摘要
【目的】阐明分离自南海雷州湾硇洲岛潮汐带沉积物的12株产铁载体细菌的系统发育多样性,并探测这些实验菌株及微泡菌属(Microbulbifer)已知物种代表性菌株的铁载体生物合成基因簇的组成、功能、遗传多样性及其演化模式。【方法】采用常规方法观察主要表型特征并进行产铁载体活性验证;利用16S r RNA基因序列分析法了解实验菌株的系统发育多样性(包括类群多样性、物种多样性和遗传多样性);测定代表性实验菌株JSM ZJ756的基因组框架图,通过比较基因组学技术对比分析实验菌株与其系统发育关系密切的典型菌株的G+C含量、平均核苷酸一致性(average nucleotide identity,ANI)和数码DNA-DNA杂交估值(digitalDNA-DNAhybridization,d DDH),以更准确地判定其系统分类地位;采用anti SMASH 7.0、BLASTn、BLASTp和MEGA 11等生物信息学工具,对次级代谢产物合成基因簇(secondary metabolite biosynthetic gene clusters,BGCs)进行快速鉴定、功能注释和序列比对,以探测其产铁载体相关功能基因簇的组成、功能、遗传多样性及其演化模式。【结果】实验菌株均为严格好氧、不产芽孢、具有产铁载体活性的革兰氏阴性杆菌。16S r RNA基因序列和全基因组序列分析结果表明,12株菌均属于微泡菌属,归为6-8个物种,与该属的典型菌株构成4个进化系(clade),其中JSMZJ756为舟山微泡菌(Microbulbifer zhoushanensis)的新成员。通过anti SMASH基因簇分析发现,JSM ZJ756及9个微泡菌属代表性菌株各具有1个铁载体生物合成基因簇(NI-siderophore),其中8个与已知同类型基因簇的相似性在40%及以下。根据与已知基因簇的种类和相似性差异,这10个铁载体基因簇可分为5个功能亚型:JSM ZJ756等3株为Ochrobactin亚型(序列号JYFX01000060.1,相似性28%),M.mangrove DD-13T和M.epialgicus DSM 18651T为Vibrioferrin亚型(AB082123.1,100%;CP005094.1,85%),M.agarilyticusGP101为Putrebactin亚型(NIBS01000001.1,40%),M.echiniJCM30400T为Aerobactin亚型(AB199785.1,22%),而M.variabilis ATCC 700307T等3株未发现与之相匹配的已知铁载体合成相关功能基因簇(未知功能亚型)。BLASTn和BLASTp搜索显示,这些基因簇的核心基因具有独特的序列,能编码新颖的功能蛋白;基于核心基因序列的遗传演化分析表明,这些NI-siderophore基因簇具有较高的遗传多样性;在核心基因发育树上,菌株M.agarilyticus GP101的铁载体合成基因簇的遗传演化路径相对孤立,而其他9个分属于3个较独立的遗传演化群。对比分析发现,这些NI-siderophore基因簇的遗传演化模式与相应菌株的16S r RNA基因序列系统发育模式基本一致。【结论】本研究表明分离自南海雷州湾硇洲岛潮汐带沉积物的12株产铁载体菌株属于微泡菌属,具有较高的系统发育多样性,其中JSM ZJ756为舟山微泡菌的新成员;JSM ZJ756及微泡菌属(代表性菌株)的NI-siderophore基因簇具有较高的多样性和新颖性,具有产生新颖多样铁载体的较大潜力;JSM ZJ756及9个微泡菌属代表性菌株的NI-siderophore基因簇的生物功能、遗传演化,以及这些菌株的系统发育之间存在较高的正向关联性。因此,包括12个产铁载体实验菌株在内的微泡菌属菌株是一类典型的新资源微生物,其系统分类、铁载体代谢机制和遗传演化模式,以及生物技术潜力值得进一步探讨。
[Objective]To reveal the phylogenetic diversity of 12 marine siderophore-producing bacteria isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea,and to unravel the components,functions,genetic diversity and genetic evolution of siderophore biosynthetic gene clusters(BGCs)of the isolates as well as representatives of Microbulbifer.[Methods]The phenotypic characteristics as well as the siderophore-producing activity of the strains were observed by conventional methods.The phylogenetic diversity(including taxon,species,and genetic diversity)of the strains was analyzed based on 16S rRNA gene sequences.Then,the exact phylogenetic status of the representative strain JSM ZJ756 was investigated comprehensively by means of comparative genomics analysis based on whole-genome sequences,including comparisons of G+C content,average nucleotide identity(ANI),and digital DNA-DNA hybridization(dDDH)estimated values.We then used multiple bioinformatic stools including antiSMASH 7.0,BLASTn,BLASTp,and MEGA 11 for rapid identification,annotation,and sequence alignment of BGCs,thus exploring the components,functions,genetic diversity,and genetic evolution of siderophore BGCs.[Results]All the 12 isolates were Gram-negative,aerobic,non-sporulating and slightly halophilic rods with siderophore-producing activity.The results of phylogenetic analyses based on 16S rRNA gene sequences as well as whole-genome sequences showed that all the 12 strains belonged to Microbulbifer,representing 6 to 8 species and forming 4 clades with Microbulbifer type strains in the phylogenetic tree.Among them,strain JSM ZJ756 should be a new member of Microbulbifer zhoushanensis.JSM ZJ756 and 9 representative strains of Microbulbifer each carried one copy of NI-siderophore BGCs,and 8 out of the 10 NI-siderophore BGCs shared the similarities≤40%with known BGCs.According to the types and the similarities of known BGCs with the NI-siderophore BGCs identified in this study,the 10 NI-siderophore BGCs could be categorized into 5 functional subtypes:Ochrobactin(JSM ZJ756 and 2 type strains,JYFX01000060.1,similarity of 28%),Vibrioferrin(M.mangrove DD-13T,AB082123.1,100%;M.epialgicus DSM 18651T,CP005094.1,85%),Putrebactin(M.agarilyticus GP101,NIBS01000001.1,40%),Aerobactin(M.echini JCM 30400T,AB199785.1,22%),and unknown functional subtype(3 type strains including M.variabilis ATCC 700307T).The results of BLASTn and BLASTp analyses showed that the core biosynthetic gene sequences of the NI-siderophore BGCs identified were unique,and encoded novel proteins.The results of genetic evolution analysis showed that the core biosynthetic genes of the NI-siderophore BGCs found exhibited high genetic diversity.In the phylogenetic tree based on core biosynthetic gene sequences,9 out of the 10 NI-siderophore BGCs were grouped to 3 clades,but M.agarilyticus GP101 exhibited an independent evolution path.The comparative analysis revealed the genetic evolution of NI-siderophore BGCs being consistent with that predicted based on the 16S rRNA sequences.[Conclusion]All the 12 siderophore-producing strains isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea belong to Microbulbifer,demonstrating high phylogenetic diversity,and JSM ZJ756 is a new member of M.zhoushanensis.The NI-siderophore BGCs of JSM ZJ756 and representative strains of Microbulbifer are novel and showcase high diversity,which indicates that those strains possess high potential of producing a variety of novel siderophores.Moreover,strong positive correlations exist between the biological functions and genetic evolution of the NI-siderophore BGCs and the phylogeny of JSM ZJ756 and 9 representative strains of Microbulbifer.Therefore,we hypothesize that the 12 marine siderophore-producing bacteria and the representatives of Microbulbifer are typical new-resource microbes,and the taxonomy of Microbulbifer,and the metabolic mechanism and genetic evolution as well as the biotechnological potential of the NI-siderophore BGCs are worth being further explored.
作者
汤倩
陈锦华
邓丽颖
达春瑶
刘祝祥
陈义光
TANG Qian;CHEN Jinhua;DENG Liying;DA Chunyao;LIU Zhuxiang;CHEN Yiguang(College of Biology and Environmental Sciences,Jishou University,Jishou,Hunan,China;Yiyang Wanyuan Mingda High School,Yiyang,Hunan,China;School of Medicine,Zhangjiajie College,Zhangjiajie,Hunan,China)
出处
《微生物学报》
北大核心
2025年第4期1616-1634,共19页
Acta Microbiologica Sinica
基金
国家自然科学基金(31660003,31460004)
湖南省自然科学基金(2020JJ4501)
湖南省教育厅科技项目(22A0770)
湖南省研究生科研创新项目(CX20231081)。
关键词
微泡菌属
产铁载体海洋细菌
系统发育多样性
铁载体合成基因簇
遗传演化
新资源微生物
Microbulbifer
marine siderophore-producing bacteria
phylogenetic diversity
siderophore biosynthetic gene cluster
genetic evolution
new-resource microbe
作者简介
Corresponding authors:陈锦华,E-mail:cjh_jsu@jsu.edu.cn;Corresponding authors:刘祝祥,E-mail:liuzhuxiang@126.com。
