The type VI secretion system (T6SS) is the most recently described and least understood of the protein secretion systems of Gram-negative bacteria. It is widely distributed and has been implicated in the virulence of various pathogens, but its mechanism and exact mode of action remain to be defined. Additionally there have been several very recent reports that some T6SSs can target bacteria rather than eukaryotic cells. Serratia marcescens is an opportunistic enteric pathogen, a class of bacteria responsible for a significant proportion of hospital-acquired infections. We describe the identification of a functional T6SS in S. marcescens strain Db10, the first report of type VI secretion by an opportunist enteric bacterium. The T6SS of S. marcescens Db10 is active, with secretion of Hcp to the culture medium readily detected, and is expressed constitutively under normal growth conditions from a large transcriptional unit. Expression of the T6SS genes did not appear to be dependent on the integrity of the T6SS. The S. marcescens Db10 T6SS is not required for virulence in three nonmammalian virulence models. It does, however, exhibit dramatic antibacterial killing activity against several other bacterial species and is required for S. marcescens to persist in a mixed culture with another opportunist pathogen, Enterobacter cloacae. Importantly, this antibacterial killing activity is highly strain specific, with the S. marcescens Db10 T6SS being highly effective against another strain of S. marcescens with a very similar and active T6SS. We conclude that type VI secretion plays a crucial role in the competitiveness, and thus indirectly the virulence, of S. marcescens and other opportunistic bacterial pathogens.

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr-and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious ldhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.

SummaryThe anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and biofilm formation during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network in response to oxygen tension and NO. Little is known about all members of the Anr and Dnr regulons and the mediated immediate response to oxygen depletion. Comprehensive transcriptome and bioinformatics analyses in combination with a limited proteome analyses were used for the investigation of the P. aeruginosa response to an immediate oxygen depletion and for definition of the corresponding Anr and Dnr regulons. We observed at first the activation of fermentative pathways for immediate energy generation followed by induction of alternative respiratory chains. A solid position weight matrix model was deduced from the experimentally identified Anr boxes and used for identification of 170 putative Anr boxes in potential P. aeruginosa promoter regions. The combination with the experimental data unambiguously identified 130 new members for the Anr and Dnr regulons. The basis for the understanding of two regulons of P. aeruginosa central to biofilm formation and infection is now defined.

Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS-secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self-resistance (‘immunity’) against T6SS-dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS-secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS-secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches.