A Genome-Wide CRISPR Interference Screen Reveals an StkP-Mediated Connection between Cell Wall Integrity and Competence in Streptococcus salivarius.

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a short time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococcus salivarius. We show that initiation of competence increases upon cell wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCE Survival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

Expanding natural transformation to improve beneficial lactic acid bacteria.

Nowadays, the growing human population exacerbates the need for sustainable resources. Inspiration and achievements in nutrient production or human/animal health might emanate from microorganisms and their adaptive strategies. Here, we exemplify the benefits of lactic acid bacteria (LAB) for numerous biotechnological applications and showcase their natural transformability as a fast and robust method to hereditarily influence their phenotype/traits in fundamental and applied research contexts. We described the biogenesis of the transformation machinery and we analyzed the genome of hundreds of LAB strains exploitable for human needs to predict their transformation capabilities. Finally, we provide a stepwise rational path to stimulate and optimize natural transformation with standard and synthetic biology techniques. A comprehensive understanding of the molecular mechanisms driving natural transformation will facilitate and accelerate the improvement of bacteria with properties that serve broad societal interests.

The CovRS Environmental Sensor Directly Controls the ComRS Signaling System To Orchestrate Competence Bimodality in Salivarius Streptococci.

In bacteria, phenotypic heterogeneity in an isogenic population compensates for the lack of genetic diversity and allows concomitant multiple survival strategies when choosing only one is too risky. This powerful tactic is exploited for competence development in streptococci where only a subset of the community triggers the pheromone signaling system ComR-ComS, resulting in a bimodal activation. However, the regulatory cascade and the underlying mechanisms of this puzzling behavior remained partially understood. Here, we show that CovRS, a well-described virulence regulatory system in pathogenic streptococci, directly controls the ComRS system to generate bimodality in the gut commensal Streptococcus salivarius and the closely related species Streptococcus thermophilus. Using single-cell analysis of fluorescent reporter strains together with regulatory mutants, we revealed that the intracellular concentration of ComR determines the proportion of competent cells in the population. We also showed that this bimodal activation requires a functional positive-feedback loop acting on ComS production, as well as its exportation and reinternalization via dedicated permeases. As the intracellular ComR concentration is critical in this process, we hypothesized that an environmental sensor could control its abundance. We systematically inactivated all two-component systems and identified CovRS as a direct repression system of comR expression. Notably, we showed that the system transduces its negative regulation through CovR binding to multiple sites in the comR promoter region. Since CovRS integrates environmental stimuli, we suggest that it is the missing piece of the puzzle that connects environmental conditions to (bimodal) competence activation in salivarius streptococci. IMPORTANCE Combining production of antibacterial compounds and uptake of DNA material released by dead cells, competence is one of the most efficient survival strategies in streptococci. Yet, this powerful tactic is energy consuming and reprograms the metabolism to such an extent that cell proliferation is transiently impaired. To circumvent this drawback, competence activation is restricted to a subpopulation, a process known as bimodality. In this work, we explored this phenomenon in salivarius streptococci and elucidated the molecular mechanisms governing cell fate. We also show that an environmental sensor controlling virulence in pathogenic streptococci is diverted to control competence in commensal streptococci. Together, those results showcase how bacteria can sense and transmit external stimuli to complex communication devices for fine-tuning collective behaviors.

Coevolution of the bacterial pheromone ComS and sensor ComR fine-tunes natural transformation in streptococci.

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semirational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodels a network of aromatic-aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor coevolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction.

Motorization of bacterial pili is key to generate traction forces to achieve cellular function. The Tad (or Type IVc) pilus from Caulobacter crescentus is a widespread motorized nanomachine crucial for bacterial survival, evolution and virulence. An unusual bifunctional ATPase motor drives Tad pilus retraction, which helps the bacteria to land on target surfaces. Here, we use a novel platform combining a fluorescence-based screening of piliated bacteria and atomic force microscopy (AFM) force-clamp spectroscopy, to monitor over time (30 s) the nanomechanics and dynamics of the Tad nanofilament retraction under a high constant tension (300 pN). We observe striking transient variations of force and height originating from two phenomena: active pilus retraction and passive hydrophobic interactions between the pilus and the hydrophobic substrate. That the Tad pilus is able to retract under high tensile loading - at a velocity of ∼150 nm s-1 - indicates that this nanomachine is stronger than previously anticipated. Our findings show that pilus retraction and hydrophobic interactions work together to mediate bacterial cell landing and surface adhesion. The motorized pilus retraction actively triggers the cell to approach the substrate. At short distances, passive hydrophobic interactions accelerate the approach phenomenon and promote strong cell-substrate adhesion. This mechanism could provide a strategy to save ATP-based energy by the retraction ATPase. Our force-clamp AFM methodology offers promise to decipher the physics of bacterial nanomotors with high sensitivity and temporal resolution.

AFM in cellular and molecular microbiology.

The unique capabilities of the atomic force microscope (AFM), including super-resolution imaging, piconewton force-sensitivity, nanomanipulation and ability to work under physiological conditions, have offered exciting avenues for cellular and molecular biology research. AFM imaging has helped unravel the fine architectures of microbial cell envelopes at the nanoscale, and how these are altered by antimicrobial treatment. Nanomechanical measurements have shed new light on the elasticity, tensile strength and turgor pressure of single cells. Single-molecule and single-cell force spectroscopy experiments have revealed the forces and dynamics of receptor-ligand interactions, the nanoscale distribution of receptors on the cell surface and the elasticity and adhesiveness of bacterial pili. Importantly, recent force spectroscopy studies have demonstrated that extremely stable bonds are formed between bacterial adhesins and their cognate ligands, originating from a catch bond behaviour allowing the pathogen to reinforce adhesion under shear or tensile stress. Here, we survey how the versatility of AFM has enabled addressing crucial questions in microbiology, with emphasis on bacterial pathogens. TAKE AWAYS: AFM topographic imaging unravels the ultrastructure of bacterial envelopes. Nanomechanical mapping shows what makes cell envelopes stiff and resistant to drugs. Force spectroscopy characterises the molecular forces in pathogen adhesion. Stretching pili reveals a wealth of mechanical and adhesive responses.

AFM Unravels the Unique Adhesion Properties of the Caulobacter Type IVc Pilus Nanomachine.

Bacterial pili are proteinaceous motorized nanomachines that play various functional roles including surface adherence, bacterial motion, and virulence. The surface-contact sensor type IVc (or Tad) pilus is widely distributed in both Gram-positive and Gram-negative bacteria. In Caulobacter crescentus, this nanofilament, though crucial for surface colonization, has never been thoroughly investigated at the molecular level. As Caulobacter assembles several surface appendages at specific stages of the cell cycle, we designed a fluorescence-based screen to selectively study single piliated cells and combined it with atomic force microscopy and genetic manipulation to quantify the nanoscale adhesion of the type IVc pilus to hydrophobic substrates. We demonstrate that this nanofilament exhibits high stickiness compared to the canonical type IVa/b pili, resulting mostly from multiple hydrophobic interactions along the fiber length, and that it features nanospring mechanical properties. Our findings may be helpful to better understand the structure-function relationship of bacterial pilus nanomachines.

How Microbes Use Force To Control Adhesion.

Microbial adhesion and biofilm formation are usually studied using molecular and cellular biology assays, optical and electron microscopy, or laminar flow chamber experiments. Today, atomic force microscopy (AFM) represents a valuable addition to these approaches, enabling the measurement of forces involved in microbial adhesion at the single-molecule level. In this minireview, we discuss recent discoveries made applying state-of-the-art AFM techniques to microbial specimens in order to understand the strength and dynamics of adhesive interactions. These studies shed new light on the molecular mechanisms of adhesion and demonstrate an intimate relationship between force and function in microbial adhesins.

Molecular dissection of pheromone selectivity in the competence signaling system ComRS of streptococci.

Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems from Streptococcus thermophilus and Streptococcus vestibularis Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system from S. vestibularis. Comparison with its ortholog from S. thermophilus reveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.

Subtle selectivity in a pheromone sensor triumvirate desynchronizes competence and predation in a human gut commensal.

Constantly surrounded by kin or alien organisms in nature, eukaryotes and prokaryotes developed various communication systems to coordinate adaptive multi-entity behavior. In complex and overcrowded environments, they require to discriminate relevant signals in a myriad of pheromones to execute appropriate responses. In the human gut commensal Streptococcus salivarius, the cytoplasmic Rgg/RNPP regulator ComR couples competence to bacteriocin-mediated predation. Here, we describe a paralogous sensor duo, ScuR and SarF, which circumvents ComR in order to disconnect these two physiological processes. We highlighted the recurring role of Rgg/RNPP in the production of antimicrobials and designed a robust genetic screen to unveil potent/optimized peptide pheromones. Further mutational and biochemical analyses dissected the modifiable selectivity toward their pheromone and operating sequences at the subtle molecular level. Additionally, our results highlight how we might mobilize antimicrobial molecules while silencing competence in endogenous populations of human microflora and temper gut disorders provoked by bacterial pathogens.

Mobilization of Microbiota Commensals and Their Bacteriocins for Therapeutics.

With the specter of resurgence of pathogens due to the propagation of antibiotic-resistance genes, innovative antimicrobial strategies are needed. In this review, we summarize the beneficial aspects of bacteriocins, a set of miscellaneous peptide-based bacterium killers, compared with classical antibiotics, and emphasize their use in cocktails to curb the emergence of new resistance. We highlight that their prey spectrum, their molecular malleability, and their multiple modes of production might lead to specific and personalized treatments to prevent systemic disorders. Complementarily, we discuss how we might exploit prevailing bacterial commensals, such as Streptococcus salivarius, and deliberately mobilize their bacteriocin arsenal 'on site' to cure multiresistant infections or finely reshape the endogenous microbiota for prophylaxis purposes.

Circuitry Rewiring Directly Couples Competence to Predation in the Gut Dweller Streptococcus salivarius.

Small distortions in transcriptional networks might lead to drastic phenotypical changes, especially in cellular developmental programs such as competence for natural transformation. Here, we report a pervasive circuitry rewiring for competence and predation interplay in commensal streptococci. Canonically, in streptococci paradigms such as Streptococcus pneumoniae and Streptococcus mutans, the pheromone-based two-component system BlpRH is a central node that orchestrates the production of antimicrobial compounds (bacteriocins) and incorporates signal from the competence activation cascade. However, the human commensal Streptococcus salivarius does not contain a functional BlpRH pair, while the competence signaling system ComRS directly couples bacteriocin production and competence commitment. This network shortcut might underlie an optimal adaptation against microbial competitors and explain the high prevalence of S. salivarius in the human digestive tract. Moreover, the broad spectrum of bacteriocin activity against pathogenic bacteria showcases the commensal and genetically tractable S. salivarius species as a user-friendly model for competence and bacterial predation.

More than a Tad: spatiotemporal control of Caulobacter pili.

The Type IV pilus (T4P) is a powerful and sophisticated bacterial nanomachine involved in numerous cellular processes, including adhesion, DNA uptake and motility. Aside from the well-described subtype T4aP of the Gram-negative genera, including Myxococcus, Pseudomonas and Neisseria, the Tad (tight adherence) pilus secretion system re-shuffles homologous parts from other secretion systems along with uncharacterized components into a new type of protein translocation apparatus. A representative of the Tad apparatus, the Caulobacter crescentus pilus assembly (Cpa) machine is built exclusively at the newborn cell pole once per cell cycle. Recent comprehensive genetic analyses unearthed a myriad of spatiotemporal determinants acting on the Tad/Cpa system, many of which are conserved in other α-proteobacteria, including obligate intracellular pathogens and symbionts.

Correction: Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS.

[This corrects the article DOI: 10.1371/journal.ppat.1005980.].

Functional dichotomy and distinct nanoscale assemblies of a cell cycle-controlled bipolar zinc-finger regulator.

Protein polarization underlies differentiation in metazoans and in bacteria. How symmetric polarization can instate functional asymmetry remains elusive. Here, we show by super-resolution photo-activated localization microscopy and edgetic mutations that the bitopic zinc-finger protein ZitP implements specialized developmental functions - pilus biogenesis and multifactorial swarming motility - while shaping distinct nanoscale (bi)polar architectures in the asymmetric model bacterium Caulobacter crescentus. Polar assemblage and accumulation of ZitP and its effector protein CpaM are orchestrated in time and space by conserved components of the cell cycle circuitry that coordinate polar morphogenesis with cell cycle progression, and also act on the master cell cycle regulator CtrA. Thus, this novel class of potentially widespread multifunctional polarity regulators is deeply embedded in the cell cycle circuitry.

Modularity and determinants of a (bi-)polarization control system from free-living and obligate intracellular bacteria.

Although free-living and obligate intracellular bacteria are both polarized it is unclear whether the underlying polarization mechanisms and effector proteins are conserved. Here we dissect at the cytological, functional and structural level a conserved polarization module from the free living α-proteobacterium Caulobacter crescentus and an orthologous system from an obligate intracellular (rickettsial) pathogen. The NMR solution structure of the zinc-finger (ZnR) domain from the bifunctional and bipolar ZitP pilus assembly/motility regulator revealed conserved interaction determinants for PopZ, a bipolar matrix protein that anchors the ParB centromere-binding protein and other regulatory factors at the poles. We show that ZitP regulates cytokinesis and the localization of ParB and PopZ, targeting PopZ independently of the previously known binding sites for its client proteins. Through heterologous localization assays with rickettsial ZitP and PopZ orthologs, we document the shared ancestries, activities and structural determinants of a (bi-)polarization system encoded in free-living and obligate intracellular α-proteobacteria.

Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS.

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci.

In-phase oscillation of global regulons is orchestrated by a pole-specific organizer.

Cell fate determination in the asymmetric bacterium Caulobacter crescentus (Caulobacter) is triggered by the localization of the developmental regulator SpmX to the old (stalked) cell pole during the G1→S transition. Although SpmX is required to localize and activate the cell fate-determining kinase DivJ at the stalked pole in Caulobacter, in cousins such as Asticcacaulis, SpmX directs organelle (stalk) positioning and possibly other functions. We define the conserved σ54-dependent transcriptional activator TacA as a global regulator in Caulobacter whose activation by phosphorylation is indirectly down-regulated by SpmX. Using a combination of forward genetics and cytological screening, we uncover a previously uncharacterized and polarized component (SpmY) of the TacA phosphorylation control system, and we show that SpmY function and localization are conserved. Thus, SpmX organizes a site-specific, ancestral, and multifunctional regulatory hub integrating the in-phase oscillation of two global transcriptional regulators, CtrA (the master cell cycle transcriptional regulator A) and TacA, that perform important cell cycle functions.

Growth control switch by a DNA-damage-inducible toxin-antitoxin system in Caulobacter crescentus.

Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. Many such TASs induce this effect through the translation-dependent RNA cleavage (RNase) activity of their toxins, which are held in check by their cognate antitoxins in the absence of stress. However, it is not always clear whether specific mRNA targets of orthologous RNase toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here, we show that the TAS HigBA of Caulobacter crescentus can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigB's mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA, and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks.

Complete Genome Sequence of Streptococcus salivarius HSISS4, a Human Commensal Bacterium Highly Prevalent in the Digestive Tract.

The human commensal bacterium Streptococcus salivarius plays a major role in the equilibrium of microbial communities of the digestive tract. Here, we report the first complete genome sequence of a Streptococcus salivarius strain isolated from the small intestine, namely, HSISS4. Its circular chromosome comprises 1,903 coding sequences and 2,100,988 nucleotides.