中国、俄罗斯和蒙古国地区传统发酵乳制品中嗜热链球菌的多位点序列分型研究
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摘要
嗜热链球菌是重要的乳制品发酵剂菌种之一，广泛的应用于各种乳制品的工业生产中。本研究采用纯培养技术对采集于中国新疆、西藏自治区、俄罗斯卡尔梅克共和国和布里亚特自治州的传统发酵乳制品中的嗜热链球菌进行分离，并通过形态学和分子生物学相结合的方法进行鉴定，之后采用多序列位点分型（MLST）技术对分离自中国和俄罗斯地区有代表性的39株嗜热链球菌和先前分离自中国甘肃省、四川省、青海省、内蒙古自治区和蒙古国12个省市的传统发酵乳中的221株嗜热链球菌进行多态性分析和遗传进化研究。
采用M17分离培养基对中国新疆伊犁哈萨克自治州和西藏自治区拉萨市当雄县以及俄罗斯卡尔梅克共和国和布里亚特自治州共223份传统发酵乳制品（包括发酵奶油、发酵奶酪、发酵牦牛乳、发酵马乳、发酵驼奶和发酵牛乳）中的嗜热链球菌进行分离，将革兰氏阳性和过氧化氢酶阴性的球状分离株采用16S rRNA基因序列分析法进行分类鉴定，通过BLAST同源性比对和系统发育树的构建将52株菌鉴定为嗜热链球菌，其中40株分离自从中国新疆伊犁哈萨克自治州的发酵牛乳中，5株分离自中国西藏自治区拉萨市当雄县的发酵牦牛乳中，5株分离自俄罗斯卡尔梅克共和国的发酵牛乳和奶油中，还有2株分离自俄罗斯布里亚特自治州发酵牛乳中。
选取了上述有代表性的39株嗜热链球菌分离株和先前分离自传统发酵乳中的221株分离株进行MLST研究，以嗜热链球菌ND03的全基因组为基础，选取10个看家基因carB、clpX、dnaA、murC、murE、pepN、pepX、pyrG、recA和rpoB作为MLST分型的靶基因。设计引物后，通过PCR扩增、双向测序技术获得靶基因的序列，将其拼接、核对后建立不同的序列型（ST），排除基因的重组交换，应用BioNumerics v6.0、START2.0、eBURST v3.0、DNAsp5.0和SplitsTree4.0等生物信息学软件对等位基因的ST型和核苷酸序列进行遗传多态性分析，结果如下：
（1）10个靶基因的等位基因的数量在7（recA和dnaA）~17（pepN和clpX）之间，多态位点在8（recA）~22（murC），嗜热链球菌存在遗传基因多样性，但核苷酸多样度（0.0036~0.0077）表明其核苷酸多态性相比其它乳酸菌菌种略小。G+C%含量范围在36.94%（dnaA）~44.20%（pepN），与嗜热链球菌ND03全基因组G+C%含量（39.1%）相接近。基因的非同义替换与同义替换的比率dN/dS值在0.0414（pyrG）~0.2624（murC）之间，显著小于1，这些基因采取负选择的进化方式，嗜热链球菌受到环境的选择压力为负选择压力。采用SplitsTree4.0软件构建的分解分析图表明10个基因无明显的重组现象。
（2）依据10个等位基因的序列将260株嗜热链球菌指定为119个ST型，其中菌株数量较多的为ST-29（16株），不同地区嗜热链球菌分离株的ST型各不相同。LIAN连接分析显示嗜热链球菌群体存在显著的连锁不平衡（SIA=0.1043）。
（3）eBURST v3.0软件将119个ST型分为58个分型组，其中31个ST型为独特型。58个分型组中有5个克隆复合体CC5，CC29，CC54，CC70和CC81。具有克隆复合体的菌株数量占总数的44.6%，属于独特型的菌株数量占总数的55.4%。相同地区的分离株形成了相应的克隆复合体，表明在不同环境下嗜热链球菌分离株发生着不同的进化进程，影响其进化的主要因素是地理位置和环境气候，而结果未显示菌株进化与乳样类型有关联性。
（4）依据119个ST型构建的最小生成树表明新疆、西藏和俄罗斯分离株的亲缘关系很近，与部分蒙古国中部、中国甘肃和四川分离株的亲缘关系较近；蒙古国不同省市的分离株的亲缘关系均很近；中国内蒙古和青海省的分离株亲缘关系较近；小部分蒙古国中部地区分离株和中国分离株关系较近。
（5）利用UPGMA法依据ST型的联合基因序列（长度为5718bp）进行的遗传聚类分析表明，地理位置不同的菌株构成了不同的亚群，各自分开向自己的方向进化。蒙古国嗜热链球菌分离株之间的遗传关系很近，中国各省分离株之间遗传关系较远。
本研究对不同地区的260株嗜热链球菌进行多位点序列分型研究，初步建立了嗜热链球菌的MLST研究方法，为嗜热链球菌分离株的基因信息库（MLST数据库）提供了数据资料。通过比较各个菌株等位基因序列的多态性，认识了菌种基因水平上的进化过程，并分析不同地区嗜热链球菌的遗传关系，从而进一步推演了嗜热链球菌的进化历程，为世界范围内嗜热链球菌的群体结构和遗传进化研究奠定了基础。
Streptococcus thermophilus is a major dairy starter used for the manufacture of dairyproducts. It is massively used for the manufacture of all kinds of dairy products. In thisstudy, S. thermophilus were isolated using pure culture method from traditionalfermented dairy products collected from Yili Kazakh Autonomous Prefecture in Xinjiangand Damxung County in Tibet of China, and Respublika Kalmykiya and Buryatiya ofRussia. Those isolates were identified by morphology and molecular biology. Meanwhile,multilocus sequence typing (MLST) was used to study the genetic polymorphism andevolutionary relationship of260isolates of S. thermophilus from fermented dairyproducts in Russia,12different regions of Mongolia, and6minority areas of China.
Streptococcus thermophilus were isolated by M17agar from223traditional fermenteddairy products colleced from Yili Kazakh Autonomous Prefecture in Xinjiang andDamxung County in Tibet of China, and Respublika Kalmykiya and Buryatiya of Russia.Those traditional fermented dairy products including fermented butter, cheese, mare milk,cow milk, yak milk and camel milk. Gram-positive and catalase-negative cocci wereidentified by16S rRNA gene sequencing. The16S rRNA gene of all the tested strains weredetermined by the BLAST program. According to the16S rRNA gene sequences,52strains were identified as S. thermophilus. Fourty S. thermophilus were isolated from YiliKazakh Autonomous Prefecture of Xinjiang,5strains were isolated from Damxung Countyof Tibet,5strains were isolated from Respublika Kalmykiya, and2strains were isolatedfrom Respublika Buryatiya of Russia.
Thirty nine representative isolates form Xinjiang and Tibet of China, and RespublikaKalmykiya and Buryatiya of Russia, and221S. thermophilus isolated from traditionalfermented dairy products in previous study were analyzed by MLST. Based on thegeneome of S. thermophilus ND03,10housekeeping genes, carB, clpX, dnaA, murC,murE, pepN, pepX, pyrG, recA and rpoB, were chose and used for MLST analysis. Thesequences of10housekeeping genes were acquired by bidirectional sequencing technology,and forward and reverse sequences were trimmed, aligned, and analyzed. For each MLST locus, an allele number was given to each distinct combination of alleles of the ten genes,and each allele number was assigned a sequence type (ST). Exclusion of generecombination, the genetic polymorphism and evolution of S. thermophilus were analyzedbased on STs and composite sequences by bioinformatics software, such as BioNumericsv6.0, START2.0, eBURST v3.0, DnaSP5.0and SplitsTree4.0, and so on. The results wereshowed as follow:
The number of10loci ranged from7(recA and dnaA) to17(pepN and clpX), and8(recA) to22(murC) polymorphic sites in each gene, indicating intragenic diversity exist inS. thermophilus. The DNA G+C%content observed for all alleles of the ten genes rangedfrom36.94%to44.20%, which were similar to the G+C%content of the S. thermophilusND03. The dS/dNratio ranged from0.0414(pyrG) to0.2624(murC), and it is less than1,suggesting potential negative selection in these genes. The split decomposition graphs ofall ten loci by SplitsTree4.0suggesting no recombination occurred during the evolution ofthese ten loci.
The260S. thermophilus isolates by MLST identified as119STs. The mostrepresented STs were ST-29, which comprised16strains. Strains from different regionswere assigned to different STs. SIAof0.1043was significantly greater than the value of0expected for a population at linkage disequilibrium. One hundred and seventeen STs weregrouped by eBURST into58groups,31STs were assigned to singleton. Among58groups,there are5clonal complexes (CCs), CC5, CC29, CC54, CC70and CC81, representing44.6%of the strains in total. The same geographical isolates belong to same CC,suggesting S. thermophilus isolates in different environments with different evolutionaryprocesses. The main factors were location, environment and climate, but not milk type.
The minimum evolution tree showed isolates of Xinjiang, Tibet in China and Russiawere closely related to Qinghai, Inner Mogulian, and Gannan region strains, but far fromMongolia strains. Strains from12different regions in Mongolia showed near relationship.Partly strains from the central region of Mongolia were close to isolates of China. Clusteranalysis of the concatenated sequences (5718bp) by using UPGMA revealed that differentgeographical isolates clustered into different clusters, and those strains evoluted towardtheir own direction.
In the present study, MLST was used to study the genetic polymorphism andevolutionary relationship of260isolates of S. thermophilus from different regions. On theone hand, an MLST scheme was developed for the study on S. thermophilus, and provideddata for MLST database. On the other hand, analysis of geographically diverse and representative collection of isolates by MLST can provide a better understanding of S.thermophilus genome evolution, and some useful information for further studies ofworldwide S. thermophilus structure and genetic evolution.
采用M17分离培养基对中国新疆伊犁哈萨克自治州和西藏自治区拉萨市当雄县以及俄罗斯卡尔梅克共和国和布里亚特自治州共223份传统发酵乳制品（包括发酵奶油、发酵奶酪、发酵牦牛乳、发酵马乳、发酵驼奶和发酵牛乳）中的嗜热链球菌进行分离，将革兰氏阳性和过氧化氢酶阴性的球状分离株采用16S rRNA基因序列分析法进行分类鉴定，通过BLAST同源性比对和系统发育树的构建将52株菌鉴定为嗜热链球菌，其中40株分离自从中国新疆伊犁哈萨克自治州的发酵牛乳中，5株分离自中国西藏自治区拉萨市当雄县的发酵牦牛乳中，5株分离自俄罗斯卡尔梅克共和国的发酵牛乳和奶油中，还有2株分离自俄罗斯布里亚特自治州发酵牛乳中。
选取了上述有代表性的39株嗜热链球菌分离株和先前分离自传统发酵乳中的221株分离株进行MLST研究，以嗜热链球菌ND03的全基因组为基础，选取10个看家基因carB、clpX、dnaA、murC、murE、pepN、pepX、pyrG、recA和rpoB作为MLST分型的靶基因。设计引物后，通过PCR扩增、双向测序技术获得靶基因的序列，将其拼接、核对后建立不同的序列型（ST），排除基因的重组交换，应用BioNumerics v6.0、START2.0、eBURST v3.0、DNAsp5.0和SplitsTree4.0等生物信息学软件对等位基因的ST型和核苷酸序列进行遗传多态性分析，结果如下：
（1）10个靶基因的等位基因的数量在7（recA和dnaA）~17（pepN和clpX）之间，多态位点在8（recA）~22（murC），嗜热链球菌存在遗传基因多样性，但核苷酸多样度（0.0036~0.0077）表明其核苷酸多态性相比其它乳酸菌菌种略小。G+C%含量范围在36.94%（dnaA）~44.20%（pepN），与嗜热链球菌ND03全基因组G+C%含量（39.1%）相接近。基因的非同义替换与同义替换的比率dN/dS值在0.0414（pyrG）~0.2624（murC）之间，显著小于1，这些基因采取负选择的进化方式，嗜热链球菌受到环境的选择压力为负选择压力。采用SplitsTree4.0软件构建的分解分析图表明10个基因无明显的重组现象。
（2）依据10个等位基因的序列将260株嗜热链球菌指定为119个ST型，其中菌株数量较多的为ST-29（16株），不同地区嗜热链球菌分离株的ST型各不相同。LIAN连接分析显示嗜热链球菌群体存在显著的连锁不平衡（SIA=0.1043）。
（3）eBURST v3.0软件将119个ST型分为58个分型组，其中31个ST型为独特型。58个分型组中有5个克隆复合体CC5，CC29，CC54，CC70和CC81。具有克隆复合体的菌株数量占总数的44.6%，属于独特型的菌株数量占总数的55.4%。相同地区的分离株形成了相应的克隆复合体，表明在不同环境下嗜热链球菌分离株发生着不同的进化进程，影响其进化的主要因素是地理位置和环境气候，而结果未显示菌株进化与乳样类型有关联性。
（4）依据119个ST型构建的最小生成树表明新疆、西藏和俄罗斯分离株的亲缘关系很近，与部分蒙古国中部、中国甘肃和四川分离株的亲缘关系较近；蒙古国不同省市的分离株的亲缘关系均很近；中国内蒙古和青海省的分离株亲缘关系较近；小部分蒙古国中部地区分离株和中国分离株关系较近。
（5）利用UPGMA法依据ST型的联合基因序列（长度为5718bp）进行的遗传聚类分析表明，地理位置不同的菌株构成了不同的亚群，各自分开向自己的方向进化。蒙古国嗜热链球菌分离株之间的遗传关系很近，中国各省分离株之间遗传关系较远。
本研究对不同地区的260株嗜热链球菌进行多位点序列分型研究，初步建立了嗜热链球菌的MLST研究方法，为嗜热链球菌分离株的基因信息库（MLST数据库）提供了数据资料。通过比较各个菌株等位基因序列的多态性，认识了菌种基因水平上的进化过程，并分析不同地区嗜热链球菌的遗传关系，从而进一步推演了嗜热链球菌的进化历程，为世界范围内嗜热链球菌的群体结构和遗传进化研究奠定了基础。
Streptococcus thermophilus is a major dairy starter used for the manufacture of dairyproducts. It is massively used for the manufacture of all kinds of dairy products. In thisstudy, S. thermophilus were isolated using pure culture method from traditionalfermented dairy products collected from Yili Kazakh Autonomous Prefecture in Xinjiangand Damxung County in Tibet of China, and Respublika Kalmykiya and Buryatiya ofRussia. Those isolates were identified by morphology and molecular biology. Meanwhile,multilocus sequence typing (MLST) was used to study the genetic polymorphism andevolutionary relationship of260isolates of S. thermophilus from fermented dairyproducts in Russia,12different regions of Mongolia, and6minority areas of China.
Streptococcus thermophilus were isolated by M17agar from223traditional fermenteddairy products colleced from Yili Kazakh Autonomous Prefecture in Xinjiang andDamxung County in Tibet of China, and Respublika Kalmykiya and Buryatiya of Russia.Those traditional fermented dairy products including fermented butter, cheese, mare milk,cow milk, yak milk and camel milk. Gram-positive and catalase-negative cocci wereidentified by16S rRNA gene sequencing. The16S rRNA gene of all the tested strains weredetermined by the BLAST program. According to the16S rRNA gene sequences,52strains were identified as S. thermophilus. Fourty S. thermophilus were isolated from YiliKazakh Autonomous Prefecture of Xinjiang,5strains were isolated from Damxung Countyof Tibet,5strains were isolated from Respublika Kalmykiya, and2strains were isolatedfrom Respublika Buryatiya of Russia.
Thirty nine representative isolates form Xinjiang and Tibet of China, and RespublikaKalmykiya and Buryatiya of Russia, and221S. thermophilus isolated from traditionalfermented dairy products in previous study were analyzed by MLST. Based on thegeneome of S. thermophilus ND03,10housekeeping genes, carB, clpX, dnaA, murC,murE, pepN, pepX, pyrG, recA and rpoB, were chose and used for MLST analysis. Thesequences of10housekeeping genes were acquired by bidirectional sequencing technology,and forward and reverse sequences were trimmed, aligned, and analyzed. For each MLST locus, an allele number was given to each distinct combination of alleles of the ten genes,and each allele number was assigned a sequence type (ST). Exclusion of generecombination, the genetic polymorphism and evolution of S. thermophilus were analyzedbased on STs and composite sequences by bioinformatics software, such as BioNumericsv6.0, START2.0, eBURST v3.0, DnaSP5.0and SplitsTree4.0, and so on. The results wereshowed as follow:
The number of10loci ranged from7(recA and dnaA) to17(pepN and clpX), and8(recA) to22(murC) polymorphic sites in each gene, indicating intragenic diversity exist inS. thermophilus. The DNA G+C%content observed for all alleles of the ten genes rangedfrom36.94%to44.20%, which were similar to the G+C%content of the S. thermophilusND03. The dS/dNratio ranged from0.0414(pyrG) to0.2624(murC), and it is less than1,suggesting potential negative selection in these genes. The split decomposition graphs ofall ten loci by SplitsTree4.0suggesting no recombination occurred during the evolution ofthese ten loci.
The260S. thermophilus isolates by MLST identified as119STs. The mostrepresented STs were ST-29, which comprised16strains. Strains from different regionswere assigned to different STs. SIAof0.1043was significantly greater than the value of0expected for a population at linkage disequilibrium. One hundred and seventeen STs weregrouped by eBURST into58groups,31STs were assigned to singleton. Among58groups,there are5clonal complexes (CCs), CC5, CC29, CC54, CC70and CC81, representing44.6%of the strains in total. The same geographical isolates belong to same CC,suggesting S. thermophilus isolates in different environments with different evolutionaryprocesses. The main factors were location, environment and climate, but not milk type.
The minimum evolution tree showed isolates of Xinjiang, Tibet in China and Russiawere closely related to Qinghai, Inner Mogulian, and Gannan region strains, but far fromMongolia strains. Strains from12different regions in Mongolia showed near relationship.Partly strains from the central region of Mongolia were close to isolates of China. Clusteranalysis of the concatenated sequences (5718bp) by using UPGMA revealed that differentgeographical isolates clustered into different clusters, and those strains evoluted towardtheir own direction.
In the present study, MLST was used to study the genetic polymorphism andevolutionary relationship of260isolates of S. thermophilus from different regions. On theone hand, an MLST scheme was developed for the study on S. thermophilus, and provideddata for MLST database. On the other hand, analysis of geographically diverse and representative collection of isolates by MLST can provide a better understanding of S.thermophilus genome evolution, and some useful information for further studies ofworldwide S. thermophilus structure and genetic evolution.
引文
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