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.

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.

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.

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.

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.

Menaquinone and iron are essential for complex colony development in Bacillus subtilis.

Cells of undomesticated species of Bacillus subtilis frequently form complex colonies during spreading on agar surfaces. Given that menaquinone is involved in another form of coordinated behavior, namely, sporulation, we looked for a possible role for menaquinone in complex colony development (CCD) in the B. subtilis strain NCIB 3610. Here we show that inhibition of menaquinone biosynthesis in B. subtilis indeed abolished its ability to develop complex colonies. Additionally some mutations of B. subtilis which confer defective CCD could be suppressed by menaquinone derivatives. Several such mutants mapped to the dhb operon encoding the genes responsible for the biosynthesis of the iron siderophore, bacillibactin. Our results demonstrate that both menaquinone and iron are essential for CCD in B. subtilis.