197 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress in the metatranscriptome, possibly due to (too) late sampling relative to the salinity increase. Dedicated RT-qPCR experiments with “Ca. Methanoperedens” specific kamA and ablB primers at six different time points and salinity levels revealed strong salinity-dependent upregulation of this gene cluster. In addition, we decided to screen for evidence of ablB expression in publicly available “Ca. Methanoperedens” metatranscriptomes. For the freshwater metatranscriptomes, the expression levels (measured as TPM) were very low, with values ranging from ∼0-2 TPM (Dalcin Martins et al., 2022, Chapter 4) to ∼0-1 TPM (Schoelmerich et al., 2022) (Fig. 3C and Supplementary Fig. 10). For the brackish “Ca. Methanoperedens” metatranscriptomes, values remained low, with ∼80 TPM (planktonic) and ∼40 TPM (granular) (McIlroy et al., 2023) (Fig. 3C and Supplementary Fig. 10). The “Ca. Methanoperedens” LAM (encoded by kamA) and AT (encoded by ablB, Fig. 3AB) cluster phylogenetically with those encoded by other ANME archaea and are distantly related to the first reported reference in methanogenic archaea (Pflüger et al., 2003) (Fig. 3AB). No LAM or AT were retrieved from ANME-1 and ANME-2b groups, suggesting either limited genome coverage of these lineages or, their use of different physiological strategies to cope with salinity stress. Several kamA and ablB genes were encoded on extrachromosomal Borg elements, as reported in the original Borg study (Al-Shayeb et al., 2022). The nature of these elements and gene assimilation process from associated chromosomes is still a conundrum. Al Shayeb et al. (2022) claimed that they found not enough evidence to prove or discard them as archaeal viruses, plasmids or, minichromosomes. Finally, these elements were classified as novel extrachromosomal elements closest probably to megaplasmids which, so far, have only been reported associated with “Ca. Methanoperedens”. In their study, “Ca. Methanoperedens” was also found to encode key functional enzymes such as the MCR complex and genes associated with stress response systems, including putative N(ε)-acetyl-β-L-lysine production potential (Al-Shayeb et al., 2022). Taking all together, the presence of N(ε)-acetyl-βL-lysine biosynthesis genes in both “Ca. Methanoperedens” chromosome and Borgs makes kamA and ablB suitable candidates for phylogenetic studies. On the one hand, we showed that Borgs might have assimilated the LAM and AT encoding kamA and ablB-gene operon from a close relative of our current study’s “Ca. Methanoperedens 6
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