DNA-uptake machinery of naturally competent Vibrio cholerae

Abstract

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.

Fig. 1.

Natural transformation and DNA uptake in strains lacking individual competence proteins. The natural transformability of a wild-type strain and derivatives lacking individual competence genes was tested in a chitin-independent assay (17) (all strains contained TntfoX). Shown are the average transformation frequencies of at least three independent biological replicates. Error bars indicate SD. All mutants were significantly impaired in natural transformation (P < 0.02). < d.l., below detection limit. The average detection limit of nontransformable strains was 7.1 × 10⁻⁸ (±3.3 × 10⁻⁸). Uptake of DNA was tested by detection of internalized tDNA in a whole-cell duplex-PCR assay (16); the results are indicated below the graph (row comEC^WT). The DNA-uptake assay was also performed in double-knockout strains from which comEC was concomitantly absent (row ΔcomEC). Labels: −, no tDNA detectable; +/++, tDNA detected by PCR; n.a., not applicable. Band intensities were judged relative to that of the wild-type strain (Fig. S1).

Fig. 2.

Visualization of the competence-induced pilus. The major pilus subunit PilA was fused to an affinity tag, and the construct (pilA-strep) was brought onto the chromosome, replacing the wild-type copy of pilA. Pili were visualized by immunofluorescence microscopy, using an Oyster488 conjugated anti–Strep-tag antibody. (A) Diverse polar and nonpolar pili structures were observed. Depicted are fluorescence images (Upper) and overlays with the phase contrast images (Lower). (B) The majority of competence-gene mutants are no longer piliated. Shown are merged images (fluorescence in green plus phase contrast). Strain details are described in Table S2 (all contained TntfoX). (Scale bars, 2 μm.)

Fig. 3.

Cellular localization of specific competence proteins. The wild-type versions of the competence genes pilQ and pilB were replaced by functional translational fusion constructs to visualize their localization. Shown are representative fluorescent images (mCherry, α-Strep, and GFP), phase contrast (Ph) images, and overlays of fluorescent channels/fluorescent and Ph channels (merge). Cells are outlined with dashed lines. V. cholerae strains carrying mCherry-pilQ (A, mCherry N terminally fused to PilQ), mCherry-pilQ and pilA-strep (B), pilB-gfp (C, GFP fused to the C terminus of PilB), and mCherry-pilQ and pilB-gfp (D) were subjected to epifluorescence microscopy (after immunostaining, B). Colocalization of mCherry-PilQ and the pilus was observed in the majority of piliated cells (B). For D, two-color time-lapse microscopy was performed. Representative fluorescent images for mCherry-PilQ (red) and PilB-GFP (green) are indicated. Observed spots for both fluorescent proteins are schematically depicted and projected on the Ph images (Bottom). Colocalization events are indicated by a white arrowhead. The time is given in minutes. (Scale bars, 2 μm.)

Fig. 4.

Model of the DNA-uptake machinery of V. cholerae. The proposed model of the DNA-uptake machinery is based on the results provided in this study and on homologies to (predicted) competence proteins from other competent bacteria or other type IV pili-containing organisms. Details are described in the text.