The type VI secretion system (T6SS) is a macromolecular machine that delivers protein effectors into host cells and/or competing bacteria. The effectors may be delivered as noncovalently bound cargo of T6SS needle proteins (VgrG/Hcp/PAAR) or as C-terminal extensions of these proteins. Many strains produce a T6SS, but little is known about the specific effectors or how they are delivered. In this study, we show that AB307-0294 encodes three loci, each containing a gene, a T6SS toxic effector gene, and an antitoxin/immunity gene. Each of the T6SS toxic effectors could kill when produced in unless the cognate immunity protein was coproduced. To determine the role of each VgrG in effector delivery, we performed interbacterial competitive killing assays using AB307-0294 mutants, together with prey cells expressing pairs of immunity genes that protected against two toxic effectors but not a third. Using this approach, we showed that AB307-0294 produces only three T6SS toxic effectors capable of killing and that each VgrG protein is specific for the carriage of one effector. Finally, we analyzed a number of genomes and identified significant diversity in the range of encoded T6SS VgrG and effector proteins, with correlations between effector types and global clone lineages.

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium However, colistin-resistant isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 μg/ml and 128 μg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element IS within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an deletion mutant in 6009-1and showed that colistin resistance increased upon the deletion of We also provided 6009-2 with an intact copy of and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant mutant 6009-2. Importantly, the expression of, encoding a second lipid A-specific pEtN transferase but not , was increased in the mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.

is a Gram-negative bacterium responsible for many important animal diseases. While a number of virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. Small RNA (sRNA) molecules are critical regulators that act by binding to specific mRNA targets, often in association with the RNA chaperone protein Hfq. In this study, transcriptomic analysis of the strain VP161 revealed a putative sRNA with high identity to GcvB from and serovar Typhimurium. High-throughput quantitative liquid proteomics was used to compare the proteomes of the VP161 wild-type strain, a mutant, and a GcvB overexpression strain. These analyses identified 46 proteins that displayed significant differential production after inactivation of , 36 of which showed increased production. Of the 36 proteins that were repressed by GcvB, 27 were predicted to be involved in amino acid biosynthesis or transport. Bioinformatic analyses of putative GcvB target mRNAs identified a strongly conserved 10 nucleotide consensus sequence, 5'-AACACAACAT-3', with the central eight nucleotides identical to the seed binding region present within GcvB mRNA targets in and Typhimurium. Using a defined set of seed region mutants, together with a two-plasmid reporter system that allowed for quantification of sRNA-mRNA interactions, this sequence was confirmed to be critical for the binding of the GcvB to the target mRNA,.