Shedding light on bacteria-host interactions with the aid of TnSeq approaches.

Bacteria are highly adaptable and grow in diverse niches, where they often interact with eukaryotic organisms. These interactions with different hosts span the entire spectrum from symbiosis to pathogenicity and thus determine the lifestyle of the bacterium. Knowledge of the genetic determinants involved in animal and plant host colonization by pathogenic and mutualistic bacteria is not only crucial to discover new drug targets for disease management but also for developing novel biostimulant strategies. In the last decades, significant progress in genome-wide high-throughput technologies such as transposon insertion sequencing has led to the identification of pathways that enable efficient host colonization. However, the extent to which similar genes play a role in this process in different bacteria is yet unclear. This review highlights the commonalities and specificities of bacterial determinants important for bacteria-host interaction.

Paraburkholderia sabiae Uses One Type VI Secretion System (T6SS-1) as a Powerful Weapon against Notorious Plant Pathogens.

Paraburkholderia sabiae LMG24235 is a nitrogen-fixing betaproteobacterium originally isolated from a root nodule of Mimosa caesalpiniifolia in Brazil. We show here that this strain effectively kills strains from several bacterial families (Burkholderiaceae, Pseudomonadaceae, Enterobacteriaceae) which include important plant pathogens in a contact-dependent manner. De novo assembly of the first complete genome of P. sabiae using long sequencing reads and subsequent annotation revealed two gene clusters predicted to encode type VI secretion systems (T6SS), which we named T6SS-1 and T6SS-3 according to previous classification methods (G. Shalom, J. G. Shaw, and M. S. Thomas, Microbiology, 153:2689-2699, 2007, https://doi.org/10.1099/mic.0.2007/006585-0). We created P. sabiae with mutations in each of the two T6SS gene clusters that abrogated their function, and the T6SS-1 mutant was no longer able to outcompete other strains in a contact-dependent manner. Notably, our analysis revealed that T6SS-1 is essential for competition against several important plant pathogens in vitro, including Burkholderia plantarii, Ralstonia solanacearum, Pseudomonas syringae, and Pectobacterium carotovorum. The 9-log reduction in P. syringae cells in the presence of P. sabiae was particularly remarkable. Importantly, in an in vivo assay, P. sabiae was able to protect potato tubers from bacterial soft rot disease caused by P. carotovorum, and this protection was partly dependent on T6SS-1. IMPORTANCE Rhizobia often display additional beneficial traits such as the production of plant hormones and the acquisition of limited essential nutrients that improve plant growth and enhance plant yields. Here, we show that the rhizobial strain P. sabiae antagonizes important phytopathogens such as P. carotovorum, P. syringae, and R. solanacearum and that this effect is due to contact-dependent killing mediated by one of two T6SS systems identified in the complete, de novo assembled genome sequence of P. sabiae. Importantly, co-inoculation of Solanum tuberosum tubers with P. sabiae also resulted in a drastic reduction of soft rot caused by P. carotovorum in an in vivo model system. This result highlights the protective potential of P. sabiae against important bacterial plant diseases, which makes it a valuable candidate for application as a biocontrol agent. It also emphasizes the particular potential of rhizobial inoculants that combine several beneficial effects such as plant growth promotion and biocontrol for sustainable agriculture.

Identification of Key Factors for Anoxic Survival of B. cenocepacia H111.

Burkholderia cenocepacia is an opportunistic pathogen that can lead to severe infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Being an obligate aerobe, B. cenocepacia is unable to grow in the absence of oxygen. In this study, we show that the CF isolate B. cenocepacia H111 can survive in the absence of oxygen. Using a transposon sequencing (Tn-seq) approach, we identified 71 fitness determinants involved in anoxic survival, including a Crp-Fnr family transcriptional regulatory gene (anr2), genes coding for the sensor kinase RoxS and its response regulator RoxR, the sigma factor for flagella biosynthesis (FliA) and subunits of a cytochrome bd oxidase (CydA, CydB and the potentially novel subunit CydP). Individual knockouts of these fitness determinants significantly reduced anoxic survival, and inactivation of both anr copies is shown to be lethal under anoxic conditions. We also show that the two-component system RoxS/RoxR and FliA are important for virulence and swarming/swimming, respectively.

Mapping of the Denitrification Pathway in Burkholderia thailandensis by Genome-Wide Mutant Profiling.

Burkholderia thailandensis is a soil saprophyte that is closely related to the pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of B. thailandensis Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon narIJHGK2K1, the aniA gene encoding a previously unknown nitrite reductase, and the petABC genes encoding a cytochrome bc1, as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of B. thailandensis and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic Burkholderia species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates N2O, which may play a role in the dispersal of B. thailandensis biofilms.IMPORTANCEBurkholderia thailandensis is a soil-dwelling saprophyte that is often used as surrogate of the closely related pathogen Burkholderia pseudomallei, the causative agent of melioidosis and a classified biowarfare agent. Both organisms are adapted to grow under oxygen-limited conditions in rice fields by generating energy through denitrification. Microoxic growth of B. pseudomallei is also considered essential for human infections. Here, we have used a Tn-Seq approach to identify the genes encoding the enzymes and regulators required for growth under denitrifying conditions. We show that a mutant that is defective in the conversion of N2O to N2, the last step in the denitrification process, is unaffected in microoxic growth but is severely impaired in biofilm formation, suggesting that N2O may play a role in biofilm dispersal. Our study identified novel targets for the development of therapeutic agents to treat meliodiosis.