ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.

The multicomponent type VI secretion system (T6SS) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell-puncturing device of tailed bacteriophages. The T6SS members VipA/VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA/VipB tubules in vitro, but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA/VipB tubules localize near-perpendicular to the inner membrane of Vibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA/VipB tubules are decorated by ClpV in vivo and become static in ΔclpV cells, indicating that ClpV is required for tubule removal. VipA/VipB tubules mislocalize in ΔclpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non-productive VipA/VipB tubules. ClpV activity is restricted to the contracted state of VipA/VipB, allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA/VipB is sufficient to replace ClpV function in vivo, suggesting that ClpV activity is autonomously regulated by VipA/VipB conformation.

Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion.

Ring-forming AAA(+) ATPases act in a plethora of cellular processes by remodeling macromolecules. The specificity of individual AAA(+) proteins is achieved by direct or adaptor-mediated association with substrates via distinct recognition domains. We investigated the molecular basis of substrate interaction for Vibrio cholerae ClpV, which disassembles tubular VipA/VipB complexes, an essential step of type VI protein secretion and bacterial virulence. We identified the ClpV recognition site within VipB, showed that productive ClpV-VipB interaction requires the oligomeric state of both proteins, solved the crystal structure of a ClpV N-domain-VipB peptide complex, and verified the interaction surface by mutant analysis. Our results show that the substrate is bound to a hydrophobic groove, which is formed by the addition of a single α-helix to the core N-domain. This helix is absent from homologous N-domains, explaining the unique substrate specificity of ClpV. A limited interaction surface between both proteins accounts for the dramatic increase in binding affinity upon ATP-driven ClpV hexamerization and VipA/VipB tubule assembly by coupling multiple weak interactions. This principle ensures ClpV selectivity toward the VipA/VipB macromolecular complex.

Tubules and donuts: a type VI secretion story.

The recently identified type VI secretion systems (T6SSs) are present in many pathogenic proteobacteria and are encoded by a conserved gene cluster. T6SSs contribute to virulence development of various pathogens and are often activated upon contact with target cells. Since the identification of the T6SS, substantial progress has been made at all levels, including gene regulation, its impact on bacterial virulence, the function of effector proteins and the mechanism of secretion. Recent structural and mechanistic studies revealed unique features of the T6SS that distinguish it from other secretion systems. Structural similarities between the T6SS-specific exoproteins Hcp and VgrG and components of the cell-puncturing device of tailed bacteriophages suggest that the T6SSs mimic a bacteriophage machinery to puncture target cell membranes and to translocate effector proteins, representing a novel mechanism of effector delivery. In bacteriophages contraction of the tail sheath, which engulfs the tail tube, causes ejection of the cell-puncturing machinery. The T6SS components VipA/VipB form tubular structures, which might function as tail sheaths by engulfing Hcp proteins. The severing of VipA/VipB complexes by the AAA+ chaperone ClpV is essential for type VI protein secretion and might be linked to VipA/VipB tubule contraction, leading to the export of Hcp and VgrG.

Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion.

The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteobacteria, including the human pathogen Vibrio cholerae. T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N-terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel-like structures that are converted by a threading activity of ClpV into small complexes. ClpV-mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion.

IncP-1-beta plasmid pGNB1 isolated from a bacterial community from a wastewater treatment plant mediates decolorization of triphenylmethane dyes.

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.

Mobilizable IncQ-related plasmid carrying a new quinolone resistance gene, qnrS2, isolated from the bacterial community of a wastewater treatment plant.

Plasmid-encoded quinolone resistance was previously reported for different bacteria isolated from patients not only in the United States and Asia but also in Europe. Here we describe the isolation, by applying a new selection strategy, of the quinolone resistance plasmid pGNB2 from an activated sludge bacterial community of a wastewater treatment plant in Germany. The hypersensitive Escherichia coli strain KAM3 carrying a mutation in the multidrug efflux system genes acrAB was transformed with total plasmid DNA preparations isolated from activated sludge bacteria and subsequently selected on medium containing the fluoroquinolone norfloxacin. This approach resulted in the isolation of plasmid pGNB2 conferring decreased susceptibility to nalidixic acid and to different fluoroquinolones. Analysis of the pGNB2 nucleotide sequence revealed that it is 8,469 bp in size and has a G+C content of 58.2%. The plasmid backbone is composed of a replication initiation module (repA-repC) belonging to the IncQ-family and a two-component mobilization module that confers horizontal mobility to the plasmid. In addition, plasmid pGNB2 carries an accessory module consisting of a transposon Tn1721 remnant and the quinolone resistance gene, qnrS2, that is 92% identical to the qnrS gene located on plasmid pAH0376 from Shigella flexneri 2b. QnrS2 belongs to the pentapeptide repeat protein family and is predicted to protect DNA-gyrase activity against quinolones. This is not only the first report on a completely sequenced plasmid mediating quinolone resistance isolated from an environmental sample but also on the first qnrS-like gene detected in Europe.