Hirschfeld, C. | Search bioRxiv

bioRxiv2020

Anaerobic sulfur oxidation underlies adaptation of a chemosynthetic symbiont to oxic-anoxic interfaces

Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by monocultures of thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. However, nothing is known about how these variations affect their physiology or metabolism. Here, by applying omics, Raman microspectroscopy and stable isotope labelling, we investigated the effect of oxygen on Candidatus Thiosymbion oneisti. Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation were upregulated in oxic conditions, together with genes involved in organic carbon assimilation, polyhydroxyalkanoate (PHA) biosynthesis, nitrogen fixation and urea utilization. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand its deleterious effects, and the symbiont appeared to proliferate less. Based on its physiological response to oxygen, we propose that Ca. T. oneisti may exploit anaerobic sulfur oxidation coupled to denitrification to proliferate in anoxic sand. However, the ectosymbiont would still profit from the oxygen available in superficial sand, as the energy-efficient aerobic respiration would facilitate carbon and nitrogen assimilation by the ectosymbiont. IMPORTANCEChemoautotrophic endosymbionts are famous for exploiting sulfur oxidization to feed marine organisms with fixed carbon. However, the physiology of thiotrophic bacteria thriving on the surface of animals (ectosymbionts) is less understood. One long standing hypothesis posits that attachment to animals that migrate between reduced and oxic environments would boost sulfur oxidation, as the ectosymbionts would alternatively access sulfide and oxygen, the most favorable electron acceptor. Here, we investigated the effect of oxygen on the physiology of Candidatus Thiosymbion oneisti, a Gammaproteobacterium which lives attached to marine nematodes inhabiting shallow water sand. Surprisingly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions. Furthermore, under anoxia, the ectosymbiont appeared to be less stressed and to proliferate more. We propose that animal-mediated access to oxygen, rather than enhancing sulfur oxidation, would facilitate assimilation of carbon and nitrogen by the ectosymbiont.

microbiology↗

bioRxiv2019

A robust metaproteomics pipeline for a holistic taxonomic and functional characterization of microbial communities from marine particles

This study aimed to establish a robust, reproducible and reliable metaproteomic pipeline for an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we compared six well-established protein extraction protocols together with different MS-sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In this optimized workflow, proteins are extracted using a combination of SDS-containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel digested and analysed by LC-MS/MS, before MASCOT search against a metagenome-based database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver.\n\nAs proof of principle, free-living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9,354 and 5,034 identified protein groups for FL and PA bacteria, respectively. Our data revealed that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. In contrast, significant functional differences including proteins involved in polysaccharide degradation, sugar- and phosphorus uptake, adhesion, motility, and stress response were detected.\n\nOriginality-Significance StatementMarine particles consist of organic particulate matter (e.g. phyto- or zooplankton) and particle-associated (PA) microbial communities, which are often embedded in a sugary matrix. A significant fraction of the decaying algal biomass in marine ecosystems is expected to be mineralized by PA heterotrophic communities, which are thus greatly contributing to large-scale carbon fluxes. Whilst numerous studies have investigated the succession of planktonic marine bacteria along phytoplankton blooms, the community structure and functionality of PA bacterial communities remained largely unexplored and knowledge on specific contributions of these microorganisms to carbon cycling is still surprisingly limited. This has been mostly been due to technical problems, i.e. to the difficulty to retrieve genomic DNA and proteins from these polysaccharide-rich entities, their enormous complexity and the high abundance of eukaryotic microorganisms.\n\nOur study presents an innovative, robust, reproducible, and reliable metaproteomics pipeline for marine particles, which will help to address and fill the above-described knowledge gap. Employing the here established workflow enabled us to identify more than 5,000 PA proteins, which is, at least to our knowledge, the largest number of protein groups ever assigned to marine particles. Notably, the novel pipeline has been validated by a first, comparative metaproteome analysis of free-living and PA bacterial communities indicating a significant functional shift enabling surface-associated bacteria to adapt to particle-specific living conditions. In conclusion, our novel metaproteomics pipeline presents a solid and promising methodological groundwork for future culture-independent analyses of seasonal taxonomic and functional successions of PA microbial communities in aquatic habitats.

microbiology↗