AUTHOR=López Marina Gil , Irla Marta , Brito Luciana F. , Wendisch Volker F. 

TITLE=Characterization of D-Arabitol as Newly Discovered Carbon Source of Bacillus methanolicus

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01725

DOI=10.3389/fmicb.2019.01725

ISSN=1664-302X

ABSTRACT=<p><italic>Bacillus methanolicus</italic> is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, <italic>B. methanolicus</italic> is also known to utilize <sc>D</sc>-mannitol, <sc>D</sc>-glucose and, as recently discovered, sugar alcohol <sc>D</sc>-arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in <italic>B. methanolicus</italic>. In this work we present the elucidation of this hitherto uncharted pathway. In order to confirm our predictions regarding genes coding for arabitol utilization, we performed differential gene expression analysis of <italic>B. methanolicus</italic> MGA3 cells grown on arabitol as compared to mannitol via transcriptome sequencing (RNA-seq). We identified a gene cluster comprising eight genes that was up-regulated during growth with arabitol as a sole carbon source. The RNA-seq results were subsequently confirmed via qRT-PCR experiments. The transcriptional organization of the gene cluster identified via RNA-seq was analyzed and it was shown that the arabitol utilization genes are co-transcribed in an operon that spans from BMMGA3_RS07325 to BMMGA3_RS07365. Since gene deletion studies are currently not possible in <italic>B. methanolicus</italic>, two complementation experiments were performed in an arabitol negative <italic>Corynebacterium glutamicum</italic> strain using the four genes discovered via RNA-seq analysis as coding for a putative PTS for arabitol uptake (BMMGA3_RS07330, BMMGA3_RS07335, and BMMGA3_RS07340 renamed to <italic>atlABC</italic>) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to <italic>atlD</italic>). <italic>C. glutamicum</italic> is a natural <sc>D</sc>-arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The <italic>C. glutamicum mtlD</italic> deletion mutant was chosen for complementation experiments. Heterologous expression of <italic>atlABCD</italic> as well as the arabitol phosphate dehydrogenase gene <italic>atlD</italic> from <italic>B. methanolicus</italic> alone restored growth of the <italic>C. glutamicum</italic> Δ<italic>mtlD</italic> mutant with arabitol. Furthermore, <sc>D</sc>-arabitol phosphate dehydrogenase activities could be detected in crude extracts of <italic>B. methanolicus</italic> and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, <italic>B. methanolicus</italic> possesses an arabitol inducible operon encoding, amongst others, a putative PTS system and an arabitol phosphate dehydrogenase for uptake and activation of arabitol as growth substrate.</p>