Methylotrophy in Azospirillum thiophilum BV-S

Abstract

Metanotrophs and methylotrophs are an ecologically significant group of microorganisms that are able to use reduced single-carbon compounds such as methane, methanol, methylated amines and methylated sulfur compounds as the single carbon source and as the energy source. These groups of microorganisms have a global significance as the main consumers of greenhouse gases and as key members of microbial communities living in environments full of organic C1-compounds. The genes encoding enzymes of methanol oxidation have also been found in the genomes of four other sequenced Azospirillum species, A. lipoferum, A. brasilense, A.oryzae and A. humicireducens. However there is still no experimental data establishing their ability to oxidize methanol at the moment.

The ability to oxidize single carbon compounds can be explained by the living conditions of this strain. A. thiophillum, unlike other representatives of the genus, that are localized in the soil, lives in hydrogen sulfide springs in oil and gas bearing area. Their metabolism leads to the production of methanol, formate and formaldehyde, that are oxidized by methylotrophic bacteria.

The aim of this work was to prove the ability of Azospirillum thiophilum BV-S to the methylotrophic growth. For this purpose the following methods were used: the cultivation of the bacteria in heterotrophic and methylotrophic conditions, annotation of the genome using the automatic annotator RAST, spectrophotometric measurement of the enzymatic activities, extraction of RNA using sorbent microcolumns, reverse transcription, qPCR. The genes encoding enzymes for methanol oxidation, tetrahydrometanopterine pathway and formate oxidation were identified in the genome of Azospirillum thiophilum BV-S. Experimental data confirmed the ability of Azospirillum thiophilum BV-S to methylotrophic growth: high activity of methanol dehydrogenase and formate dehydrogenase in methylotrophic conditions; an increase in the expression of mdh2 and xoxF genes in methylotrophic cultivation as compared to heterotrophic was shown.