BACKGROUND: Microorganisms living in saline environments are forced to regulate turgor via the synthesis of organic osmoprotective compounds. Microbial adaptation to fluctuations in external salinity includes degradation of compatible solutes. Here we have examined the biochemical pathway of degradation of the cyclic imino acid ectoine, the major osmoprotector in halotolerant methane-utilizing bacteria. METHODS: The BLAST search of the genes involved in ectoine degradation in the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z was performed with the reference sequences of Halomonas elongata. The genes for the key enzymes of the pathway were disrupted by insertion mutagenesis and the cellular metabolites in the methanol extracts of mutant cells were analyzed by HPLC. The doeA gene from Mm. alcaliphilum 20Z was heterologously expressed in Escherichia coli to identify the product of ectoine hydrolysis catalyzed by ectoine hydrolase DoeA. RESULTS: We have shown that the halotolerant methanotroph Mm. alcaliphilum 20Z possesses the doeBDAC gene cluster coding for putative ectoine hydrolase (DoeA), Nα-acetyl-L-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD) and aspartate-semialdehyde dehydrogenase (DoeC). The deletion of the doeA gene resulted in accumulation of the higher level of ectoine compared to the wild type strain. Nγ-acetyl-L-2,4-diaminobutyrate (Nγ-acetyl-DAB), a substrate for ectoine synthase, was found in the cytoplasm of the wild type strain. Nα-acetyl-L-2,4-diaminobutyrate (Nα-acetyl-DAB), a substrate for the DoeB enzyme, appeared in the cells as a result of exposure of the doeB mutant to low osmotic pressure. The genes for the enzymes involved in ectoine degradation were found in all aerobic methylotrophs capable of ectoine biosynthesis. These results provide the first evidence for the in vivo operation of the ectoine degradation pathway in methanotrophs and thus expand our understanding of the regulation mechanisms of bacterial osmoadaptation. CONCLUSIONS: During adaptation to the changes in external osmolarity, halophilic and halotolerant methylotrophs cleave ectoine, thereby entering the carbon and nitrogen of the compatible solute to the central metabolic pathways. The biochemical route of ectoine degradation in the halotolerant methanotroph Mm. alcaliphilum 20Z is similar to that in heterotrophic halophiles. We have shown that ectoine hydrolase DoeA in this methanotroph hydrolyzes ectoine with the formation of the only isomer: Nα-acetyl-DAB. All aerobic methylotrophs capable of ectoine biosynthesis harbor the genetic determinants for ectoine degradation.
BACKGROUND: Microorganisms living in saline environments are forced to regulate turgor via the synthesis of organic osmoprotective compounds. Microbial adaptation to fluctuations in external salinity includes degradation of compatible solutes. Here we have examined the biochemical pathway of degradation of the cyclic imino acidectoine, the major osmoprotector in halotolerantmethane-utilizing bacteria. METHODS: The BLAST search of the genes involved in ectoine degradation in the halotolerantmethanotroph Methylotuvimicrobium alcaliphilum 20Z was performed with the reference sequences of Halomonas elongata. The genes for the key enzymes of the pathway were disrupted by insertion mutagenesis and the cellular metabolites in the methanol extracts of mutant cells were analyzed by HPLC. The doeA gene from Mm. alcaliphilum 20Z was heterologously expressed in Escherichia coli to identify the product of ectoine hydrolysis catalyzed by ectoine hydrolase DoeA. RESULTS: We have shown that the halotolerantmethanotroph Mm. alcaliphilum 20Z possesses the doeBDAC gene cluster coding for putative ectoine hydrolase (DoeA), Nα-acetyl-L-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD) and aspartate-semialdehyde dehydrogenase (DoeC). The deletion of the doeA gene resulted in accumulation of the higher level of ectoine compared to the wild type strain. Nγ-acetyl-L-2,4-diaminobutyrate (Nγ-acetyl-DAB), a substrate for ectoine synthase, was found in the cytoplasm of the wild type strain. Nα-acetyl-L-2,4-diaminobutyrate (Nα-acetyl-DAB), a substrate for the DoeB enzyme, appeared in the cells as a result of exposure of the doeB mutant to low osmotic pressure. The genes for the enzymes involved in ectoine degradation were found in all aerobic methylotrophs capable of ectoine biosynthesis. These results provide the first evidence for the in vivo operation of the ectoine degradation pathway in methanotrophs and thus expand our understanding of the regulation mechanisms of bacterial osmoadaptation. CONCLUSIONS: During adaptation to the changes in external osmolarity, halophilic and halotolerant methylotrophs cleave ectoine, thereby entering the carbon and nitrogen of the compatible solute to the central metabolic pathways. The biochemical route of ectoine degradation in the halotolerantmethanotroph Mm. alcaliphilum 20Z is similar to that in heterotrophic halophiles. We have shown that ectoine hydrolase DoeA in this methanotroph hydrolyzes ectoine with the formation of the only isomer: Nα-acetyl-DAB. All aerobic methylotrophs capable of ectoine biosynthesis harbor the genetic determinants for ectoine degradation.
Authors: Lucas Hermann; Felix Dempwolff; Wieland Steinchen; Sven-Andreas Freibert; Sander H J Smits; Andreas Seubert; Erhard Bremer Journal: Front Microbiol Date: 2021-12-24 Impact factor: 5.640