Type VI protein secretion system (T6SS) is important for bacterial competition through contact-dependent killing of competitors. T6SS delivers effectors to neighboring cells and corresponding antagonistic proteins confer immunity against effectors that are delivered by sister cells. Although T6SS has been found in more than 100 gram-negative bacteria including many important human pathogens, few T6SS-dependent effector and immunity proteins have been experimentally determined. Here we report a high-throughput approach using transposon mutagenesis and deep sequencing (Tn-seq) to identify T6SS immunity proteins in Vibrio cholerae . Saturating transposon mutagenesis was performed in wild type and a T6SS null mutant. Genes encoding immunity proteins were predicted to be essential in the wild type but dispensable in the T6SS mutant. By comparing the relative abundance of each transposon mutant in the mutant library using deep sequencing, we identified three immunity proteins that render protection against killing by T6SS predatory cells. We also identified their three cognate T6SS-secreted effectors and show these are important for not only antibacterial and antieukaryotic activities but also assembly of T6SS apparatus. The lipase and muramidase T6SS effectors identified in this study underscore the diversity of T6SS-secreted substrates and the distinctly different mechanisms that target these for secretion by the dynamic T6SS organelle.

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.

Determining how an organism responds to its environment by altering gene expression is key to understanding its ecology. Here, we used RNA-seq to comprehensively and quantitatively assess the transcriptional response of the bacterial opportunistic cystic fibrosis (CF) pathogen and endemic soil dweller, Burkholderia cenocepacia, in conditions mimicking these 2 environments. By sequencing 762 million bases of cDNA from 2 closely related B. cenocepacia strains (one isolated from a CF patient and one from soil), we identified a number of potential virulence factors expressed under CF-like conditions, whereas genes whose protein products are involved in nitrogen scavenging and 2-component sensing were among those induced under soil-like conditions. Interestingly, 13 new putative noncoding RNAs were discovered using this technique, 12 of which are preferentially induced in the soil environment, suggesting that ncRNAs play an important role in survival in the soil. In addition, we detected a surprisingly large number of regulatory differences between the 2 strains, which may represent specific adaptations to the niches from which each strain was isolated, despite their high degree of DNA sequence similarity. Compared with the CF strain, the soil strain shows a stronger global gene expression response to its environment, which is consistent with the need for a more dynamic reaction to the heterogeneous conditions of soil.cystic fibrosis ͉ RNA-seq ͉ soil ͉ transcriptomics

High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200–1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.