DNA-Uptake Machinery of Naturally Competent Vibrio Cholerae
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DNA-uptake machinery of naturally competent Vibrio cholerae Patrick Seitz and Melanie Blokesch1 Laboratory of Molecular Microbiology, Global Health Institute, School of Life Sciences, Swiss Federal Institute of Technology Lausanne (École Polytechnique Fédérale de Lausanne), CH-1015 Lausanne, Switzerland Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved September 19, 2013 (received for review August 22, 2013) Natural competence for transformation is a mode of horizontal tweezers, Hahn et al. showed that DNA binding and uptake also gene transfer that is commonly used by bacteria to take up DNA occur preferentially at the cell pole (9). It is unknown whether from their environment. As part of this developmental program, a polar localization pattern of the DNA-uptake machinery is so-called competence genes, which encode the components of universal for all naturally competent bacteria and essential for its a DNA-uptake machinery, are expressed. Several models have functionality. We addressed this question and demonstrate that been proposed for the DNA-uptake complexes of competent bacte- upon competence induction, V. cholerae cells produce a type ria, and most include a type IV (pseudo)pilus as a core component. IV pilus (Tfp)-like appendage that extends beyond the outer However, cell-biology–based approaches to visualizing compe- membrane. We also visualized other components of the DNA- tence proteins have so far been restricted to Gram-positive bacte- uptake complex, using fluorescently labeled fusion proteins, ria. Here, we report the visualization of a competence-induced and showed that those components and the pilus are not pilus in the Gram-negative bacterium Vibrio cholerae. We show strictly associated with the cell poles of V. cholerae.Further- that piliated cells mostly contain a single pilus that is not biased more, we identified a minimal set of competence genes re- toward a polar localization and that this pilus colocalizes with the quired for efficient transformation of V. cholerae. We show outer membrane secretin PilQ. PilQ, on the other hand, forms sev- that most gene products within this competence regulon contrib- eral foci around the cell and occasionally colocalizes with the dy- ute to DNA uptake and efficient transformation, even though the fi namic cytoplasmic-traf c ATPase PilB, which is required for pilus Tfp-related competence proteins are not entirely essential for extension. We also determined the minimum competence regulon transformation. These data provide unique insight into the func- MICROBIOLOGY V. cholerae of , which includes at least 19 genes. Bacteria with tion of the competence proteins with respect to DNA transfer mutations in those genes were characterized with respect to the across the outer membrane, the periplasm, or the inner mem- presence of surface-exposed pili, DNA uptake, and natural trans- brane and suggest an at least two-step DNA-uptake process in formability. Based on these phenotypes, we propose that DNA the Gram-negative bacterium V. cholerae. uptake in naturally competent V. cholerae cells occurs in at least two steps: a pilus-dependent translocation of the incoming DNA Results across the outer membrane and a pilus-independent shuttling of Identification of Components of the DNA-Uptake Complex in Naturally the DNA through the periplasm and into the cytoplasm. Competent V. cholerae Cells. Although the regulation of natural competence differs widely between Gram-negative bacteria (3, atural competence for genetic transformation is one of three 13), the core components of the DNA-uptake machinery are Nmodes of horizontal gene transfer (HGT) in prokaryotes often conserved, and the uptake process might be close to uni- – and is often tightly regulated (1 3). Large pieces of DNA con- versal; however, major knowledge gaps still exist with respect to taining a series of genes can be transferred by natural trans- the mechanistic aspects of DNA uptake, as also noted recently by formation without the need for direct interaction with other Rosemary Redfield and coworkers [“our knowledge of the proteins microbes or mobile genetic elements. This process can foster responsible for DNA uptake and transformation is piecemeal” rapid evolution, and HGT is known to be involved in the spread of antibiotic resistance, adaptation to new environmental niches, Significance 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 Transformation allows naturally competent bacteria to take up DNA from the environment and integrate the DNA into this bacterium, competence is induced upon growth on chitinous the chromosome by recombination. In Gram-negative bacte- surfaces (3, 4), the natural habitat of V. cholerae (5). Although ria, the DNA-uptake machinery shuttles the incoming DNA we have gained a reasonably clear understanding of the regula- across the outer membrane, the periplasmic space, and the inner tory network driving competence induction in this organism (for membrane. This study investigates the DNA-uptake complex of a review, see ref. 3), almost nothing is known about its DNA- the human pathogen Vibrio cholerae, using a cellular biology- uptake machinery. Indeed, the sophisticated DNA-uptake com- based approach. We visualized different components of this plexes used by naturally competent bacteria during transformation multicomponent complex, including a type IV pilus appendage, are still poorly characterized (6), especially in Gram-negative determined their (co)localization within the bacterial cell, and bacteria in which the transforming DNA (tDNA) must cross two conducted an analysis of competence-gene mutants. We con- membranes and the periplasmic space (including the peptidogly- clude that the uptake of DNA occurs via (at least) a two- can layer) to enter the cytoplasm and recombine with the chro- step process. mosome (the latter step is not required if the tDNA consists of plasmid DNA). Interestingly, the majority of competence-protein Author contributions: P.S. and M.B. designed research; P.S. and M.B. performed research; localization studies using cellular microbiology approaches are P.S. and M.B. analyzed data; and M.B. wrote the paper. based on studies performed with the Gram-positive bacterium The authors declare no conflict of interest. Bacillus subtilis.ForB. subtilis, a multicomponent protein ma- This article is a PNAS Direct Submission. chine may be responsible for DNA uptake (1, 7, 8), as many 1To whom correspondence should be addressed. E-mail: melanie.blokesch@epfl.ch. – transformation proteins colocalize to the pole(s) of the cell (9 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 12). Furthermore, using single-molecule experiments with laser 1073/pnas.1315647110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1315647110 PNAS | October 29, 2013 | vol. 110 | no. 44 | 17987–17992 Downloaded by guest on September 27, 2021 (ref. 14, p. 5245)]. Thus, the aim of this study was to better un- 10-3 derstand DNA uptake in naturally competent V. cholerae. First, 10-4 Tfp biogenesis or function Tfp-unrelated we examined genes (including neighboring genes in the same -5 operon) that were significantly and reproducibly up-regulated 10 upon growth of the bacterium on chitin (4, 15) or upon chitin- 10-6 independent competence induction (through expression of the -7 10 < d.l. transformation regulator gene tfoX) (4, 16, 17) and excluded 10-8 those genes with a potential function in chitin colonization and Transformation frequency WT pilA pilB pilP pilT pilC pilN pilO pilQ degradation or in a general stress response (including protein- pilM recA comF comEA comEC VC0861 VC0859 VC0858 VC0860 VC1612 VC0857 folding chaperones). Next, we screened for genes with homologs detectable WT + - - - - - - - - - - - - - - - - n.a. ++ + comEC in other naturally transformable bacteria (Table S1) and dem- tDNA ++ - - - - - - - - - - - - - - - - ++++ ++ comEC onstrated that all these competence genes were also strictly uptake conserved in other naturally transformable species of the genus Fig. 1. Natural transformation and DNA uptake in strains lacking individual Vibrio (3, 18) (Table S1). The majority of this gene set was competence proteins. The natural transformability of a wild-type strain and predicted to encode components of a Tfp, as previously sug- derivatives lacking individual competence genes was tested in a chitin- gested (15). We also identified a cluster of genes (VC0857–0861) independent assay (17) (all strains contained TntfoX). Shown are the aver- encoding (among other products) putative type IV pilins, which age transformation frequencies of at least three independent biological replicates. Error bars indicate SD. All mutants were significantly impaired in we included in further analysis even though no clear homologs natural transformation (P < 0.02). < d.l., below detection limit. The average fi − − were identi ed in naturally competent bacteria such as Neisseria detection limit of nontransformable strains was 7.1 × 10 8 (±3.3 × 10 8). fl gonorrheae, Haemophilus in uenzae,orB. subtilis (Table S1). On Uptake of DNA was tested by detection of internalized tDNA in a whole-cell the basis of the data provided below, we define this set of 19 genes duplex-PCR assay (16); the results are indicated below the graph (row (Table S1) as the minimum (but most likely still incomplete) comECWT). The DNA-uptake assay was also performed in double-knockout