Deep-sea hydrothermal vents at mid-oceanic ridges occur due to seawater circulation between the cold ocean water column and the heated mantle, resulting in thermal and redox gradients, which in turn support a chemosynthesis-powered ecosystem. These chemosynthetic bacteria form biofilms on basalt substrates and chimneys, acting as primary producers that facilitate the settlement of diverse bacteria and macrofauna. The microorganisms within these complex biofilms possess an assortment of genes for a variety of metabolic processes and resistance to adverse environmental factors. Such genes are generally widely propagated via Horizontal Gene Transfer (HGT), especially under selective pressure in dynamic ecosystems. The extensive occurrence of Mobile Genetic Elements (MGEs), acquired through transduction and conjugation, has been identified in organisms inhabiting the vents, indicating the occurrence of HGT. But genetic transformation, another major mode of HGT, which involves the uptake of free environmental DNA via natural competence, has not been studied in deep-sea hydrothermal vent biofilms. The aim of this work is to further our understanding of natural transformation caused by bacteria in these environments. Our investigation of nine distinct hydrothermal vent biofilms revealed the abundance and taxonomic distribution of the genes associated with natural transformation. Consistent with the 16S taxonomic data, which showed an age- and temperature-dependent distribution of bacterial classes, class Campylobacteria constitutes the most abundant class in young biofilms that possessed and expressed the majority of the transformation genes involved in extracellular DNA (eDNA) uptake, translocation, and homologous recombination. The metatranscriptomic analysis of seafloor and subseafloor biofilms from the Riftia bioregime further validated our hypothesis that the transformation pathway genes are being actively expressed in situ in these biofilms. Since Campylobacteria constitute the most abundant and active fraction of several of these biofilm communities, we aimed to characterize the genetic machinery of natural competence in two pure cultures of Campylobacteria, isolated in our laboratory from the deep-sea hydrothermal vents. Vent isolates Caminibacter mediatlanticus and Sulfurovum riftiae, belonging to different orders with disparate metabolic lifestyles and environmental requirements, were chosen for comparative genome analysis with Campylobacter jejuni and Helicobacter pylori. The latter two campylobacterial organisms exhibit pathogenic lifestyles and are well characterized as naturally competent, and hence we used their transformation machinery as models to construct a hypothetical model of the transformation pathway in deep-sea vent Campylobacteria. We were able to detect homologs of most transformation proteins that have been demonstrated to be necessary for transformation in diderm bacteria in the two vent representatives. Reconstruction of the phylogeny of some of the key proteins revealed monophyly. Gene synteny analysis of key transformation components showed the conserved arrangement of gene clusters between the vent organisms and their pathogenic relatives, while also revealing some novel proteins unique to these bacteria, which may play a role in their transformation. The comparative genomic analysis shows that the transformation pathway of the pathogenic Campylobacteria has likely evolved from the hydrothermal vent ancestors, which prompted us to hypothesize that the vent Campylobacteria, like their pathogenic relatives, are naturally competent in their optimal growth conditions. A protocol to visualize DNA uptake in Caminibacter mediatlanticus and Sulfurovum riftiae was designed, involving exposure of labelled eDNA fragments to log and stationary phase cultures, with which we demonstrated that C. mediatlanticus was able to take up eDNA in both growth phases. Exposure to the antimicrobial, mitomycin C (MMC), increased the uptake efficiency in C. mediatlanticus by ~2.6 fold, likely demonstrating a DNA-damage-associated DNA uptake response. Proteomic analysis comparing cultures with and without exposure to eDNA in the presence and absence of MMC detected two novel uncharacterized proteins that were induced, along with the upregulation of proteins that, in turn, affect transcriptional regulation of several proteins. This is the first study that shows natural transformation as an actively expressed process in the hydrothermal vents, while also demonstrating in vitro eDNA uptake in an obligately anaerobic, thermophilic, chemolithoautotrophic campylobacterial pure culture from a hydrothermal vent.
Avanthika Bharath (Thu,) studied this question.