Adaptation strategies of iron-oxidizing bacteria <i>Gallionella</i> and Zetaproteobacteria crossing the marine–freshwater barrier

Iron-oxidizing Betaproteobacteria and Zetaproteobacteria are generally associated with freshwater and marine environments, respectively. Despite repeated cross-environment observations of these taxa, there has been no focused exploration of the genomes of marine <i>Gallionella</i> (Betaproteobacteria) to understand transitions between freshwater and marine habitats. Consequently, their roles in these environments remain uncertain. Here, we present strong evidence for the co-occurrence of <i>Gallionella</i> and Zetaproteobacteria at deep-sea hydrothermal vents at the Arctic Mid-Ocean Ridges through metagenomic analyses. Phylogenomic analysis of <i>Gallionella</i> metagenome-assembled genomes (MAGs) suggests that seawater adaptation is an evolutionary event that occurred multiple times in distinct lineages. Similarly, several distinct evolutionary events for freshwater and terrestrial <i>Mariprofundus</i> and other Zetaproteobacteria are predicted. The presence of <i>c</i>-type cytochrome <i>cyc2</i> iron oxidation genes in co-occurring marine Betaproteobacteria and Zetaproteobacteria implies an overlap in niches of these iron-oxidizers. Functional enrichment analyses reveal genetic differences between marine MAGs of both iron-oxidizing groups and their terrestrial aquatic counterparts linked to salinity adaptation. Though scanning electron microscopy confirms the presence of Fe(III) oxyhydroxide stalks where <i>Gallionella</i> and <i>Mariprofundus</i> co-occur, <i>Gallionella</i> MAGs from hydrothermal vents lack evidence of putative stalk formation genes. <i>Mariprofundus</i> is therefore the likely sole stalk-producing iron-oxidizer in this environment. Conversely, the discovery of putative stalk formation genes in <i>Mariprofundus</i> MAGs across the marine-freshwater barrier suggests that Fe(III) oxyhydroxide stalks might not be an exclusive signature for single iron-oxidizing taxa in marine and freshwater environments. Our research provides novel insights into the iron-oxidizing capacities, stalk production, environmental adaptation, and evolutionary transitions between marine and freshwater habitats for <i>Gallionella</i> and Zetaproteobacteria.IMPORTANCEIron-oxidizing bacteria (FeOB) play an important role in the global cycling of iron, carbon, and other metals. While it has previously been assumed that bacterial evolution does not frequently involve crossing the salinity barrier, recent studies indicate that such occurrences are more common than previously thought. Our study offers strong evidence that this also happens among FeOB, with new insights into how these bacteria adapt to the new environment, including hydrothermal vents and freshwater habitats. In addition, we emphasize the importance of accurate iron-oxidizing taxa identification through sequencing, rather than relying solely on the morphology of Fe(III) oxyhydroxides and environment. On a larger scale, microorganisms within established communities need to respond to changes in salinity due to events like seawater intrusion in coastal aquifers, and thus, our findings underscore the importance of knowledge of transitions across habitat types with different salt concentrations.