNase E (RNase Y in streptococci). Far
fewer GFP-positive cells were visualized in the SMu.1781 mutant in the presence of CSP
than in the strain with the wild-type genetic background, but the mutant had a
response to XIP that mirrored that of the wild type (Fig. 4C). Several transposon
insertions leading to decreased PcomX-lacZ activity were identified as insertions in genes
and noncoding regions downstream of cipB (SMu.1914c) (Table S1). A strain carrying a
deletion of the SMu.1904 to SMu.1913 genes showed greatly reduced proportions of
PcomX-gfp-positive cells in the presence of CSP, but the deletion of this cluster of genes
had no impact on comX expression when cells were exposed to exogenous XIP (Fig. 4C),
providing further evidence that there are gene products that appear essential for
coupling the CSP-ComDE circuit with the ComRS-XIP pathway.
The divIB gene is predicted to encode a protein that participates in the regulation
of cell division, and potential divIB homologues have been studied in Streptococcus
pneumoniae (31) and Escherichia coli (divIB is designated ftsQ in Gram-negative bacteria)

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(32). Tn insertions in divIB decreased PcomX-lacZ activity (Table S1) and led to improved
growth in BHI broth containing CSP (Table S2). The divIB mutant was virtually non-
transformable, either in BHI broth with CSP or in FMC with XIP. Interestingly, the
deletion of divIB did not result in improved growth in the presence of XIP in FMC (Fig.
2 and 3). Compared to that in S. mutans UA159, the proportion of cells that exhibited
PcomX-gfp activity in the divIB mutant was reduced when cultured in BHI broth con-
taining CSP. Only modest reductions in comX expression were seen in FMC supple-
mented with XIP, and PcomX-gfp activity remained unimodal (Fig. 5A). Intriguingly, this
mutant produces bacteriocins at a level similar to that of the wild-type S. mutans when
treated with CSP, indicating the ComDE signaling pathway is fully intact (Fig. 5B). We
observed noticeable cell division defects in this mutant, evident from the increased
chaining and a subgroup of cells that appeared to bulge (Fig. 5C). As in Bacillus subtilis
(33), cells carrying a deletion of divIB displayed thermosensitivity, with no growth
evident at 42°C even after 20 h of incubation (Fig. 5D). We sought to compare the
above phenotypes with those of Streptococcus gordonii, an organism with a true
comCDE system controlling genetic competence (ComE regulates comX expression
directly). S. gordonii ΔdivIB shared similar cell division and thermosensitive phenotypes
with S. mutans, but divIB deletion had no impact on the transformation efficiency in S.
gordonii (Fig. 5E to G).

DISCUSSION
In this study, we describe the first application of transposon sequencing to S.
mutans, employing Tn-seq and a classical genetic screen to identify genes that influ-

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FIG 5 DivIB is required for comX activation in S. mutans in response to CSP. (A) PcomX-gfp activity was visualized by fluorescence microscopy when 200 nM
exogenous CSP or XIP was added to rich medium (RM; BHI broth) or defined medium (DM; FMC), respectively. Several phenotypes of the S. mutans ΔdivIB strain
were also assessed (B to D). (B) Bacteriocin production (using a Streptococcus sanguinis indicator strain) was not altered in the ΔdivIB mutant compared with
that in the S. mutans UA159. (C) The ΔdivIB mutant forms longer chains than S. mutans UA159, with occasional cell bulging (arrowheads). Cells were stained
with the LIVE/DEAD BacLight bacterial viability kit according to the manufacturer’s instructions and visualized with fluorescence microscopy (63⫻ objective).
(D) The ΔdivIB mutant (red line) grew poorly in BHI broth at 42°C. Growth was measured using a Bioscreen C automated growth curve analysis system. (E to
G) The impacts of a divIB deletion on the following S. gordonii phenotypes were also characterized: growth at 42°C in BHI broth (E), cell chaining using a similar
assay to that used for S. mutans (F), and transformation efficiency (G). For the transformation efficiency assay, 200 nM S. gordonii CSP (DVRSNKIRLWWENIFFNKK)
or scrambled CSP (same amino acid composition, different sequence [DKRFKWWILKVFNSNEINR]) was added to cultures at an OD600 of 0.1 in BHI broth along
with 100 ng of purified pDL278 plasmid (Spr). After incubating for 2.5 h, samples were plated on BHI-Sp agar or BHI agar, and plates were incubated for 2 days
and the transformation efficiency was calculated.

ence genetic competence. Tn-seq enabled us to assess in a fairly comprehensive

manner the relative contribution of gene products to the responses to competence
peptides. This systematic approach, confirmed using targeted gene knockouts, has led
to the discovery of 20 additional genes that have substantial impacts on the regulation
of competence and competence-related phenotypes. We further expanded our analysis
by exploring comX expression dynamics at the single-cell level. Since many of the genes
that we discovered have not been studied in the context of genetic competence, these
investigations contribute in novel and important ways to the overall understanding of
cell-cell communication in S. mutans.

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Shields et al. Journal of Bacteriology

Overview of the transposon screens. The addition of CSP or XIP to growth media
causes global changes in gene expression (16, 18, 19). However, the comprehensive
mutational analysis performed here indicates that the total number of genes that are
required for optimal comX expression is relatively small, approximately 35 genes. By
comparison, sporulation in B. subtilis requires ⬎170 of the approximately 4,100 genes
in the typical B. subtilis genome (21). The relatively small number of genes affecting
optimal comX transcription and ComX levels may reflect the evolutionary pressures that
also severely restricted the conditions under which S. mutans is able to activate comX
transcription and progress to the competent state. For example, even in the presence
of sufficient exogenously supplied CSP or XIP, the activation of comX occurs only within
a very narrow pH range; for CSP this is between 6.7 and 7.7 and for XIP the range is 6.3
to 7.9, although the basis for this narrow window is not yet defined (11). Notably,
genetic competence in S. mutans appears to be more intimately intertwined with
physiologic homeostasis and stress tolerance than in many other bacteria that have the
capability to acquire natural genetic competence. In fact, regulators of competence in
S. mutans have potent impacts on biofilm formation (34), the stringent response (8, 10),
oxidative stress responses (13, 35), and persistence (36). Thus, a long-term goal of this
research is to understand how competence and other essential biological processes are
coordinated and whether this coordination evolved to enhance the persistence and
virulence of S. mutans. The studies presented here have identified new genes in the
competence circuit and reveal possible pathways for the integration of the ComRS and
ComDE pathways, and many of the genes identified are likely to influence stress
tolerance and play essential roles in other homeostatic processes. Future studies will be
oriented toward determining the extent to which these newly identified gene products
integrate stress and competence. Likewise, analyses of these mutants should clarify
how activation of bacteriocin gene expression influences cellular physiology in a way
that modifies the development of competence.
While the quality of the transposon library and the results obtained were such that
the likelihood was high that we could identify all the genes participating in the
processes of interest, there are probably additional gene products and possibly non-
coding regions of the genome that were not identified by the mutant screen or by
Tn-seq. While genes that impact comX expression on solid surfaces (LacZ screen on

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agar) and in liquid media in a microaerophilic environment (Tn-seq) were isolated, it is
also easy to envision that additional genes could participate in the modulation of comX
expression in the presence of higher concentrations of oxygen, on carbohydrate
sources other than glucose, or perhaps in response to other known factors that
influence genetic competence (12, 13, 35). Tn-seq is somewhat limited in its ability to
enable the discovery of small genes or small RNAs, where transposon density may not
be sufficiently high to detect during screening. Likewise, genes that encode products
with impacts on comX expression that can be fulfilled by other gene products (redun-
dancy) would probably not have been identified in our screens. However, now that this
technology has been established in S. mutans, it is possible to begin to probe how
screening in different environmental conditions or screening strains carrying certain
preexisting mutations might influence the ability to identify other genes that may
contribute to competence.
Cell division and cell envelope integrity shape competence decisions. Interest-
ingly, the majority of the genes that we identified that had no previous links to comX
regulation are those that function to control cell division or cell wall metabolism.
Single-cell comX-GFP fluorescence microscopy studies facilitated the discovery of genes
that perturb the ability of the ComDE TCS system to relay information to the ComRS
pathway or genes that act at the ComRS regulatory level. The cell division protein
DivIB is a notable example of a gene that specifically reduces the proportion of cells
activating comX in response to CSP without having a profound effect on the XIP
pathway. The machinery involved in S. mutans cell division is not well studied, but in
S. pneumoniae, DivIB has a role in septal peptidoglycan synthesis during the late stages

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Novel comX Regulators in Streptococcus mutans Journal of Bacteriology

of cell division (31). DivIB interacts with two other cell division proteins, FtsL and DivIC.
In S. pneumoniae, DivIB is important for stabilizing FtsL, with both FtsL and DivIC being
essential; mutations in ftsL or divIC appear lethal in S. pneumoniae, and we were not
able to obtain transformants when we attempted to knockout the ftsL or divIC genes
(data not shown) (31). Interestingly, the mutation of divIB in S. gordonii had no impact
on transformation efficiency, despite the fact that loss of DivIB in this commensal
altered cell morphology and thermosensitivity in a manner similar to that observed in
the divIB mutant of S. mutans, reinforcing the idea that factors that control competence
development are in some cases species specific. We are presently contrasting other
behaviors of the S. mutans and S. gordonii divIB mutants to understand why DivIB is
integrated with competence in S. mutans, with one goal being to unmask the molecular
link(s) between the ComDE and ComRS networks in S. mutans.
Many of the gene products identified as influencing comX expression participate in
cell envelope biogenesis or in monitoring the integrity of the cell envelope. Among
these were proteins with somewhat well-defined roles, including signal transduction
involved in cell division (PknB) (23), cell envelope stress regulation (LiaS and PspC) (28,
29), and D-alanylation of teichoic acids (DltA) (37, 38). Also identified were genes for a
glycosyltransferase enzyme (RgpI) involved in side chain formation during rhamnose-
glucose polysaccharide synthesis (30), for a phosphoglycerol transferase (LtaS), and for
a protein with a putative peptidoglycan-binding domain (SMu.695). The dltA, rgpI, and
ltaS mutants had severe defects in XIP-dependent activation of comX but showed
less-severe impairment in their ability to activate comX when provided with CSP; an
unusual finding considering that the CSP circuit appears to work through ComR. One
possible explanation for these observations is that the mechanism by which these gene
products affect XIP signaling is related to the mutants having alterations in the cell
envelope that compromise peptide transport and thus internalization of XIP, perhaps
by influencing the translocation or membrane insertion of transport proteins (39, 40).
However, it is possible that the enzymes that alter cell wall characteristics (DltA, RgpI,
LtaS, and SMu.695) have the observed phenotypes because they impact cell
membrane-associated sensing and signal transduction systems like LiaS and PknB.
Homologues of PknB in other bacterial species sense cell wall precursors required
during cell division (41, 42). PknB homologues also regulate the VicRK two-component

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system (also known as WalRK) by phosphorylating the response regulator VicR (43).
VicRK positively regulates cell wall metabolism in S. mutans (44). In agreement with
the cross talk between these signaling systems, the deletion of pknB in S. mutans
caused severe cell morphology defects, as determined by transmission electron mi-
croscopy, and the PknB regulon was shown to overlap that of VicK (23). The liaFSR
two-component system of S. mutans also has a regulatory role in cell envelope
biogenesis and is important for sensing cell envelope stress (28). Therefore, the
monitoring of and responding to changes in the cell envelope by LiaS and PknB could
play critical regulatory roles in the reception of competence signal inputs and/or the
progression to the competent state. It must also be considered that the mutants simply
have a diminished capacity to take up DNA, since much of the machinery required for
the active transport of single-stranded DNA (ssDNA) is membrane associated. Such
overlaps in the regulatory inputs could have evolved to enable cells to integrate
environmental stimuli, including the reductions in pH during carbohydrate metabolism,
the production of cationic bacteriocins (known to be regulated by comDE), and the
modifications induced by the competence-induced murein hydrolase LytF (regulated
by ComX), into the control of the activation of, or the exit from, the competent state.
Novel components of comX regulation. We also identified genes that have an
impact on comX expression with functions that are more difficult to predict, including
SMu.1781 and several genes that are in the cipB operon. Mutations in either of these
regions greatly diminished comX expression and increased growth rates when exoge-
nous CSP was added to BHI broth. SMu.1781 has a domain that is found in proteins
associated with the endoribonuclease enzymes RNase G and RNase E (RNase Y in

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Shields et al. Journal of Bacteriology

Gram-positive bacteria). If the predicted function of the product of SMu.1781 is

accurate, it may modulate the stability of RNAs in S. mutans. In Gram-positive bacteria,
the protein complex responsible for RNA decay (known as the RNA degradosome) has
not been well studied, but the core components are RNase Y, RNase J1, RNase J2, and
polynucleotide phosphorylase (PNPase [pnpA]), along with enolase, phosphofructoki-
nase, and a DEAD box helicase (45). Notably, a PNPase (pnpB, orthologous to pnpA) and
a DEAD-like helicase (SMu.1388) were also identified in our screens and induced
significant competence defects when mutated. It is unknown whether SMu.1781, pnpB,
and SMu.1388 interact with one another and/or the RNA degradosome in S. mutans.
However, RNA stability could be an intriguing avenue for genetic competence research
for two reasons. First, it is possible that interactions between glycolytic enzymes or
intermediates and the RNA degradation machinery explain the finding that the carbo-
hydrate source can affect comX expression in S. mutans (12, 46). Additionally, RNA
stability is a neglected biological process that may be an important aspect of CSP-
mediated bimodal comX expression, fine-tuning of ComX levels, or exiting the compe-
tent state, perhaps by influencing the stability of comRS or comX transcripts.
Concluding remarks. This study provides candidate interactions which, with de-
tailed investigations, could link competence and stress pathways with the regulation of
cell division, the monitoring of envelope integrity, and signal transduction. By validat-
ing the mariner-based approach in S. mutans, we demonstrate that Tn-seq can now be
applied as a powerful tool for discovering genes that are critical for other key functions,
such as the colonization and persistence of this organism, in the human oral micro-
biome.

MATERIALS AND METHODS

Strains and growth conditions. Streptococcus mutans strains were cultured from single colonies in
BHI broth (Difco). Unless otherwise stated (here or in Text S1 in the supplemental material), S. mutans was
routinely cultured at 37°C in a 5% CO2 aerobic atmosphere. Escherichia coli strains were routinely cultured
in LB broth (10 g/liter tryptone, 5 g/liter yeast extract, and 5 g/liter NaCl) at 37°C with aeration. Antibiotics
were added to growth media at the following concentrations: spectinomycin, 1.0 mg/ml for S. mutans
and 50 ␮g/ml for E. coli; kanamycin, 1.0 mg/ml for S. mutans, 500 ␮g/ml for S. gordonii, and 50 ␮g/ml for
E. coli; ampicillin, 100 ␮g/ml for E. coli. Lists of strains and plasmids (Table S3) and oligonucleotide primers
(Table S4) can be found in the supplemental material.

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Transposon library construction. An in vitro transposon mutagenesis protocol was utilized to
randomly mutagenize purified chromosomal DNA from S. mutans UA159 (20). Briefly, MarC9 transposase
(47, 48) was purified from E. coli pMalC9 (see Text S1). MarC9, S. mutans genomic DNA, and pMagellan6
plasmid, harboring the mariner Himar1 minitransposon derivative magellan6, were combined and
incubated for 1 h at 30°C. After transposon junctions were repaired, the fragments of genomic DNA
containing magellan6 insertions were transformed into competent S. mutans. Transposon mutants were
selected on BHI agar containing relevant antibiotics after 48 h of incubation. Colonies were removed
from the surface of the agar, resuspended in BHI broth, and centrifuged at 3,500 ⫻ g for 10 min at 4°C
and then resuspended in 5 ml BHI broth with glycerol (25% glycerol) and aliquoted in 250-␮l volumes
for long-term storage at ⫺80°C.
Plate-based screen for mutants that fail to activate comX transcription. Glycerol stocks of the
library were thawed on ice and then diluted 1:50 in fresh BHI broth. Samples were incubated until the
OD600 was equal to 0.1 and then were diluted to 1:105. One hundred-microliter aliquots were spread on
TYG–X-Gal agar (2 g/liter glucose, 10 g/liter tryptone, 5 g/liter yeast extract, 3 g/liter K2HPO4, 15 g/liter
agarose, 50 ␮g/ml X-Gal). Plates were incubated for 2 days at 37°C in a 5% CO2 incubator. White colonies
were picked and cultured overnight in BHI broth, and glycerol stocks were prepared and stored at ⫺80°C.
Strains with apparently low PcomX-lacZ activity were then screened for genetic competence defects, and
strains with phenotypes different from that of S. mutans UA159 were sequenced with arbitrary PCR to
determine the sites of transposon insertion (for details on the method, see Text S1).
Tn-seq. An aliquot of the transposon library pool was thawed and diluted 1:100 in 50 ml BHI broth
or in 50 ml BHI broth plus 2 ␮M CSP. Samples were cultured to an OD600 of 1.0 and then transferred to
fresh BHI broth or BHI-CSP for a total of 5 passages. The total viable counts before and after incubation
were enumerated by plating on BHI agar. After each passage, a 10-ml sample was harvested for genomic
DNA preparation. Genomic DNA was extracted using the MasterPure Gram Positive DNA purification kit
(Epicentre Biotechnologies) in accordance with the supplier’s instructions, with minor modifications (49).
Genomic DNA restriction digestion, Illumina adapter ligation, library PCR amplification, and Illumina
sequencing were performed as previously described (20, 22). Libraries were sequenced at the NextGen
DNA sequencing core facility at the University of Florida on a NextSeq 500 (Illumina) with a 75-bp
single-end sequencing read (1 ⫻ 50 cycles). See Text S1 for Tn-seq bioinformatics analyses.

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Novel comX Regulators in Streptococcus mutans
Phenotypic assays and clustering analysis. Text S1 details the criteria and methodologies em-
ployed to validate putative genetic competence-regulating genes, including transformation assays,
growth studies, and single-cell PcomX-gfp reporter investigations.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/JB
.00508-17.
SUPPLEMENTAL FILE 1, PDF file, 13.0 MB.
ACKNOWLEDGMENTS
We thank Andrew Camilli for providing the transposon sequencing plasmids pMalC9
and pMagellan6. We also thank Sara Palmer for helpful discussions on bioinformatics
and Jeong Nam Kim for technical advice.
This study was supported by NIDCR R01 grants DE13239 and DE23339.
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