Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines have an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyze the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, 10 T6SS effector–immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device, which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens, which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol, which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.

The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1–3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier.

Significance: Bacteria evolved molecular weapons to help them thrive in polymicrobial environments. The type VI secretion system (T6SS) is a gun loaded with a great diversity of bacterial toxins. On contact with neighboring cells, toxins are fired, and in the absence of immunity, the prey is killed, allowing the attacker to prevail. Each bacterium can be equipped with several distinct T6SSs, and it is unclear whether they are simultaneously active or whether each has a specific role in a particular environment. Here we showed that production of the three Pseudomonas aeruginosa T6SSs is orchestrated by global regulators. We suggest it may be possible for simultaneous assembly of multiple T6SSs within a single cell, priming it to fight a wide variety of organisms.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease. The emergence and rapid dissemination of new bacterial pathogens presents multiple challenges to healthcare systems, including the need for rapid detection, precise diagnostics, effective transmission control and effective treatment. E. coli ST131 is an example of a recently emerged multidrug resistant pathogen that is capable of causing urinary tract and bloodstream infections with limited available treatment options. In order to increase our molecular understanding of E. coli ST131, we developed a high-throughput transposon mutagenesis system in combination with next generation sequencing to test every gene for its essential role in growth and for its contribution to serum resistance. We identified 315 essential genes, 270 of which were conserved among all currently available complete E. coli genomes. Fifty-six genes that define the serum resistome of E. coli ST131 were identified, including genes encoding membrane proteins, proteins involved in LPS biosynthesis, regulators and several novel proteins with previously unknown function. This study therefore provides an inventory of essential and serum resistance genes that could form a framework for the future development of targeted therapeutics to prevent disease caused by multidrug-resistant E. coli ST131 strains.

Significance: The type VI secretion system (T6SS) is a molecular weapon used for interbacterial competition. It acts like a crossbow bolt to deliver toxic effectors into target cells. Here we characterize a T6SS toxin which we call Tse7. We show that it is a DNase and that its two-domain PAAR-nuclease structure allows coupling to the T6SS VgrG tip via the PAAR domain. Disruption of this interface abrogates toxin delivery and results in the loss of interbacterial killing ability. We identify the corresponding immunity protein, Tsi7, and show it directly interacts with the Tse7 toxin. Finally, as Tse7–Tsi7 sequences vary significantly between Pseudomonas aeruginosa strains, and as Tsi7 protection is strain specific, we show that this toxin/immunity pair contributes to self- nonself-recognition.