The type VI secretion system (T6SS) of Gram-negative bacteria inhibits competitor cells through contact-dependent translocation of toxic effector proteins. In Proteobacteria, the T6SS is anchored to the cell envelope through a megadalton-sized membrane complex (MC). However, the genomes of Bacteroidota with T6SSs appear to lack genes encoding homologs of canonical MC components. Here, we identify five genes in Bacteroides fragilis ( tssNQOPR ) that are essential for T6SS function and encode a Bacteroidota-specific MC. We purify this complex, reveal its dimensions using electron microscopy, and identify a protein-protein interaction network underlying the assembly of the MC including the stoichiometry of the five TssNQOPR components. Protein TssN mediates the connection between the Bacteroidota MC and the conserved baseplate. Although MC gene content and organization varies across the phylum Bacteroidota, no MC homologs are detected outside of T6SS loci, suggesting ancient co-option and functional convergence with the non-homologous MC of Pseudomonadota.

Understanding the relationship between the composition of the human gut microbiota and the ecological forces shaping it is of great importance; however, knowledge of the biogeographical and ecological relationships between physically interacting taxa is limited. Interbacterial antagonism may play an important role in gut community dynamics, yet the conditions under which antagonistic behaviour is favoured or disfavoured by selection in the gut are not well understood. Here, using genomics, we show that a species-specific type VI secretion system (T6SS) repeatedly acquires inactivating mutations in Bacteroides fragilis in the human gut. This result implies a fitness cost to the T6SS, but we could not identify laboratory conditions under which such a cost manifests. Strikingly, experiments in mice illustrate that the T6SS can be favoured or disfavoured in the gut depending on the strains and species in the surrounding community and their susceptibility to T6SS antagonism. We use ecological modelling to explore the conditions that could underlie these results and find that community spatial structure modulates interaction patterns among bacteria, thereby modulating the costs and benefits of T6SS activity. Our findings point towards new integrative models for interrogating the evolutionary dynamics of type VI secretion and other modes of antagonistic interaction in microbiomes.

Bacteria have evolved systems dedicated to interbacterial competition. Here we highlight defenses utilized by Gram-negative cells against type VI secretion system (T6SS)-wielding competitors, including physical barriers, genetically encoded antidotes, and stress responses. Further investigation of specific and general defenses will reveal the interbacterial selective pressures impacting bacterial survival in nature.

SummaryPseudomonas aeruginosa exploits several types of motility behaviours to colonize diverse environments. One of these is swarming motility, a coordinated group movement on a semi‐solid surface. This bacterium needs to express a functional flagellum and produce rhamnolipids to display this type of social motility. A ΔhptB mutant, a gene part of the Gac/Rsm signalling pathway, produces rhamnolipids and expresses a functional flagellum but has an important swarming defect. Experimental‐directed evolution was performed on this mutant under swarming conditions to obtain compensatory mutations and thus identify genes responsible for its deficient swarming phenotype. Unexpectedly, a gain‐of‐function subpopulation emerged from this evolution with mutations in lasR, which codes for a key quorum‐sensing transcriptional regulator. Furthermore, we found that lasR− mutants even emerge at high frequencies in the wild‐type strain when using the same experimental evolution strategy. The resulting evolved population, largely composed of LasR‐defective mutants, is fitter than the original strain in swarming motility. We also established that lasR− mutants have a growth advantage under swarming conditions when compared with wild‐type. Our results demonstrate that a social phenotype, that is, swarming motility, favours the emergence of mutants deficient in a quorum‐sensing regulatory pathway to the benefit of the whole population.

Bacteria can colonize a variety of different environments by modulating their gene regulation using two-component systems. The versatile opportunistic pathogen Pseudomonas aeruginosa has been studied for its capacity to adapt to a broad range of environmental conditions. The Gac/Rsm pathway is composed of the sensor kinase GacS, that detects environmental cues, and the response regulator GacA, that modulates the expression of a specific genes. This system, through the sRNA repressors RsmY and RsmZ, negatively controls the activity of the protein RsmA, which is centrally involved in the transition from chronic to acute infections by post-transcriptionally regulating several virulence functions. RsmA positively regulates swarming motility, a social surface behaviour. Through a poorly defined mechanism, RsmA is also indirectly regulated by HptB, and a Δ hptB mutant exhibits a severe swarming defect. Since a Δ hptB mutant retains all the known functions required for that type of motility, we used an experimental evolution approach to identify elements responsible for its swarming defect. After a few passages under swarming conditions, the defect of the Δ hptB mutant was rescued by the emergence of spontaneous single nucleotide substitutions in the gacA and rsmA genes. Since GacA indirectly represses RsmA activity, it was coherent that an inactivating mutation in gacA would compensate the Δ hptB swarming defect. However, the effect of the mutation in rsmA was unexpected since RsmA promotes swarming; indeed, using expression reporters, we found that the mutation that does not abolish its activity. Instead, using electrophoretic mobility shift assays and molecular simulations, we show that this variant of RsmA is actually less amenable to titration by its cognate repressor RsmY, supporting the other phenotypes observed for this mutant. These results confirm the central role of RsmA as a regulator of swarming motility in P. aeruginosa and identify residues crucial for RsmA function in social motility.Author summary Bacteria need to readily adapt to their environment. Two-component systems (TCS) allow such adaption by triggering bacterial regulation changes through the detection of environmental cues. The opportunistic pathogen Pseudomonas aeruginosa possesses more than 60 TCS in its genome. The Gac/Rsm is a TCS extensively studied for its implication in virulence regulation. This system regulates the transition between chronic and acute bacterial infection behaviours. To acquire a better understanding of this regulation, we performed a directed experimental evolution on a swarming-deficient mutant in a poorly understood regulatory component of the Gac/Rsm pathway. We observed single nucleotide substitutions that allowed restoration of a swarming phenotype similar to the wild-type behaviour. More specifically, mutations were found in the gacA and rsmA genes. Interestingly, the observed mutation in rsmA does not result in loss of function of the protein but rather alters its susceptibility to repression by its cognate interfering sRNA. Since modification in the RNA sequence of RsmA results in the rescue of swarming motility, we confirm the central role of this posttranscriptional repressor in this social lifestyle.

AbstractBacteria can colonize a variety of different environments by modulating their gene regulation using two-component systems. The versatile opportunistic pathogenPseudomonas aeruginosahas been studied for its capacity to adapt to a broad range of environmental conditions. The Gac/Rsm pathway is composed of the sensor kinase GacS, that detects environmental cues, and the response regulator GacA, that modulates the expression of a specific genes. This system, through the sRNA repressors RsmY and RsmZ, negatively controls the activity of the protein RsmA, which is centrally involved in the transition from chronic to acute infections by post-transcriptionally regulating several virulence functions. RsmA positively regulates swarming motility, a social surface behaviour. Through a poorly defined mechanism, RsmA is also indirectly regulated by HptB, and a ΔhptBmutant exhibits a severe swarming defect. Since a ΔhptBmutant retains all the known functions required for that type of motility, we used an experimental evolution approach to identify elements responsible for its swarming defect. After a few passages under swarming conditions, the defect of the ΔhptBmutant was rescued by the emergence of spontaneous single nucleotide substitutions in thegacAandrsmAgenes. Since GacA indirectly represses RsmA activity, it was coherent that an inactivating mutation ingacAwould compensate the ΔhptBswarming defect. However, the effect of the mutation inrsmAwas unexpected since RsmA promotes swarming; indeed, using expression reporters, we found that the mutation that does not abolish its activity. Instead, using electrophoretic mobility shift assays and molecular simulations, we show that this variant of RsmA is actually less amenable to titration by its cognate repressor RsmY, supporting the other phenotypes observed for this mutant. These results confirm the central role of RsmA as a regulator of swarming motility inP. aeruginosaand identify residues crucial for RsmA function in social motility.Author summaryBacteria need to readily adapt to their environment. Two-component systems (TCS) allow such adaption by triggering bacterial regulation changes through the detection of environmental cues. The opportunistic pathogenPseudomonas aeruginosapossesses more than 60 TCS in its genome. The Gac/Rsm is a TCS extensively studied for its implication in virulence regulation. This system regulates the transition between chronic and acute bacterial infection behaviours. To acquire a better understanding of this regulation, we performed a directed experimental evolution on a swarming-deficient mutant in a poorly understood regulatory component of the Gac/Rsm pathway. We observed single nucleotide substitutions that allowed restoration of a swarming phenotype similar to the wild-type behaviour. More specifically, mutations were found in thegacAandrsmAgenes. Interestingly, the observed mutation inrsmAdoes not result in loss of function of the protein but rather alters its susceptibility to repression by its cognate interfering sRNA. Since modification in the RNA sequence of RsmA results in the rescue of swarming motility, we confirm the central role of this posttranscriptional repressor in this social lifestyle.