Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin-antitoxin defence system.

Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage ϕ3T modulates the host-encoded MazEF toxin-antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in ϕ3T and other Spß-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin-antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.

Characterization of a unique repression system present in arbitrium phages of the SPbeta family.

Arbitrium-coding phages use peptides to communicate and coordinate the decision between lysis and lysogeny. However, the mechanism by which these phages establish lysogeny remains unknown. Here, focusing on the SPbeta phage family's model phages phi3T and SPß, we report that a six-gene operon called the "SPbeta phages repressor operon" (sro) expresses not one but two master repressors, SroE and SroF, the latter of which folds like a classical phage integrase. To promote lysogeny, these repressors bind to multiple sites in the phage genome. SroD serves as an auxiliary repressor that, with SroEF, forms the repression module necessary for lysogeny establishment and maintenance. Additionally, the proteins SroABC within the operon are proposed to constitute the transducer module, connecting the arbitrium communication system to the activity of the repression module. Overall, this research sheds light on the intricate and specialized repression system employed by arbitrium SPß-like phages in making lysis-lysogeny decisions.

Abortive infection antiphage defense systems: separating mechanism and phenotype.

Bacteria have evolved a wide array of mechanisms that allow them to eliminate phage infection. 'Abortive infection' (abi) systems are an expanding category of such mechanisms, defined as those which induce programmed cell death (or dormancy) upon infection, and thus halt phage propagation within a bacterial population. This definition entails two requirements - a phenotypic observation (cell death upon infection), and a mechanistic determination of its sources (system-induced death). The phenotypic and mechanistic aspects of abi are often implicitly assumed to be tightly linked, and studies regularly tend to establish one and deduce the other. However, recent evidence points to a complicated relationship between the mechanism of defense and the phenotype observed upon infection. We argue that rather than viewing the abi phenotype as an inherent quality of a set of defense systems, it should be more appropriately thought of as an attribute of interactions between specific phages and bacteria under given conditions. Consequently, we also point to potential pitfalls in the prevailing methods for ascertaining the abi phenotype. Overall, we propose an alternative framework for parsing interactions between attacking phages and defending bacteria.

Biophysical aspects underlying the swarm to biofilm transition.

Bacteria organize in a variety of collective states, from swarming-rapid surface exploration, to biofilms-highly dense immobile communities attributed to stress resistance. It has been suggested that biofilm and swarming are oppositely controlled, making this transition particularly interesting for understanding the ability of bacterial colonies to adapt to challenging environments. Here, the swarm to biofilm transition is studied in Bacillus subtilis by analyzing the bacterial dynamics both on the individual and collective scales. We show that both biological and physical processes facilitate the transition. A few individual cells that initiate the biofilm program cause nucleation of large, approximately scale-free, stationary aggregates of trapped swarm cells. Around aggregates, cells continue swarming almost unobstructed, while inside, trapped cells are added to the biofilm. While our experimental findings rule out previously suggested purely physical effects as a trigger for biofilm formation, they show how physical processes, such as clustering and jamming, accelerate biofilm formation.

Dormant phages communicate via arbitrium to control exit from lysogeny.

Temperate bacterial viruses (phages) can transition between lysis-replicating and killing the host-and lysogeny, that is, existing as dormant prophages while keeping the host viable. Recent research showed that on invading a naïve cell, some phages communicate using a peptide signal, termed arbitrium, to control the decision of entering lysogeny. Whether communication can also serve to regulate exit from lysogeny (known as phage induction) is unclear. Here we show that arbitrium-coding prophages continue to communicate from the lysogenic state by secreting and sensing the arbitrium signal. Signalling represses DNA damage-dependent phage induction, enabling prophages to reduce the induction rate when surrounded by other lysogens. We show that in certain phages, DNA damage and communication converge to regulate the expression of the arbitrium-responsive gene aimX, while in others integration of DNA damage and communication occurs downstream of aimX expression. Additionally, signalling by prophages tilts the decision of nearby infecting phages towards lysogeny. Altogether, we find that phages use small-molecule communication throughout their entire life cycle to sense the abundance of lysogens in the population, thus avoiding lysis when they are likely to encounter established lysogens rather than permissive uninfected hosts.

Short-range quorum sensing controls horizontal gene transfer at micron scale in bacterial communities.

In bacterial communities, cells often communicate by the release and detection of small diffusible molecules, a process termed quorum-sensing. Signal molecules are thought to broadly diffuse in space; however, they often regulate traits such as conjugative transfer that strictly depend on the local community composition. This raises the question how nearby cells within the community can be detected. Here, we compare the range of communication of different quorum-sensing systems. While some systems support long-range communication, we show that others support a form of highly localized communication. In these systems, signal molecules propagate no more than a few microns away from signaling cells, due to the irreversible uptake of the signal molecules from the environment. This enables cells to accurately detect micron scale changes in the community composition. Several mobile genetic elements, including conjugative elements and phages, employ short-range communication to assess the fraction of susceptible host cells in their vicinity and adaptively trigger horizontal gene transfer in response. Our results underscore the complex spatial biology of bacteria, which can communicate and interact at widely different spatial scales.

A mobile genetic element increases bacterial host fitness by manipulating development.

Horizontal gene transfer is a major force in bacterial evolution. Mobile genetic elements are responsible for much of horizontal gene transfer and also carry beneficial cargo genes. Uncovering strategies used by mobile genetic elements to benefit host cells is crucial for understanding their stability and spread in populations. We describe a benefit that ICEBs1, an integrative and conjugative element of Bacillus subtilis, provides to its host cells. Activation of ICEBs1 conferred a frequency-dependent selective advantage to host cells during two different developmental processes: biofilm formation and sporulation. These benefits were due to inhibition of biofilm-associated gene expression and delayed sporulation by ICEBs1-containing cells, enabling them to exploit their neighbors and grow more prior to development. A single ICEBs1 gene, devI (formerly ydcO), was both necessary and sufficient for inhibition of development. Manipulation of host developmental programs allows ICEBs1 to increase host fitness, thereby increasing propagation of the element.

Many bacteria can 'have sex' ­ that is, they can share their genetic information and trade off segments of DNA. While these mobile genetic elements can be parasites that use the resources of their host to make more of themselves, some carry useful genes which, for example, help bacteria to fight off antibiotics. Integrative and conjugative elements (or ICEs) are a type of mobile segments that normally stay inside the genetic information of their bacterial host but can sometimes replicate and be pumped out to another cell. ICEBs1 for instance, is an element found in the common soil bacterium Bacillus subtilis. Scientists know that ICEBs1 can rapidly spread in biofilms ­ the slimly, crowded communities where bacteria live tightly connected ­ but it is still unclear whether it helps or hinders its hosts. Using genetic manipulations and tracking the survival of different groups of cells, Jones et al. show that carrying ICEBs1 confers an advantage under many conditions. When B. subtilis forms biofilms, the presence of the devI gene in ICEBs1 helps the cells to delay the production of the costly mucus that keeps bacteria together, allowing the organisms to 'cheat' for a little while and benefit from the tight-knit community without contributing to it. As nutrients become scarce in biofilms, the gene also allows the bacteria to grow for longer before they start to form spores ­ the dormant bacterial form that can weather difficult conditions. Mobile elements can carry genes that make bacteria resistant to antibiotics, harmful to humans, or able to use new food sources; they could even be used to artificially introduce genes of interest in these cells. The work by Jones et al. helps to understand the way these elements influence the fate of their host, providing insight into how they could be harnessed for the benefit of human health.

Competition between social cheater viruses is driven by mechanistically different cheating strategies.

Cheater viruses, also known as defective interfering viruses, cannot replicate on their own yet replicate faster than the wild type upon coinfection. While there is growing interest in using cheaters as antiviral therapeutics, the mechanisms underlying cheating have been rarely explored. During experimental evolution of MS2 phage, we observed the parallel emergence of two independent cheater mutants. The first, a point deletion mutant, lacked polymerase activity but was advantageous in viral packaging. The second synonymous mutant cheater displayed a completely different cheating mechanism, involving an altered RNA structure. Continued evolution revealed the demise of the deletion cheater and rise of the synonymous cheater. A mathematical model inferred that while a single cheater is expected to reach an equilibrium with the wild type, cheater demise arises from antagonistic interactions between coinfecting cheaters. These findings highlight layers of parasitism: viruses parasitizing cells, cheaters parasitizing intact viruses, and cheaters may parasitize other cheaters.

A Bacterial Tower of Babel: Quorum-Sensing Signaling Diversity and Its Evolution.

Quorum sensing is a process in which bacteria secrete and sense a diffusible molecule, thereby enabling bacterial groups to coordinate their behavior in a density-dependent manner. Quorum sensing has evolved multiple times independently, utilizing different molecular pathways and signaling molecules. A common theme among many quorum-sensing families is their wide range of signaling diversity-different variants within a family code for different signal molecules with a cognate receptor specific to each variant. This pattern of vast allelic polymorphism raises several questions-How do different signaling variants interact with one another? How is this diversity maintained? And how did it come to exist in the first place? Here we argue that social interactions between signaling variants can explain the emergence and persistence of signaling diversity throughout evolution. Finally, we extend the discussion to include cases where multiple diverse systems work in concert in a single bacterium.

Clonality and non-linearity drive facultative-cooperation allele diversity.

Kin discrimination describes the differential interaction of organisms with kin versus non-kin. In microorganisms, many genetic loci act as effective kin-discrimination systems, such as kin-directed help and non-kin-directed harm. Another important example is facultative cooperation, where cooperators increase their investment in group-directed cooperation with the abundance of their kin in the group. Many of these kin-discrimination loci are highly diversified, yet it remains unclear what evolutionary mechanisms maintain this diversity, and how it is affected by population structure. Here, we demonstrate the unique dependence of kin-discriminative interactions on population structure, and how this could explain facultative-cooperation allele-diversity. We show mathematically that low relatedness between microbes in non-clonal social groups is needed to maintain the diversity of facultative-cooperation alleles, while high clonality is needed to stabilize this diversity against cheating. Interestingly, we demonstrate with simulations that such population structure occurs naturally in expanding microbial colonies. Finally, analysis of experimental data of quorum-sensing mediated facultative cooperation, in Bacillus subtilis, demonstrates the relevance of our results to realistic microbial interactions, due to their intrinsic non-linear frequency dependence. Our analysis therefore stresses the impact of clonality on the interplay between exploitation and kin discrimination and portrays a way for the evolution of facultative cooperation.

Selection for increased quorum-sensing cooperation in Pseudomonas aeruginosa through the shut-down of a drug resistance pump.

The opportunistic pathogen Pseudomonas aeruginosa employs a hierarchical quorum-sensing network to regulate virulence factor production that cooperatively benefit the population at a cost to the individual. It has been argued that the evolution of a cooperative mutant in a quorum sensing-suppressed population would be hampered through its exploitation by neighboring non-mutant cells. It remains unclear whether mechanisms which overcome this exploitation exist. Here we investigate the regain of quorum-sensing cooperation by evolving a mutant of the lasR master quorum-sensing regulator. The mutant regained partial cooperative growth through null mutations in mexT, which codes for an activator of the MexEF-OprN multidrug-resistant pump. We find that these mutations enhance cooperative growth in both the lasR mutant and wild-type backgrounds through the activation of the RhlIR system. We show that the regain of cooperation in mexT mutants is mediated by the reduction in MexEF-OprN activity, whereas an additional source of private benefit is mostly mexEF-oprN-independent. Finally, we show that addition of antibiotics for which resistance is mediated by MexEF-OprN prevents the selection of increased cooperation at sub-MIC concentrations. MexT, therefore, not only links private and public goods, but also exposes conflicts between selection for antibiotic resistance and enhanced cooperation.

Self-sensing in Bacillus subtilis quorum-sensing systems.

Bacterial cell-cell signalling, or quorum sensing, is characterized by the secretion and groupwide detection of small diffusible signal molecules called autoinducers. This mechanism allows cells to coordinate their behaviour in a density-dependent manner. A quorum-sensing cell may directly respond to the autoinducers it produces in a cell-autonomous and quorum-independent manner, but the strength of this self-sensing effect and its impact on bacterial physiology are unclear. Here, we explore the existence and impact of self-sensing in the Bacillus subtilis ComQXP and Rap-Phr quorum-sensing systems. By comparing the quorum-sensing response of autoinducer-secreting and non-secreting cells in co-culture, we find that secreting cells consistently show a stronger response than non-secreting cells. Combining genetic and quantitative analyses, we demonstrate this effect to be a direct result of self-sensing and rule out an indirect regulatory effect of the autoinducer production genes on response sensitivity. In addition, self-sensing in the ComQXP system affects persistence to antibiotic treatment. Together, these findings indicate the existence of self-sensing in the two most common designs of quorum-sensing systems of Gram-positive bacteria.

Transient Duplication-Dependent Divergence and Horizontal Transfer Underlie the Evolutionary Dynamics of Bacterial Cell-Cell Signaling.

Evolutionary expansion of signaling pathway families often underlies the evolution of regulatory complexity. Expansion requires the acquisition of a novel homologous pathway and the diversification of pathway specificity. Acquisition can occur either vertically, by duplication, or through horizontal transfer, while divergence of specificity is thought to occur through a promiscuous protein intermediate. The way by which these mechanisms shape the evolution of rapidly diverging signaling families is unclear. Here, we examine this question using the highly diversified Rap-Phr cell-cell signaling system, which has undergone massive expansion in the genus Bacillus. To this end, genomic sequence analysis of >300 Bacilli genomes was combined with experimental analysis of the interaction of Rap receptors with Phr autoinducers and downstream targets. Rap-Phr expansion is shown to have occurred independently in multiple Bacillus lineages, with >80 different putative rap-phr alleles evolving in the Bacillius subtilis group alone. The specificity of many rap-phr alleles and the rapid gain and loss of Rap targets are experimentally demonstrated. Strikingly, both horizontal and vertical processes were shown to participate in this expansion, each with a distinct role. Horizontal gene transfer governs the acquisition of already diverged rap-phr alleles, while intralocus duplication and divergence of the phr gene create the promiscuous intermediate required for the divergence of Rap-Phr specificity. Our results suggest a novel role for transient gene duplication and divergence during evolutionary shifts in specificity.

Social Evolution Selects for Redundancy in Bacterial Quorum Sensing.

Quorum sensing is a process of chemical communication that bacteria use to monitor cell density and coordinate cooperative behaviors. Quorum sensing relies on extracellular signal molecules and cognate receptor pairs. While a single quorum-sensing system is sufficient to probe cell density, bacteria frequently use multiple quorum-sensing systems to regulate the same cooperative behaviors. The potential benefits of these redundant network structures are not clear. Here, we combine modeling and experimental analyses of the Bacillus subtilis and Vibrio harveyi quorum-sensing networks to show that accumulation of multiple quorum-sensing systems may be driven by a facultative cheating mechanism. We demonstrate that a strain that has acquired an additional quorum-sensing system can exploit its ancestor that possesses one fewer system, but nonetheless, resume full cooperation with its kin when it is fixed in the population. We identify the molecular network design criteria required for this advantage. Our results suggest that increased complexity in bacterial social signaling circuits can evolve without providing an adaptive advantage in a clonal population.

Facultative cheating supports the coexistence of diverse quorum-sensing alleles.

Bacterial quorum sensing enables bacteria to cooperate in a density-dependent manner via the group-wide secretion and detection of specific autoinducer molecules. Many bacterial species show high intraspecific diversity of autoinducer-receptor alleles, called pherotypes. The autoinducer produced by one pherotype activates its coencoded receptor, but not the receptor of another pherotype. It is unclear what selection forces drive the maintenance of pherotype diversity. Here, we use the ComQXPA system of Bacillus subtilis as a model system, to show that pherotype diversity can be maintained by facultative cheating--a minority pherotype exploits the majority, but resumes cooperation when its frequency increases. We find that the maintenance of multiple pherotypes by facultative cheating can persist under kin-selection conditions that select against "obligate cheaters" quorum-sensing response null mutants. Our results therefore support a role for facultative cheating and kin selection in the evolution of quorum-sensing diversity.

Tobramycin and nebramine as pseudo-oligosaccharide scaffolds for the development of antimicrobial cationic amphiphiles.

Antimicrobial cationic amphiphiles derived from aminoglycoside pseudo-oligosaccharide antibiotics interfere with the structure and function of bacterial membranes and offer a promising direction for the development of novel antibiotics. Herein, we report the design and synthesis of cationic amphiphiles derived from the pseudo-trisaccharide aminoglycoside tobramycin and its pseudo-disaccharide segment nebramine. Antimicrobial activity, membrane selectivity, mode of action, and structure-activity relationships were studied. Several cationic amphiphiles showed marked antimicrobial activity, and one amphiphilic nebramine derivative proved effective against all of the tested strains of bacteria; furthermore, against several of the tested strains, this compound was well over an order of magnitude more potent than the parent antibiotic tobramycin, the membrane-targeting antimicrobial peptide mixture gramicidin D, and the cationic lipopeptide polymyxin B, which are in clinical use.

A complex path for domestication of B. subtilis sociality.

Microorganisms adapt to the lab environment by eliminating unnecessary genetic systems. In Bacillus subtilis, such adaptation resulted in the lab strain being unable to form complex, matrix-associated structures known as biofilms. We recently showed that the ancestor of the lab strain, which is considered by the research community to be a stereotypical 'wild' strain, carries an atypical mutation in the RapP-PhrP quorum-sensing system. We have found that this mutation has profound effects on the biofilm phenotype of the ancestral strain. Here we discuss these recent findings and present more data that focuses on the lessons that can be learned from this work on the domestication of microorganisms.

The RapP-PhrP quorum-sensing system of Bacillus subtilis strain NCIB3610 affects biofilm formation through multiple targets, due to an atypical signal-insensitive allele of RapP.

The genome of Bacillus subtilis 168 encodes eight rap-phr quorum-sensing pairs. Rap proteins of all characterized Rap-Phr pairs inhibit the function of one or several important response regulators: ComA, Spo0F, or DegU. This inhibition is relieved upon binding of the peptide encoded by the cognate phr gene. Bacillus subtilis strain NCIB3610, the biofilm-proficient ancestor of strain 168, encodes, in addition, the rapP-phrP pair on the plasmid pBS32. RapP was shown to dephosphorylate Spo0F and to regulate biofilm formation, but unlike other Rap-Phr pairs, RapP does not interact with PhrP. In this work we extend the analysis of the RapP pathway by reexamining its transcriptional regulation, its effect on downstream targets, and its interaction with PhrP. At the transcriptional level, we show that rapP and phrP regulation is similar to that of other rap-phr pairs. We further find that RapP has an Spo0F-independent negative effect on biofilm-related genes, which is mediated by the response regulator ComA. Finally, we find that the insensitivity of RapP to PhrP is due to a substitution of a highly conserved residue in the peptide binding domain of the rapP allele of strain NCIB3610. Reversing this substitution to the consensus amino acid restores the PhrP dependence of RapP activity and eliminates the effects of the rapP-phrP locus on ComA activity and biofilm formation. Taken together, our results suggest that RapP strongly represses biofilm formation through multiple targets and that PhrP does not counteract RapP due to a rare mutation in rapP.