168 Chapter 6 One of the most common microbial osmoregulation strategies consists of intracellular osmolyte accumulation. Osmolytes are water-soluble organic compounds that accumulate intracellularly to counter hyperosmotic pressure (Beblo-Vranesevic et al., 2017; Gregory & Boyd, 2021; Guan et al., 2017; Mukhtar et al., 2020). Osmolytes are categorized into sugars, amino acids, polyols and their derivatives. A special case are polyhydroxyalkanoates (PHA), well-described storage polymers, that confer cell stability to halophilic microorganisms (Cui et al., 2017; Salgaonkar et al., 2013). In this vein, “Candidatus (Ca.) Methanoperedens nitroreducens” has been reported to accumulate PHAs (Cai et al., 2019; Frank et al., 2023; McIlroy et al., 2023). Protein glycosylation has been found to be another possible strategy to adapt to saline stress conditions. For example, post translational protein modifications are used as a response to changes in salinity, where for example the S‐layer glycoprotein of Haloferax volcanii was found to show a distinct glycan decoration for either cells grown at high (3.4 M NaCl) or low (1.75 M NaCl) salt concentrations (Guan et al., 2012). Furthermore, sialic acids, negatively charged monosaccharides which can modify surface layer proteins, flagella or, other extracellular proteins such as extracellular polymeric substances (EPS), are yet another potential salinity-stress related adaptation in microorganisms (Kleikamp et al., 2020; Merino & Tomás, 2014; Pabst et al., 2022; Verma et al., 2020; Vikram et al., 2016). Along these lines, de Graaff et al. (2019) presented evidence that sialic acids are found on the EPS of environmental bacteria in sea water-adapted aerobic granular sludge (de Graaff et al., 2019). Here, we evaluated the metabolic and physiological response of a bioreactor enriched in “Ca. Methanoperedens” ANME to salinity stress. A complementary array of methods including bioreactor enrichment, methane oxidation activity assays, PHA visualization and quantification, fluorescent in situ hybridization (FISH) microscopy as well as metagenomics, metatranscriptomics, metaproteomics, and non-targeted metabolomics, revealed that the “Ca. Methanoperedens” produces the osmolyte N(ε)-acetyl-β-L-lysine. We revealed that the responsible genes were probably horizontally acquired from bacterial taxa and can be also encoded on extrachromosomal elements termed Borgs (Al-Shayeb et al., 2022).
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