DNA-uptake machinery of naturally competent Vibrio cholerae

Natural competence for transformation is a mode of horizontal gene transfer that is commonly used by bacteria to take up DNA from their environment. As part of this developmental program, so-called competence genes, which encode the components of a DNA-uptake machinery, are expressed. Several models have been proposed for the DNA-uptake complexes of competent bacteria, and most include a type IV (pseudo)pilus as a core component. However, cell-biology–based approaches to visualizing competence proteins have so far been restricted to Gram-positive bacteria. Here, we report the visualization of a competence-induced pilus in the Gram-negative bacterium Vibrio cholerae. We show that piliated cells mostly contain a single pilus that is not biased toward a polar localization and that this pilus colocalizes with the outer membrane secretin PilQ. PilQ, on the other hand, forms several foci around the cell and occasionally colocalizes with the dynamic cytoplasmic-traffic ATPase PilB, which is required for pilus extension. We also determined the minimum competence regulon of V. cholerae, which includes at least 19 genes. Bacteria with mutations in those genes were characterized with respect to the presence of surface-exposed pili, DNA uptake, and natural transformability. Based on these phenotypes, we propose that DNA uptake in naturally competent V. cholerae cells occurs in at least two steps: a pilus-dependent translocation of the incoming DNA across the outer membrane and a pilus-independent shuttling of the DNA through the periplasm and into the cytoplasm.Natural competence for genetic transformation is one of three modes of horizontal gene transfer (HGT) in prokaryotes and is often tightly regulated (1–3). Large pieces of DNA containing a series of genes can be transferred by natural transformation without the need for direct interaction with other microbes or mobile genetic elements. This process can foster rapid evolution, and HGT is known to be involved in the spread of antibiotic resistance, adaptation to new environmental niches, and the emergence of new pathogens.Many bacterial species are able to enter a state of natural competence, including the human pathogen Vibrio cholerae. In this bacterium, competence is induced upon growth on chitinous surfaces (3, 4), the natural habitat of V. cholerae (5). Although we have gained a reasonably clear understanding of the regulatory network driving competence induction in this organism (for a review, see ref. 3), almost nothing is known about its DNA-uptake machinery. Indeed, the sophisticated DNA-uptake complexes used by naturally competent bacteria during transformation are still poorly characterized (6), especially in Gram-negative bacteria in which the transforming DNA (tDNA) must cross two membranes and the periplasmic space (including the peptidoglycan layer) to enter the cytoplasm and recombine with the chromosome (the latter step is not required if the tDNA consists of plasmid DNA). Interestingly, the majority of competence-protein localization studies using cellular microbiology approaches are based on studies performed with the Gram-positive bacterium Bacillus subtilis. For B. subtilis, a multicomponent protein machine may be responsible for DNA uptake (1, 7, 8), as many transformation proteins colocalize to the pole(s) of the cell (9–12). Furthermore, using single-molecule experiments with laser tweezers, Hahn et al. showed that DNA binding and uptake also occur preferentially at the cell pole (9). It is unknown whether a polar localization pattern of the DNA-uptake machinery is universal for all naturally competent bacteria and essential for its functionality. We addressed this question and demonstrate that upon competence induction, V. cholerae cells produce a type IV pilus (Tfp)-like appendage that extends beyond the outer membrane. We also visualized other components of the DNA-uptake complex, using fluorescently labeled fusion proteins, and showed that those components and the pilus are not strictly associated with the cell poles of V. cholerae. Furthermore, we identified a minimal set of competence genes required for efficient transformation of V. cholerae. We show that most gene products within this competence regulon contribute to DNA uptake and efficient transformation, even though the Tfp-related competence proteins are not entirely essential for transformation. These data provide unique insight into the function of the competence proteins with respect to DNA transfer across the outer membrane, the periplasm, or the inner membrane and suggest an at least two-step DNA-uptake process in the Gram-negative bacterium V. cholerae.