Metabolic strategies of marine subseafloor Chloroflexi inferred from genome reconstructions

Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood–Ljungdahl pathway, electron bifurcation, and ferredoxin-dependent transport-coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO2 reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H2 consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class

A novel family of functional operons encoding methane/ammonia monooxygenase-related proteins in gammaproteobacterial methanotrophs

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In-Situ Activity of the Heterotrophic Microbial Community in Seep Sediments from the Santa Monica Basin

The role of microbes in marine sediments is of great importance for the understanding of processes and elemental cycles in the ocean. However, many microbes inhabiting the seafloor remain unculturable because the in-situ conditions are difficult to mimic in a laboratory setting. Incubations of sediment slurries are likewise limited as the retrieval of sample material from the seafloor is associated with depressurization and disruption of the sedimentary matrix, with still unknown effects on the microbial community and its activity. To overcome some limitations of in vitro studies, we used an in-situ injector coring system deployed in the deep sea by a remotely operated vehicle. Specially designed push cores enable the injection of different substrates, including stable isotope labeled compounds, into the sediment, allowing an incubation directly in the deep-sea environment with minimal disturbance of the microbial community. For this study, an injector core and a reference neighboring push core without injected fluid were inserted in the center of an orange-colored sulfur-oxidizing microbial mat at an active methane seep in the Santa Monica Basin at 800 m water depth (Fig. 1a, b). 13C-labeled glucose, deuterated water and homopropargylglycine (HPG), an alkyne modified methionine analog, were injected through multiple needles aligned along the vertical core axis into the sediment. The core was incubated directly at the seafloor with the goal of better understanding the activity and diversity of heterotrophic microorganisms within this seep setting under in situ conditions. A similar injector core approach with 13C-labeled glucose (Takano et al., 2010) demonstrated archaeal activity after 9 days through the labeling of the glycerol backbone of archaeal membrane lipids. Here, we expand the scope of injected stable isotope labels and combine them with single cell resolved biorthogonal non-canonical amino acid tagging (BONCAT; Hatzenpichler et al., 2016) and fluorescence in situ hybridization microscopy (FISH) over a relatively short incubation period

Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function

We have coupled fluorescence in situ hybridization (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of 13C incorporation into microbial cells. Highly resolved Raman confocal spectra were generated for individual cells which were grown in minimal medium where the ratio of 13C to 12C content of the sole carbon source was incrementally varied. Cells which were 13C-labelled through anabolic incorporation of the isotope exhibited key red-shifted spectral peaks, the calculated ‘red shift ratio’ (RSR) being highly correlated with the 13C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of Fluos, Cy3 and Cy5-labelled microbial populations at the single cell level. Cellular 13C-content determinations exhibited good congruence between fresh cells and FISH hybridized cells indicating that spectral peaks, including phenylalanine resonance, which were used to determine 13C-labelling, were preserved during fixation and hybridization. In order to demonstrate the suitability of this technology for structure–function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseudomonas populations during naphthalene degradation in groundwater microcosms. Raman-FISH extends and complements current technologies such as FISH-microautoradiography and stable isotope probing in that it can be applied at the resolution of single cells in complex communities, is quantitative if suitable calibrations are performed, can be used with stable isotopes and has analysis times of typically 1 min per cell