Interbacterial predation as a strategy for DNA acquisition in naturally competent bacteria

Natural competence enables bacteria to take up exogenous DNA. The evolutionary function of natural competence remains controversial, as imported DNA can act as a source of substrates or can be integrated into the genome. Exogenous homologous DNA can also be used for genome repair. In this Opinion article, we propose that predation of non-related neighbouring bacteria coupled with competence regulation might function as an active strategy for DNA acquisition. Competence-dependent kin-discriminated killing has been observed in the unrelated bacteria Vibrio cholerae and Streptococcus pneumoniae. Importantly, both the regulatory networks and the mode of action of neighbour predation differ between these organisms, with V. cholerae using a type VI secretion system and S. pneumoniae secreting bacteriocins. We argue that the forced release of DNA from killed bacteria and the transfer of non-clonal genetic material have important roles in bacterial evolution.

Neighbor predation linked to natural competence fosters the transfer of large genomic regions in Vibrio cholerae

Melanie Blokesch, Nicolas Jean Philippe Guex, Christian Iseli, Noémie Matthey, Candice Stoudmann, Sandrine Stutzmann

Natural competence for transformation is a primary mode of horizontal gene transfer (HGT). Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. And while it is known that transformation contributes to evolution and pathogen emergence in bacteria, there are still questions regarding the general prevalence of non-degraded DNA with sufficient coding capacity. In this context, we previously showed that the naturally competent bacterium Vibrio cholerae uses its type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors under chitin-colonizing conditions. We therefore sought to further explore the role of the T6SS in acquiring DNA, the condition of the DNA released through T6SS-mediated killing versus passive cell lysis, and the extent of the transfers that occur due to these conditions. To do this, we herein measured the frequency and the extent of genetic exchanges in bacterial co-cultures on competence-inducing chitin under various DNA-acquisition conditions. We show that competent V. cholerae strains acquire DNA fragments with an average and maximum length exceeding 50 kbp and 150 kbp, respectively, and that the T6SS is of prime importance for such HGT events. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters HGT and that the coding capacity of the exchanged genetic material is sufficient to significantly accelerate bacterial evolution.

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

The physiological state of competence for natural transformation allows bacteria to take up free DNA from the environment and recombine it into their genome. As one of three modes of horizontal gene transfer, natural transformation has hence contributed substantially to the enormous diversity and plasticity of bacterial genomes. With its potential to promote the spread of antibiotic resistances and virulence factors of a variety of pathogens, natural transformation also has an important impact on human health. Even though conserved components required to undergo natural transformation have been identified in many bacterial species, our knowledge on the underlying mechanism of DNA transport remains very limited. To better understand this aspect, we established a first model of DNA uptake in the naturally competent human pathogen Vibrio cholerae. We showed that at least twenty proteins are required for efficient uptake, protection and recombination of transforming DNA in a non-species-specific manner. 14 of these proteins are needed to produce a type IV pilus, which we were able to visualize for the first time. We showed that this competence pilus is not restricted to cell poles, as has been shown for other competent bacterial species (e.g. Bacillus subtilis). Furthermore, we compared the position of this pilus to the localization of other competence proteins and determined their role in the assembly of a surface exposed pilus fiber. While this type IV pilus is important for efficient DNA uptake, we also observed rare transformation events in its absence. In contrast, three pilus-unrelated proteins that are likely involved in the transport process were absolutely required for transformation. Two of them were implicated in DNA transport across the inner membrane. Deletion of the corresponding genes led to a strong accumulation of transforming DNA within the periplasm. However, one of them, ComEA, was essential for DNA uptake across the outer membrane, independently of competence pili. We further characterized the role of this protein by showing that it is localized to the periplasm of competent V. cholerae cells and that it binds to DNA in vivo. Based on our results, we proposed that ComEA directly contributes to DNA transport. Although we showed that DNA uptake across the outer membrane and translocation of DNA into the cytoplasm are functionally uncoupled, our results suggest that the two events coincide spatially. In summary, these findings allow us to present a first mechanistic model of the DNA uptake process in V. cholerae. Many aspects of this model likely apply to other naturally competent bacteria, and therefore represent an important contribution to the general understanding of this basic biological process.

EPFL2014

Two nanomachines drive evolution in diverse Vibrio species

Noémie Matthey

Horizontal gene transfer (HGT) has a major impact on bacterial evolution, leading to acquisition or deletion of genes and gene clusters, including those encoding antibiotic resistances and virulence factors. HGT therefore contributes to pathogen emergence, which can have a major impact on human health. As a mode of HGT, natural competence for transformation allows bacteria to directly acquire DNA from the environment and to integrate parts of this genetic material within their genomes. The human pathogen Vibrio cholerae is naturally competent when it grows in association with the chitinous exoskeletons of zooplankton. Under this condition, the competence state is initiated by the regulatory protein TfoX, which co-induces a molecular killing device known as the type VI secretion system (T6SS). The co-regulation of the T6SS and competence results in the lysis of non-immune neighboring bacteria and the subsequent acquisition of their DNA, which fosters HGT. In V. cholerae, the T6SS is also activated by the TfoX homologue protein, named TfoY. In this study, we aimed at studying the regulatory pathways and the co-regulation of interbacterial predation and DNA uptake in order to provide new insights into the environmental lifestyle of V. cholerae and other vibrios. We first investigated the conserved function of TfoX- and TfoY in V. fischeri, V. alginolyticus, and V. parahaemolyticus using diverse methods such as qRT-PCR, interbacterial killing assays, and motility assessments. We demonstrated that TfoX-mediated T6SS and competence induction and TfoY-induced motility are conserved phenotypes in the tested Vibrio species, whereas the TfoY-mediated T6SS regulation varied. These variations might reflect diverse defense mechanisms, as these different species have adapted to cope with their environmental niches. Next, we tested the outcome of the TfoX-mediated co-regulation of the T6SS and competence in V. cholerae. Under conditions mimicking the bacteriumâs natural habitat, we showed for the first time the extent of DNA that was integrated on V. choleraeâs genome after T6SS-mediated interbacterial predation. Indeed, we demonstrated that T6SS-dependent bacterial predation not only increased the transformation rates but also fostered the exchange of multiple and huge fragments of DNA. We also showed the necessity of an exquisite co-regulation between the T6SS, the competence-related DNA uptake machinery, and the competence-repressed nuclease. Finally, we disclosed that prey-released DNA is not a private good of the attacking bacterium but also accessible to other surrounding cells that have likewise entered the competence state. Altogether, our findings shed light on the conservation of the TfoX- and TfoY-driven phenotypes in several Vibrio species. We also contributed to a better understanding of the interplay between neighbor predation and DNA uptake and the consequences that this interplay has on genome plasticity.