Competence shut-off by intracellular pheromone degradation in salivarius streptococci.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation.

In the process of natural transformation bacteria import extracellular DNA molecules for integration into their genome. One strand of the incoming DNA molecule is degraded, whereas the remaining strand is transported across the cytoplasmic membrane. The DNA transport channel is provided by the protein ComEC. Many ComEC proteins have an extracellular C-terminal domain (CTD) with homology to the metallo-ß-lactamase fold. Here we show that this CTD binds Mn2+ ions and exhibits Mn2+ -dependent phosphodiesterase and nuclease activities. Inactivation of the enzymatic activity of the CTD severely inhibits natural transformation in Bacillus subtilis. These data suggest that the ComEC CTD is a nuclease responsible for degrading the nontransforming DNA strand during natural transformation and that this process is important for efficient DNA import.

One-Step Bacterial Artificial Chromosome (BAC) Modification: Cloning of the A Homology Arm into Reporter-Shuttle Vector.

In this protocol, the homology arm sequence for one-step bacterial artificial chromosome (BAC) modification is introduced by ligation into the shuttle vector carrying the reporter sequence to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector. To provide further assurance that the homology box has been successfully integrated into the plasmid, the enzyme digestion pattern of the modified plasmid is compared with that of the unmodified plasmid.

Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics.

Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the development of competence, a specialized physiological state during which proteins involved in DNA uptake and chromosomal integration are produced. In Streptococcus pneumoniae, competence is transient. It is controlled by a secreted peptide pheromone, the competence-stimulating peptide (CSP) that triggers the sequential transcription of two sets of genes termed early and late competence genes, respectively. Here, we used a microfluidic system with fluorescence microscopy to monitor pneumococcal competence development and transformation, in live cells at the single cell level. We present the conditions to grow this microaerophilic bacterium under continuous flow, with a similar doubling time as in batch liquid culture. We show that perfusion of CSP in the microfluidic chamber results in the same reduction of the growth rate of individual cells as observed in competent pneumococcal cultures. We also describe newly designed fluorescent reporters to distinguish the expression of competence genes with temporally distinct expression profiles. Finally, we exploit the microfluidic technology to inject both CSP and transforming DNA in the microfluidic channels and perform near real time-tracking of transformation in live cells. We show that this approach is well suited to investigating the onset of pneumococcal competence together with the appearance and the fate of transformants in individual cells.

Diversity in Natural Transformation Frequencies and Regulation across Vibrio Species.

In Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in Vibrio campbellii strains DS40M4 and NBRC 15631 enables high natural transformation frequencies. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4 or Vibrio parahaemolyticus in contrast to the established regulatory pathway in Vibrio cholerae in which quorum sensing is required to activate the competence regulator QstR. Similar to V. cholerae, expression of both QstR and TfoX is necessary for transformation in DS40M4. There is a wide disparity in transformation frequencies among even closely related Vibrio strains, with V. vulnificus having the lowest functional transformation frequency. Ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus To explore differences in competence regulation, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. We find that transformation capability cannot necessarily be predicted by the level of gene conservation but rather correlates with competence gene expression following TfoX induction. Thus, we have uncovered notable species- and strain-level variations in the competence gene regulation pathway across the Vibrio genus.IMPORTANCE Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus.

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans.

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.

Gram-Positive Type IV Pili and Competence.

Type IV pili (T4P) are remarkable bacterial surface appendages that carry out a range of functions. Various types of T4P have been identified in bacteria and archaea, making them almost universal structures in prokaryotes. T4P are best characterized in Gram-negative bacteria, in which pilus biogenesis and T4P-mediated functions have been studied for decades. Recent advances in microbial whole-genome sequencing have provided ample evidence for the existence of T4P also in many Gram-positive species. However, comparatively little is known, and T4P in Gram-positive bacteria are just beginning to be dissected. So far, they have mainly been studied in Clostridium and Streptococcus spp. and are involved in diverse cellular processes such as adhesion, motility, and horizontal gene transfer. Here we summarize the current understanding of T4P in Gram-positive species and their functions, with particular focus on the type IV competence pilus produced by the human pathogen Streptococcus pneumoniae and its role in natural transformation.

Human fluids alter DNA-acquisition in Acinetobacter baumannii.

Transformation is one of the mechanisms of acquisition of foreign genetic material leading to the emergence of multidrug resistant (MDR) bacteria. Recently, human serum albumin (HSA) was shown to specifically increase transformation frequency in the nosocomial pathogen Acinetobacter baumannii. To further assess the relevance of HSA as a possible modulator of A. baumannii transformation in host-pathogen interactions, in this work we examined the effect of different human fluids. We observed a significant increase in transformation frequencies in the presence of pleural fluid, whole blood cells and liquid ascites, and to a lesser extent with urine. The observed effects correlate with both HSA and bacterial content found in the assayed patient fluids. Taken together, these results are in agreement with our previous findings that highlight HSA as a possible host signal with the ability to trigger natural transformation in A. baumannii.

Competence inhibition by the XrpA peptide encoded within the comX gene of Streptococcus mutans.

Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first described negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ΔxrpA strain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.

Impaired competence in flagellar mutants of Bacillus subtilis is connected to the regulatory network governed by DegU.

The competent state is a developmentally distinct phase, in which bacteria are able to take up and integrate exogenous DNA into their genome. Bacillus subtilis is one of the naturally competent bacterial species and the domesticated laboratory strain 168 is easily transformable. In this study, we report a reduced transformation frequency of B. subtilis mutants lacking functional and structural flagellar components. This includes hag, the gene encoding the flagellin protein forming the filament of the flagellum. We confirm that the observed decrease of the transformation frequency is due to reduced expression of competence genes, particularly of the main competence regulator gene comK. The impaired competence is due to an increase in the phosphorylated form of the response regulator DegU, which is involved in regulation of both flagellar motility and competence. Altogether, our study identified a close link between motility and natural competence in B. subtilis suggesting that hindrance in motility has great impact on differentiation of this bacterium not restricted only to the transition towards sessile growth stage.

A positive feedback loop mediated by Sigma X enhances expression of the streptococcal regulator ComR.

Natural transformation is used by bacteria to take up DNA from their surroundings and incorporate it into their genomes. Streptococci do so during a transient period of competence, triggered by pheromones that they produce, secrete and sense under conditions influenced by the environment. In Streptococcus mutans, Streptococcus suis, and species of the bovis, salivarius and pyogenic groups of streptococci, the pheromone XIP is sensed by the intra-cellular regulator ComR, that in turn activates the transcription of comS, encoding the XIP precursor, and of sigX, encoding the only known alternative sigma factor in streptococci. Although induction of comR during competence has been known for more than fifteen years, the mechanism regulating its expression remains unidentified. By a combination of directional RNA-sequencing, optimal competence conditions, stepwise deletions and marker-less genome editing, we found that SigX is the missing link in overproduction of ComR. In the absence of comR induction, both sigX expression and transformation were significantly reduced. Placing comR and comS transcripts under the control of different regulators so as to form two interlocked positive feedback circuits may enable S. mutans to fine-tune the kinetics and magnitude of the competence response according to their need.

Interbacterial predation as a strategy for DNA acquisition in naturally competent bacteria.

Natural competence enables bacteria to take up exogenous DNA. The evolutionary function of natural competence remains controversial, as imported DNA can act as a source of substrates or can be integrated into the genome. Exogenous homologous DNA can also be used for genome repair. In this Opinion article, we propose that predation of non-related neighbouring bacteria coupled with competence regulation might function as an active strategy for DNA acquisition. Competence-dependent kin-discriminated killing has been observed in the unrelated bacteria Vibrio cholerae and Streptococcus pneumoniae. Importantly, both the regulatory networks and the mode of action of neighbour predation differ between these organisms, with V. cholerae using a type VI secretion system and S. pneumoniae secreting bacteriocins. We argue that the forced release of DNA from killed bacteria and the transfer of non-clonal genetic material have important roles in bacterial evolution.

K+ modulates genetic competence and the stress regulon of Streptococcus mutans.

Potassium (K+) is the most abundant cation in dental plaque fluid. Previously, we reported the link between K+ transport via Trk2 in Streptococcus mutans and its two critical virulence attributes: acid tolerance and surface adhesion. Herein, we build further on the intimate link between K+ levels and S. mutans biology. High (>25 mM) versus low (≤5 mM) K+ concentrations in the growth medium affected conformational epitopes of cell surface-localized adhesin P1. At low K+, the expression of stress response elements gcrR and codY, cell-adhesion-associated genes such as spaP and metabolism-associated genes such as bglP was induced at stationary phase (P<0.05), suggesting that K+-mediated regulation is growth phase-dependent and stress-sensitive. Production of the newly discovered secretory protein encoded by SMU_63c was strongly dependent on the availability of K+ and growth phase. This protein is a newly discovered regulator of genetic competence and biofilm cell density. Thus, the influence of K+ on DNA transformation efficiency was also examined. Compared with 25 mM K+ concentration, the presence of low K+ reduced the transformation frequency by 100-fold. Genetic transformation was abolished in a strain lacking a Trk2 system under all K+ concentrations tested. Consistent with these findings, repression of competence-associated genes, comS and comX, was observed under low environmental K+ conditions and in the strain lacking Trk2. Taken together, these results highlight a pivotal role for environmental K+ as a regulatory cation that modulates stress responses and genetic transformation in S. mutans.

Overexpression of the fratricide immunity protein ComM leads to growth inhibition and morphological abnormalities in Streptococcus pneumoniae.

The important human pathogen Streptococcus pneumoniae is a naturally transformable species. When developing the competent state, it expresses proteins involved in DNA uptake, DNA processing and homologous recombination. In addition to the proteins required for the transformation process, competent pneumococci express proteins involved in a predatory DNA acquisition mechanism termed fratricide. This is a mechanism by which the competent pneumococci secrete a muralytic fratricin termed CbpD, which lyses susceptible sister cells or closely related streptococcal species. The released DNA can then be taken up by the competent pneumococci and integrated into their genomes. To avoid committing suicide, competent pneumococci produce an integral membrane protein, ComM, which protects them against CbpD by an unknown mechanism. In the present study, we show that overexpression of ComM results in growth inhibition and development of severe morphological abnormalities, such as cell elongation, misplacement of the septum and inhibition of septal cross-wall synthesis. The toxic effect of ComM is tolerated during competence because it is not allowed to accumulate in the competent cells. We provide evidence that an intra-membrane protease called RseP is involved in the process of controlling the ComM levels, since △rseP mutants produce higher amounts of ComM compared to wild-type cells. The data presented here indicate that ComM mediates immunity against CbpD by a mechanism that is detrimental to the pneumococcus if exaggerated.

Pneumococcal Competence Coordination Relies on a Cell-Contact Sensing Mechanism.

Bacteria have evolved various inducible genetic programs to face many types of stress that challenge their growth and survival. Competence is one such program. It enables genetic transformation, a major horizontal gene transfer process. Competence development in liquid cultures of Streptococcus pneumoniae is synchronized within the whole cell population. This collective behavior is known to depend on an exported signaling Competence Stimulating Peptide (CSP), whose action generates a positive feedback loop. However, it is unclear how this CSP-dependent population switch is coordinated. By monitoring spontaneous competence development in real time during growth of four distinct pneumococcal lineages, we have found that competence shift in the population relies on a self-activated cell fraction that arises via a growth time-dependent mechanism. We demonstrate that CSP remains bound to cells during this event, and conclude that the rate of competence development corresponds to the propagation of competence by contact between activated and quiescent cells. We validated this two-step cell-contact sensing mechanism by measuring competence development during co-cultivation of strains with altered capacity to produce or respond to CSP. Finally, we found that the membrane protein ComD retains the CSP, limiting its free diffusion in the medium. We propose that competence initiator cells originate stochastically in response to stress, to form a distinct subpopulation that then transmits the CSP by cell-cell contact.

Serum Albumin and Ca2+ Are Natural Competence Inducers in the Human Pathogen Acinetobacter baumannii.

The increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition. Acinetobacter baumannii is a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca(2+)or albumin. We show that comEA and pilQ are involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence in A. baumannii Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen.

A Low Dimensional Approximation For Competence In Bacillus Subtilis.

The behaviour of a high dimensional stochastic system described by a chemical master equation (CME) depends on many parameters, rendering explicit simulation an inefficient method for exploring the properties of such models. Capturing their behaviour by low-dimensional models makes analysis of system behaviour tractable. In this paper, we present low dimensional models for the noise-induced excitable dynamics in Bacillus subtilis, whereby a key protein ComK, which drives a complex chain of reactions leading to bacterial competence, gets expressed rapidly in large quantities (competent state) before subsiding to low levels of expression (vegetative state). These rapid reactions suggest the application of an adiabatic approximation of the dynamics of the regulatory model that, however, lead to competence durations that are incorrect by a factor of 2. We apply a modified version of an iterative functional procedure that faithfully approximates the time-course of the trajectories in terms of a two-dimensional model involving proteins ComK and ComS. Furthermore, in order to describe the bimodal bivariate marginal probability distribution obtained from the Gillespie simulations of the CME, we introduce a tunable multiplicative noise term in a two-dimensional Langevin model whose stationary state is described by the time-independent solution of the corresponding Fokker-Planck equation.

The Thermus thermophilus†comEA/comEC operon is associated with DNA binding and regulation of the DNA translocator and type IV pili.

Natural transformation systems and type IV pili are linked in many naturally competent bacteria. In the Gram-negative bacterium Thermus thermophilus, a leading model organism for studies of DNA transporters in thermophilic bacteria, seven competence proteins play a dual role in both systems, whereas two competence genes, comEA and comEC, are suggested to represent unique DNA translocator proteins. Here we show that the T. thermophilus ComEA protein binds dsDNA and is anchored in the inner membrane. comEA is co-transcribed with the flanking comEC gene, and transcription of this operon is upregulated by nutrient limitation and low temperature. To our surprise, a comEC mutant was impaired in piliation. We followed this observation and uncovered that the impaired piliation of the comEC mutant is due to a transcriptional downregulation of pilA4 and the pilN both playing a dual role in piliation and natural competence. Moreover, the comEC mutation resulted in a dramatic decrease in mRNA levels of the pseudopilin gene pilA1, which is unique for the DNA transporter. We conclude that ComEC modulates transcriptional regulation of type IV pili and DNA translocator components thereby mediating a response to extracellular parameters.

Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios.

TfoX (Sxy) and CRP are two important competence activators. The link between tfoX and CRP has been shown in H. influenza but lacking evidence of direct interaction. Recently a Sxy-dependent CRP (CRP-S) site autoregulating Sxy was reported in E. coli. Here, we show that the cAMP-CRP complex transcriptionally regulates tfoX expression through multiple canonical CRP (CRP-N) sites in Vibrios. This conclusion is supported by an analysis of the tfoX mRNA levels and tfoX transcriptional reporter fusions. The reduced expression of tfoX(VC) was restored by trans-complementation of crp in ∆crp and by exogenous cAMP in ∆cya. A promoter deletion analysis and the site-directed mutagenesis of the putative CRP-N sites revealed the presence of two functional CRP-N sites. The direct binding of cAMP-CRP to the tfoX(VC)promoter was demonstrated by EMSA assays. Additionally, the transcriptional start site (TSS) of tfoX(VF) in V. fluvialis was determined, and -10/-35 regions were predicted. Further comparison of the tfoX promoter in Vibrios revealed the existence of similar -10 motifs and putative CRP-N sites, indicating the conserved mechanism of CRP regulation on tfoX. Our study demonstrates the direct binding of the cAMP-CRP complex to tfoX promoter, and broadens the understanding of the molecular mechanism regulating tfoX in Vibrios.