Expression of an alcohol dehydrogenase gene in a heterotrophic bacterium induces carbon dioxide-dependent high-yield growth under oligotrophic conditions

Abstract

<jats:p> <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.9624" xlink:type="simple">Sphingobium japonicum</jats:ext-link> </jats:named-content> </jats:italic> strain UT26, whose γ-hexachlorocyclohexane-degrading ability has been studied in detail, is a typical aerobic and heterotrophic bacterium that needs organic carbon sources for its growth, and cannot grow on a minimal salt agar medium prepared without adding any organic carbon sources. Here, we isolated a mutant of UT26 with the ability to grow to visible state on such an oligotrophic medium from a transposon-induced mutant library. This high-yield growth under oligotrophic conditions (HYGO) phenotype was CO<jats:sub>2</jats:sub>-dependent and accompanied with CO<jats:sub>2</jats:sub> incorporation. In the HYGO mutant, a transposon was inserted just upstream of the putative Zn-dependent alcohol dehydrogenase (ADH) gene (<jats:italic>adhX</jats:italic>) so that the <jats:italic>adhX</jats:italic> gene was constitutively expressed, probably by the transposon-derived promoter. The <jats:italic>adhX</jats:italic>-deletion mutant (UT26DAX) harbouring a plasmid carrying the <jats:italic>adhX</jats:italic> gene under the control of a constitutive promoter exhibited the HYGO phenotype. Moreover, the HYGO mutants spontaneously emerged among the UT26-derived hypermutator strain cells, and <jats:italic>adhX</jats:italic> was highly expressed in these HYGO mutants, while no HYGO mutant appeared among UT26DAX-derived hypermutator strain cells, indicating the necessity of <jats:italic>adhX</jats:italic> for the HYGO phenotype. His-tagged AdhX that was expressed in <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3093" xlink:type="simple">Escherichia coli</jats:ext-link> </jats:named-content> </jats:italic> and purified to homogeneity showed ADH activity towards methanol and other alcohols. Mutagenesis analysis of the <jats:italic>adhX</jats:italic> gene indicated a correlation between the ADH activity and the HYGO phenotype. These results demonstrated that the constitutive expression of an <jats:italic>adhX</jats:italic>-encoding protein with ADH activity in UT26 leads to the CO<jats:sub>2</jats:sub>-dependent HYGO phenotype. Identical or nearly identical <jats:italic>adhX</jats:italic> orthologues were found in other sphingomonad strains, and most of them were located on plasmids, suggesting that the <jats:italic>adhX</jats:italic>-mediated HYGO phenotype may be an important adaptation strategy to oligotrophic environments among sphingomonads.</jats:p>

Oligotrophic Gene Expression in Rhodococcus erythropolis N9T-4 under Various Nutrient Conditions

“…Furthermore, DNA microarray analysis revealed that two genes, aldA and mnoA, encoding NAD-ALDH and NDMA-MDH, respectively, were strongly expressed under oligotrophic conditions [6]. All the super oligotrophs that we isolated previously belonged to the genera Rhodococcus and Streptomyces [1,5], and Nagata found that a transposon-induced mutant of Sphingobium japonicum UT26 showed oligotrophic features, and a Zn-dependent alcohol dehydrogenase was essential for oligotrophic growth [7].…”