Data_Sheet_5_Comprehensive Comparative Genomics and Phenotyping of Methylobacterium Species.ZIP

The pink-pigmented facultative methylotrophs (PPFMs), a major bacterial group found in the plant phyllosphere, comprise two genera: Methylobacterium and Methylorubrum. They have been separated into three major clades: A, B (Methylorubrum), and C. Within these genera, however, some species lack either pigmentation or methylotrophy, which raises the question of what actually defines the PPFMs. The present study employed a comprehensive comparative genomics approach to reveal the phylogenetic relationship among the PPFMs and to explain the genotypic differences that confer their different phenotypes. We newly sequenced the genomes of 29 relevant-type strains to complete a dataset for almost all validly published species in the genera. Through comparative analysis, we revealed that methylotrophy, nitrate utilization, and anoxygenic photosynthesis are hallmarks differentiating the PPFMs from the other Methylobacteriaceae. The Methylobacterium species in clade A, including the type species Methylobacterium organophilum, were phylogenetically classified into six subclades, each possessing relatively high genomic homology and shared phenotypic characteristics. One of these subclades is phylogenetically close to Methylorubrum species; this finding led us to reunite the two genera into a single genus Methylobacterium. Clade C, meanwhile, is composed of phylogenetically distinct species that share relatively higher percent G+C content and larger genome sizes, including larger numbers of secondary metabolite clusters. Most species of clade C and some of clade A have the glutathione-dependent pathway for formaldehyde oxidation in addition to the H4MPT pathway. Some species cannot utilize methanol due to their lack of MxaF-type methanol dehydrogenase (MDH), but most harbor an XoxF-type MDH that enables growth on methanol in the presence of lanthanum. The genomes of PPFMs encode between two and seven (average 3.7) genes for pyrroloquinoline quinone-dependent alcohol dehydrogenases, and their phylogeny is distinctly correlated with their genomic phylogeny. All PPFMs were capable of synthesizing auxin and did not induce any immune response in rice cells. Other phenotypes including sugar utilization, antibiotic resistance, and antifungal activity correlated with their phylogenetic relationship. This study provides the first inclusive genotypic insight into the phylogeny and phenotypes of PPFMs.

粉色菌落兼性甲基营养菌（pink-pigmented facultative methylotrophs, PPFMs）是栖息于植物叶圈（plant phyllosphere）的主要细菌类群，包含两个属：甲基杆菌属（Methylobacterium）与甲基红球菌属（Methylorubrum）。它们被划分为三个主要演化支：A、B（即甲基红球菌属）与C。然而，在这些属中，部分物种既缺乏色素沉着也不具备甲基营养能力，这一现象引发了关于PPFMs的精准界定标准的学术疑问。本研究采用全面的比较基因组学分析策略，阐明了PPFMs之间的系统发育关系，并揭示了赋予其差异化表型的基因型基础。本研究新测序了29株相关模式菌株的基因组，以此构建覆盖该两属几乎所有正式发表物种的完整数据集。通过比较基因组分析，本研究发现甲基营养能力、硝酸盐利用能力与不产氧光合作用是区分PPFMs与其他甲基杆菌科（Methylobacteriaceae）物种的核心特征。演化支A中的甲基杆菌属物种（包括模式种嗜有机甲基杆菌Methylobacterium organophilum）在系统发育层面被划分为6个亚支，每个亚支均具有较高的基因组同源性且共享统一的表型特征。其中一个亚支与甲基红球菌属物种的系统发育关系极为接近，这一发现促使我们将这两个属重新合并为单一的甲基杆菌属。与此同时，演化支C由系统发育独特的物种组成，这类物种具有相对更高的G+C碱基占比与更大的基因组规模，且包含更多的次级代谢产物基因簇。演化支C的多数物种与演化支A的部分物种，除了拥有H4MPT途径外，还具备谷胱甘肽依赖型甲醛氧化途径。部分物种因缺乏MxaF型甲醇脱氢酶（methanol dehydrogenase, MDH）而无法利用甲醇，但多数PPFMs携带XoxF型甲醇脱氢酶，使其可在镧元素存在的条件下以甲醇为唯一碳源进行生长。PPFMs的基因组编码2至7个（平均3.7个）吡咯喹啉醌（pyrroloquinoline quinone, PQQ）依赖型乙醇脱氢酶基因，其系统发育关系与基因组系统发育呈现显著相关性。所有PPFMs均能够合成生长素，且不会在水稻细胞中引发免疫应答。其余表型特征包括糖类利用能力、抗生素抗性与抗真菌活性，均与其系统发育关系密切相关。本研究首次为PPFMs的系统发育与表型特征提供了全面的基因型解析视角。