Natural competence for transformation is a common mode of horizontal gene transfer and contributes to bacterial evolution. Transformation occurs through the uptake of external DNA and its integration into the genome. Here we show that the type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae. The T6SS-encoding gene cluster is under the positive control of the competence regulators TfoX and QstR and is induced by growth on chitinous surfaces. Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae.

SummaryType VI secretion systems (T6SSs) are nanomachines used for interbacterial killing and intoxication of eukaryotes. Although Vibrio cholerae is a model organism for structural studies on T6SSs, the underlying regulatory network is less understood. A recent study showed that the T6SS is part of the natural competence regulon in V. cholerae and is activated by the regulator TfoX. Here, we identify the TfoX homolog TfoY as a second activator of the T6SS. Importantly, despite inducing the same T6SS core machinery, the overall regulons differ significantly for TfoX and TfoY. We show that TfoY does not contribute to competence induction. Instead, TfoY drives the production of T6SS-dependent and T6SS-independent toxins, together with an increased motility phenotype. Hence, we conclude that V. cholerae uses its sole T6SS in response to diverse cues and for distinctive outcomes: either to kill for the prey’s DNA, leading to horizontal gene transfer, or as part of a defensive escape reaction.

The general stress response in Alphaproteobacteria was recently described to depend on the alternative sigma factor EcfG , whose activity is regulated by its anti-sigma factor NepR. The response regulator PhyR, in turn, regulates NepR activity in a partner-switching mechanism according to which phosphorylation of PhyR triggers sequestration of NepR by the sigma factor-like effector domain of PhyR. Although genes encoding predicted histidine kinases can often be found associated with phyR, little is known about their role in modulation of PhyR phosphorylation status. We demonstrate here that the PhyR-NepREcfG cascade is important for multiple stress resistance and competitiveness in the phyllosphere in a naturally abundant plant epiphyte, Sphingomonas sp. strain Fr1, and provide evidence that the partner switching mechanism is conserved. We furthermore identify a gene, designated phyP, encoding a predicted histidine kinase at the phyR locus as essential. Genetic epistasis experiments suggest that PhyP acts upstream of PhyR, keeping PhyR in an unphosphorylated, inactive state in nonstress conditions, strictly depending on the predicted phosphorylatable site of PhyP, His-341. In vitro experiments show that Escherichia coli inner membrane fractions containing PhyP disrupt the PhyR-P/NepR complex. Together with the fact that PhyP lacks an obvious ATPase domain, these results are in agreement with PhyP functioning as a phosphatase of PhyR, rather than a kinase.

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Summary Bacteria of the genus Vibrio are common members of aquatic environments where they compete with other prokaryotes and defend themselves against grazing predators. A macromolecular protein complex called the type VI secretion system (T6SS) is used for both purposes. Previous research showed that the sole T6SS of the human pathogen V. cholerae is induced by extracellular (chitin) or intracellular (low c‐di‐GMP levels) cues and that these cues lead to distinctive signalling pathways for which the proteins TfoX and TfoY serve as master regulators. In this study, we tested whether the TfoX‐ and TfoY‐mediated regulation of T6SS, concomitantly with natural competence or motility, was conserved in non‐cholera Vibrio species, and if so, how these regulators affected the production of individual T6SSs in double‐armed vibrios. We show that, alongside representative competence genes, TfoX regulates at least one T6SS in all tested Vibrio species. TfoY, on the other hand, fostered motility in all vibrios but had a more versatile T6SS response in that it did not foster T6SS‐mediated killing in all tested vibrios. Collectively, our data provide evidence that the TfoX‐ and TfoY‐mediated signalling pathways are mostly conserved in diverse Vibrio species and important for signal‐specific T6SS induction.

The needle length of the Yersinia spp. injectisome is determined by Yop secretion protein P (YscP), an early substrate of the injectisome itself. There is a linear correlation between the length of YscP and the length of the needle, suggesting that YscP acts as a molecular ruler. However, it is not known whether one single molecule of YscP suffices to control the length of one needle or whether several molecules of YscP are exported in alternation with the needle subunit YscF until the needle length matches the ruler length, which would stop needle growth. To address this question, three different strains expressing simultaneously a short and a long version of YscP were engineered. The experimentally obtained needle length distribution was compared with the distributions predicted by stochastic modeling of the various possible scenarios. The experimental data are compatible with the single ruler model and not with the scenarios involving more than one ruler per needle.he injectisome or needle complex allows pathogenic bacteria to inject effector proteins across eukaryotic cell membranes, a process called type III secretion (T3S). This nanomachine, evolutionary related to the flagellum, has a basal body made of several rings embedded in the two bacterial membranes (1-4) and including integral membrane proteins constituting the core of the T3S export apparatus (reviewed by refs. 5-8). The Yersinia enterocolitica E40 Ysc injectisome terminates with a 65-nm-long hollow needle, made of ∼140 copies of the 9-kDa YscF protein. At the tip of the needle, a pentamer of LcrV (9, 10) forms a structure serving as an assembly platform for the translocation pore (11).During morphogenesis, the needle components, like those of the hook and filament of the flagellum, are sequentially exported by the T3S apparatus itself (12), traveling through the growing structure and polymerizing at its distal end (13,14). There is no clear hierarchy in the synthesis of the injectisome components and substrates. Thus, the export apparatus is expected to switch its substrate specificity over time so that needle subunits (early substrates) are exported before LcrV (intermediate substrates) and the translocators and effectors (late substrates). The switch between early and intermediate substrates determines the arrest of needle growth and hence the needle length (15).The control of the needle length and the switch to export intermediate substrates involves a protein, which is itself exported, Yop secretion protein P (YscP) for the Yersinia spp injectisome (16-18) and FliK for the flagellum (19-21). In the absence of this protein, injectisomes have extra-long needles (deregulated phenotype) and do not secrete LcrV, translocators, and effectors, whereas the flagellum has extralong hooks and no filament (polyhook phenotype). The switch function is assigned to the C-terminal domains of YscP and FliK (21,22). This domain, called type III secretion substrate specificity switch (T3S4) (22), is thought to interact with YscU (FlhB in the flagellum), a compo...

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Natural competence for transformation is a developmental program that allows certain bacteria to take up free extracellular DNA from the environment and integrate this DNA into their genome. Thereby, natural transformation acts as mode of horizontal gene transfer and impacts bacterial evolution. The number of genes induced upon competence induction varies significantly between organisms. However, all of the naturally competent bacteria possess competence genes that encode so-called DNA-uptake machineries. Some components of these multi-protein complexes resemble subunits of type IV pili and type II secretion systems. However, knowledge on the mechanistic aspects of such DNA-uptake complexes is still very limited. Here, we discuss some new findings regarding the DNA-uptake machinery of the naturally transformable human pathogen Vibrio cholerae. The potential of this organism to initiate the competence program was discovered less than a decade ago. However, recent studies have provided new insight into both the regulatory pathways of competence induction and into the DNA uptake dynamics.