The pan-genome of the dominant human gut-associated archaeon, Methanobrevibacter smithii. The pan-genome of the dominant human gut-associated archaeon, Methanobrevibacter smithii

The human gut microbiota harbors methanogens represented by the dominant archaeon, Methanobrevibacter smithii, a polyphyletic group of acetogens, and sulfate-reducing bacteria. Defining their roles in the H2-economy of the gut has potential therapeutic importance for modulating the efficiency of fermentation of dietary components. We quantified methanogens in fecal samples from 40 healthy adult female monozygotic(MZ) and 28 dizygotic(DZ) twin pairs, analyzed bacterial 16S rRNA datasets generated from their fecal samples to identify taxa that co-occur with methanogens, sequenced the genomes of 20 M. smithii strains isolated from families of MZ and DZ twins, and performed RNA-Seq of a subset of strains to identify their responses to varied formate concentrations. The concordance rate for methanogen carriage was significantly higher for MZ versus DZ twin pairs. Co-occurrence analysis revealed 22 bacterial species-level taxa positively correlated with methanogens: all but two were members of the Clostridiales, with several being, or related to, known hydrogen-producing and -consuming bacteria. The M. smithii pan-genome contains 987 genes conserved in all strains, and 1860 variably represented genes. Strains from MZ and DZ twin pairs had a similar degree of shared genes and SNPs, and were significantly more similar than strains isolated from mothers or members of other families. The 101 adhesin-like proteins(ALPs) in the pan-genome (45±6/strain) exhibit strain-specific differences in expression and responsiveness to formate. We hypothesize that M. smithii strains use their different repertoires of ALPs to create diversity in their metabolic niches, by allowing them to establish syntrophic relationships with bacterial partners with differing metabolic capabilities and patterns of co-occurrence These strains were isolated from human feces, but they are in pure culture now. All the information about each species is associated with the genome accession number Overall design: Fecal samples from 40 healthy adult female monozygotic(MZ) and 28 dizygotic(DZ) twin pairs, analyzed bacterial 16S rRNA datasets generated from their fecal samples to identify taxa that co-occur with methanogens, sequenced the genomes of 20 M. smithii strains isolated from families of MZ and DZ twins, and performed RNA-Seq of a subset of strains to identify their responses to varied formate concentrations. Strains of Methanobrevibacter smithii were grown in vitro (modified MBC media) to mid-log phase, at 37°C in serum bottles pressurized with 80% hydrogen, 20% CO2 gasses at 30psi. Cells were harvested by centrifugation, and DNA was isolated by phenol-chloroform and ethanol precipitation.

人体肠道微生物组中栖息有以优势古菌史氏甲烷短杆菌（Methanobrevibacter smithii）为代表的产甲烷古菌、多系群产乙酸菌以及硫酸盐还原菌。明确这类微生物在肠道氢代谢中的作用，对于调控膳食组分的发酵效率具有潜在治疗学意义。我们对40对健康成年女性同卵双生子（MZ）与28对异卵双生子（DZ）的粪便样本中的产甲烷古菌进行定量检测；对粪便样本来源的细菌16S rRNA测序数据集进行分析，以鉴定与产甲烷古菌共存的分类类群；对从同卵双生子及异卵双生子家族中分离得到的20株史氏甲烷短杆菌进行基因组测序；并对部分菌株开展RNA测序（RNA-Seq），以明确其对不同浓度甲酸盐的响应情况。同卵双生子组中产甲烷古菌定植的一致率显著高于异卵双生子组。共存分析共鉴定出22个与产甲烷古菌呈正相关的细菌物种级类群，其中除2个类群外，其余均隶属于梭菌目（Clostridiales），部分类群为已知产氢及耗氢细菌，或与其亲缘关系密切。史氏甲烷短杆菌的泛基因组包含987个所有菌株均保守的基因，以及1860个存在差异的基因。同卵双生子与异卵双生子来源的菌株，在共享基因与单核苷酸多态性（SNPs）的分布程度上相近，且其相似性显著高于来自母亲或其他家族成员的菌株。泛基因组中的101个黏素样蛋白（ALPs，平均每株菌含45±6个）在表达水平及对甲酸盐的响应上存在菌株特异性差异。我们推测，史氏甲烷短杆菌菌株可通过其不同的黏素样蛋白谱，与具备不同代谢能力及共存模式的细菌伙伴建立互养关系，从而在代谢生态位中形成多样性。本研究分离得到的菌株虽源自人类粪便，目前均已获得纯培养。所有物种相关信息均与基因组登录号相关联。总体实验设计：采集40对健康成年女性同卵双生子（MZ）与28对异卵双生子（DZ）的粪便样本，对粪便样本来源的细菌16S rRNA测序数据集进行分析以鉴定与产甲烷古菌共存的分类类群；对从上述双生子家族中分离得到的20株史氏甲烷短杆菌进行基因组测序；并对部分菌株开展RNA测序（RNA-Seq）以明确其对不同浓度甲酸盐的响应情况。将史氏甲烷短杆菌菌株置于改良MBC培养基中体外培养，于37℃、充入80%氢气与20%二氧化碳混合气并加压至30psi的血清瓶中培养至对数中期。通过离心法收集细胞，采用酚-氯仿法与乙醇沉淀法提取基因组DNA。