'Omics analyses of the hydraulically fractured shale isolate Halanaerobium highlights membrane modifications that underpin adaptation under deep subsurface biogeochemical drivers

The Gram-positive Halanaerobium spp. is a dominant bacterial genus across different fractured shale formations, which are increasingly used for natural resource extraction. These bacteria encounter harsh physicochemical conditions in the deep terrestrial biosphere, including high temperatures, brine-level salinities, and elevated pressures. Microbial membranes act as the first line of defense against these environmental stressors, and maintaining membrane functionality during changing environmental conditions requires careful regulation of intact lipid composition and membrane-embedded proteins. To investigate membrane response to varying growth rates and temperatures, we cultivated Halanaerobium congolense WG10 in continuous culture (chemostats) for the first time. 'Omics analysis of metabolites, proteins and lipids for steady state cells revealed that H. congolese WG10 alters its cell membrane to maintain membrane fluidity while maximizing growth rate. H. congolense WG10 modulates the ratio of phospholipid headgroups in response to changes in temperature, and increases cardiolipin and phosphatidylethanolamine polar lipid abundance with increasing carbon availability. We also observed higher abundance of neutrally charged simple glycerol lipids at lower temperature and growth rates, while glycerophospholipids with larger polar heads and zwitterionic lipids prevail at warmer temperatures and faster growth rates. Proteomics analysis identified a total of 2,227 out of 2,800 predicted protein-coding genes. Among these, 356 proteins were significantly higher in abundance in one or more treatments, including known stress regulators involved in cellular envelope homeostasis such as cold shock proteins (CspA), a typA, bipA GTP binding protein involved in stress response, and a nucleotide-binding universal stress protein (UspA). We also identified lipid-A synthesis proteins at lower temperature or high growth rate, a lipopolysaccharide endotoxin uncommonly found in Gram-positive bacteria. Collectively, our 'omics continuous culture approach sheds new light on the metabolism and membrane features of the halotolerant bacterium Halanaerobium under biogeochemical drivers relevant to engineered shale, with implications on membrane charge, permeability, and metabolism.