Data_Sheet_1_Characterization of D-Arabitol as Newly Discovered Carbon Source of Bacillus methanolicus.pdf

Bacillus methanolicus is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, B. methanolicus is also known to utilize D-mannitol, D-glucose and, as recently discovered, sugar alcohol D-arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in B. methanolicus. 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 B. methanolicus 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 B. methanolicus, two complementation experiments were performed in an arabitol negative Corynebacterium glutamicum 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 atlABC) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to atlD). C. glutamicum is a natural D-arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The C. glutamicum mtlD deletion mutant was chosen for complementation experiments. Heterologous expression of atlABCD as well as the arabitol phosphate dehydrogenase gene atlD from B. methanolicus alone restored growth of the C. glutamicum ΔmtlD mutant with arabitol. Furthermore, D-arabitol phosphate dehydrogenase activities could be detected in crude extracts of B. methanolicus and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, B. methanolicus 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.

甲醇芽孢杆菌（Bacillus methanolicus）是一类革兰氏阳性（Gram-positive）、嗜热的利用甲醇的细菌。作为兼性甲基营养型菌，该菌除已知可利用D-甘露醇、D-葡萄糖外，近期还被发现可利用糖醇D-阿拉伯糖醇。目前学界已阐明甲醇、甘露醇及葡萄糖的代谢途径，但甲醇芽孢杆菌中阿拉伯糖醇的分解代谢机制仍未得到解析。本研究完成了这一迄今未被探明的代谢途径的解析工作。为验证我们针对阿拉伯糖醇利用相关编码基因的预测，本研究以甘露醇为对照，对以阿拉伯糖醇为唯一碳源培养的甲醇芽孢杆菌MGA3菌株开展了转录组测序（RNA-seq）的差异基因表达分析。我们鉴定得到一个由8个基因组成的基因簇，该基因簇在以阿拉伯糖醇为唯一碳源生长时呈上调表达状态。后续通过实时定量逆转录PCR（qRT-PCR）实验验证了转录组测序的结果。我们对转录组测序鉴定得到的基因簇的转录组织结构进行了分析，结果显示阿拉伯糖醇利用相关基因以操纵子形式共转录，其转录区间从BMMGA3_RS07325延伸至BMMGA3_RS07365。由于目前甲醇芽孢杆菌尚无法开展基因敲除研究，本研究利用转录组分析筛选得到的4个候选基因——编码阿拉伯糖醇摄取磷酸转移酶系统（PTS）的BMMGA3_RS07330、BMMGA3_RS07335和BMMGA3_RS07340（三者重新命名为atlABC），以及编码阿拉伯糖醇磷酸脱氢酶的BMMGA3_RS07345（重新命名为atlD），在阿拉伯糖醇利用缺陷型谷氨酸棒杆菌（Corynebacterium glutamicum）菌株中开展了两项互补实验。谷氨酸棒杆菌是天然的D-阿拉伯糖醇利用菌，其阿拉伯糖醇分解代谢依赖阿拉伯糖醇脱氢酶MtlD。本研究选用谷氨酸棒杆菌mtlD基因缺失突变株作为互补实验宿主。将atlABCD基因簇以及单独的甲醇芽孢杆菌来源阿拉伯糖醇磷酸脱氢酶基因atlD进行异源表达后，均可恢复谷氨酸棒杆菌ΔmtlD突变株在阿拉伯糖醇培养基中的生长能力。此外，我们在甲醇芽孢杆菌的粗提取物中检测到了阿拉伯糖醇磷酸脱氢酶活性，且该酶活性在阿拉伯糖醇培养的菌体中显著高于甲醇或甘露醇培养的菌体。综上，甲醇芽孢杆菌携带一个可被阿拉伯糖醇诱导的操纵子，该操纵子编码包括磷酸转移酶系统及阿拉伯糖醇磷酸脱氢酶在内的功能蛋白，负责摄取并活化阿拉伯糖醇作为生长底物。