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The human gastrointestinal tract consists of a dense and diverse microbial community, the composition of which is intimately linked to health. Extrinsic factors such as diet and host immunity are insufficient to explain the constituents of this community, and direct interactions between co-resident microorganisms have been implicated as important drivers of microbiome composition. The genomes of bacteria derived from the gut microbiome contain several pathways that mediate contact-dependent interbacterial antagonism 1 – 3 . Many members of the Gram-negative order Bacteroidales encode the type VI secretion system (T6SS), which facilitates the delivery of toxic effector proteins into adjacent cells 4 , 5 . Here we report the occurrence of acquired interbacterial defence (AID) gene clusters in Bacteroidales species that reside within the human gut microbiome. These clusters encode arrays of immunity genes that protect against T6SS-mediated intra- and inter-species bacterial antagonism. Moreover, the clusters reside on mobile elements, and we show that their transfer is sufficient to confer resistance to toxins in vitro and in gnotobiotic mice. Finally, we identify and validate the protective capability of a recombinase-associated AID subtype (rAID-1) that is present broadly in Bacteroidales genomes. These rAID-1 gene clusters have a structure suggestive of active gene acquisition and include predicted immunity factors of toxins derived from diverse organisms. Our data suggest that neutralization of contact-dependent interbacterial antagonism by AID systems helps to shape human gut microbiome ecology. An interbacterial defence strategy, involving clusters of immunity genes against toxins released by the type VI secretion system of the same or different species, is widespread among Bacteroides species, and transfer of these gene clusters confers resistance to toxins in vitro and in the mammalian gut.

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis. Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses, trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.

The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes—one of two major phyla in the gut—also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.

Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail–like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus. The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.

Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis . The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds. Pseudomonas and Bacillus can promote plant growth but their mutual interactions are unclear. Here, the authors show that the extracellular matrix protects Bacillus colonies from infiltration by Pseudomonas cells, while the Pseudomonas type VI secretion system stimulates Bacillus sporulation.

The type VI secretion system (T6SS) is a weapon of bacterial warfare and host cell subversion. The Gram-negative pathogen Pseudomonas aeruginosa has three T6SSs involved in colonization, competition, and full virulence. H1-T6SS is a molecular gun firing seven toxins, Tse1–Tse7, challenging survival of other bacteria and helping P. aeruginosa to prevail in specific niches. The H1-T6SS characterization was facilitated through studying a P. aeruginosa strain lacking the RetS sensor, which has a fully active H1-T6SS, in contrast to the parent. However, study of H2-T6SS and H3-T6SS has been neglected because of a poor understanding of the associated regulatory network. Here we performed a screen to identify H2-T6SS and H3-T6SS regulatory elements and found that the posttranscriptional regulator RsmA imposes a concerted repression on all three T6SS clusters. A higher level of complexity could be observed as we identified a transcriptional regulator, AmrZ, which acts as a negative regulator of H2-T6SS. Overall, although the level of T6SS transcripts is fine-tuned by AmrZ, all T6SS mRNAs are silenced by RsmA. We expanded this concept of global control by RsmA to VgrG spike and T6SS toxin transcripts whose genes are scattered on the chromosome. These observations triggered the characterization of a suite of H2-T6SS toxins and their implication in direct bacterial competition. Our study thus unveils a central mechanism that modulates the deployment of all T6SS weapons that may be simultaneously produced within a single cell.

The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins-TssA, TssE, TssF, TssG, TssK and VgrG-are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.

Bacterial type VI secretion systems (T6SSs) are puncturing molecular machines that transport effector proteins to kill microbes, manipulate eukaryotic cells, or facilitate nutrient uptake. How and why T6SS machines and effectors differ within a species is not fully understood. Here, we applied molecular population genetics to the T6SSs in a global population of the opportunistic pathogen Pseudomonas aeruginosa . We reveal varying occurrence of up to four distinct T6SS machines. Moreover, we define conserved core T6SS effectors, likely critical for the biology of P. aeruginosa , and accessory effectors that can exhibit mutual exclusivity between strains. By ancestral reconstruction, we observed dynamic changes in the gain and loss of effector genes in the species’ evolutionary history. Our work highlights the potential importance of T6SS intraspecific diversity in bacterial ecology and evolution. Populations of a single bacterial species can possess a great diversity of type VI secretion systems (T6SSs) and secreted effectors. Here, Habich et al. apply molecular population genetics to the T6SSs of Pseudomonas aeruginosa , highlighting the potential importance of T6SS intraspecific diversity in bacterial ecology and evolution.

Background Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems employed by Gram negative bacteria to intoxicate other bacteria or eukaryotic cells. T6SSs were recently discovered in a few Bacteroidetes strains, thereby extending the presence of these systems beyond Proteobacteria. The present study was designed to analyze in a global nature the diversity, abundance, and properties of T6SSs in the Bacteroidales, the most predominant Gram negative bacterial order of the human gut. Results By performing extensive bioinformatics analyses and creating hidden Markov models for Bacteroidales Tss proteins, we identified 130 T6SS loci in 205 human gut Bacteroidales genomes. Of the 13 core T6SS proteins of Proteobacteria, human gut Bacteroidales T6SS loci encode orthologs of nine, and an additional five other core proteins not present in Proteobacterial T6SSs. The Bacteroidales T6SS loci segregate into three distinct genetic architectures with extensive DNA identity between loci of a given genetic architecture. We found that divergent DNA regions of a genetic architecture encode numerous types of effector and immunity proteins and likely include new classes of these proteins. TheT6SS loci of genetic architecture 1 are contained on highly similar integrative conjugative elements (ICEs), as are the T6SS loci of genetic architecture 2, whereas the T6SS loci of genetic architecture 3 are not and are confined to Bacteroides fragilis. Using collections of co-resident Bacteroidales strains from human subjects, we provide evidence for the transfer of genetic architecture 1 T6SS loci among co-resident Bacteroidales species in the human gut. However, we also found that established ecosystems can harbor strains with distinct T6SS of all genetic architectures. Conclusions This is the first study to comprehensively analyze of the presence and diversity of T6SS loci within an order of bacteria and to analyze T6SSs of bacteria from a natural community. These studies demonstrate that more than half of our gut Bacteroidales, equivalent to about ¼ of the bacteria of this ecosystem, encode T6SSs. The data reveal several novel properties of these systems and suggest that antagonism between or distributed defense among these abundant intestinal bacteria may be common in established human gut communities.

Type VI secretion systems (T6SSs) are nanomachines widely used by bacteria to deliver toxic effector proteins directly into neighbouring cells. However, the modes of action of many effectors remain unknown. Here we report that Ssp6, an anti-bacterial effector delivered by a T6SS of the opportunistic pathogen Serratia marcescens , is a toxin that forms ion-selective pores. Ssp6 inhibits bacterial growth by causing depolarisation of the inner membrane in intoxicated cells, together with increased outer membrane permeability. Reconstruction of Ssp6 activity in vitro demonstrates that it forms cation-selective pores. A survey of bacterial genomes reveals that genes encoding Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 and similar proteins represent a new family of T6SS-delivered anti-bacterial effectors. Type VI secretion systems (T6SSs) are used by bacteria to inject toxic effector proteins into neighbouring cells. Here, Mariano et al. show that an antibacterial effector from Serratia marcescens forms cation-selective pores that lead to inner-membrane depolarisation and increased outer-membrane permeability.