Date Published: March 14, 2019
Publisher: Public Library of Science
Author(s): Mio Takeuchi, Haruka Ozaki, Satoshi Hiraoka, Yoichi Kamagata, Susumu Sakata, Hideyoshi Yoshioka, Wataru Iwasaki, Ivan A. Berg.
Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M. caenitepidi Gela4 co-cultured with M. marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M. caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M. caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M. marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M. caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M. marinum S8 produced acetate (< 16 μM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M. caenitepidi Gela4 cross-fed by M. marinum S8. Co-culture of M. caenitepidi Gela4 and M. marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria.
Microbial methane oxidation plays an important role in the global methane cycle. Aerobic methane-oxidizing bacteria (methanotrophs) are key players in aerobic and micro-aerobic environments. The development of stable isotope probing has highlighted that methane-derived carbon is incorporated not only by methanotrophs but also by non-methanotrophic bacteria (methylotrophs or others) in diverse environments, and suggests the global importance of cross-feeding interactions in methane oxidation [1, 2, 3, 4]. Methanol is synthesized in the periplasm of methanotrophs, and therefore, may diffuse out of the cell . Hence, it is generally speculated that the co-existence of methanotrophs and non-methanotrophic bacteria mainly involves the cross-feeding of methanol excreted by methanotrophs to non-methanotrophic bacteria . Methanol-dependent cross-feeding between methanotrophs and non-methanotrophic bacteria has been studied since the 1970s [6, 7]. Using isolates from lake sediments, Krause et al.  demonstrated that methanol excreted by a methanotroph is indeed the sole carbon and energy source for the co-existing obligate methylotroph that can utilize methanol or methylamine exclusively.
In the current study, we compared gene expression of the facultative methylotroph M. caenitepidi Gela4 grown in pure culture with methanol as the sole carbon source or co-cultured with the methanotroph M. marinum S8 with methane as the sole carbon source. The cross-feeding compound from methanotrophs to non-methanotrophic bacteria has been often considered as methanol excreted by methanotrophs. Krause et al. (2017)  has examined this interrelationship in detail. The authors conducted transcriptome analysis of two obligate methylotroph strains in co-culture with the methanotroph Methylobacter tundripaludum and demonstrated that the obligate methylotroph relies solely on methanol in co-culture with M. tundripaludum. In the current study, we examined for the first time the response of a facultative methylotroph, M. caenitepidi Gela4, which can assimilate a wide range of substrates in addition to methanol, such as acetate, ethanol, formate and succinate , grown in co-culture with a methanotroph by transcriptome analysis.