多位点序列和比较基因组学分析对盐单胞菌科菌株JSM 104105的系统发育学研究

Phylogenetics of Halomonadaceae strain JSM 104105 based on multilocus sequence analysis and comparative genomics analysis

【目的】为了精准地判定产铁载体嗜盐新菌株JSM 104105在盐单胞菌科(Halomonadaceae)中的系统发育地位。【方法】采用基于16SrRNA基因、DNA促旋酶B亚基基因(DNAgyraseB subunit gene,gyrB)和核糖核酸聚合酶σ因子RpoD基因(RNA polymerase sigma factor RpoD gene,rpoD)序列的多位点序列分析方法(multilocus sequence analysis,MLSA),对菌株JSM 104105的系统发育地位进行初步分析;采用比较基因组学分析(comparative genomics analysis),分析该菌株与其系统发育关系密切的典型菌株之间的GC含量、平均核苷酸一致性(average nucleotide identity,ANI)和数字DNA-DNA杂交值(digital DNA-DNA hybridization,dDDH),并进行基因组系统发育学分析(phylogenomic analysis),更准确地判定菌株JSM 104105在盐单胞菌科中的系统发育地位。【结果】MLSA分析结果表明,无论是单基因序列分析,还是多基因串联序列分析,对盐单胞菌科各分类单元以及菌株JSM104105的系统发育地位都能提供较为一致的结果。分析表明,菌株JSM104105归属于盐单胞菌科盐单胞菌属(Halomonas),与该属的Halomonas gudaonensis、Halomonas azerbaijanica和Halomonas lysinitropha的系统发育关系较密切,它们在系统进化树上形成稳定亚簇(subcluster),其中菌株JSM 104105占据稳定的独立进化分支。尽管它们之间的16S rRNA基因序列相似性较高,为97.3%-98.9%,但菌株JSM104105很难归属于其中任何已知物种。比较基因组学分析结果确认了这一判断。菌株JSM104105与系统发育关系密切的H.gudaonensis CGMCC 1.16133T、H.azerbaijanica TBZ202T和H.lysinitropha 3(2)T的ANI值(78.9%-91.6%)和dDDH值(22.1%-43.7%),均显著低于界定原核生物物种的阈值(ANI,95%-96%;dDDH≥70%)。基因组系统发育分析结果表明,菌株JSM104105明确构成盐单胞菌属独立的亚分支(subclade)。【结论】从分子系统发育学视角精准地判定产铁载体嗜盐细菌JSM104105归属于盐单胞菌科盐单胞菌属,与该属的已知物种H.gudaonensis、H.azerbaijanica和H.lysinitropha的系统发育关系最密切,但不能归属于该属的已知种,应该代表了一个新的基因种(genospecies)。

[Objective]To investigate the phylogenetic position of the siderophore-producing halophilic bacterium JSM 104105 in the family Halomonadaceae.[Methods]Molecular phylogenetic analysis of strain JSM 104105 was performed by means of multilocus sequence analysis(MLSA)based on 3 housekeeping genes,i.e.,16S rRNA gene,DNA gyrase B subunit gene(gyrB)and RNA polymerase sigma factor RpoD gene(rpoD),and then its exact systematic position was investigated by comparative genomics analysis based on whole-genome sequences,including comparisons of G+C content,average nucleotide identity(ANI),and digital DNA-DNA hybridization(dDDH)values,and phylogenomic analysis.[Results]The MLSA results showed that the data both from single-gene(16S rRNA,gyrB,or rpoD)sequence comparisons and concatenated 16S rRNA-gyrB-rpoD sequence comparisons were consistent,and could indicate the phylogeny of strain JSM 104105 and related taxa of Halomonadaceae,and that strain JSM 104105 belonged to the genus Halomonas,being in closestly genetic relationship with Halomonas gudaonensis CGMCC 1.16133T(16S rRNA gene similarity,98.9%),Halomonas azerbaijanica TBZ202T(98.6%)and Halomonas lysinitropha 3(2)T(97.3%),and strain JSM 104105 formed a coherent branch in the phylogenetic trees which were generated based on either single-gene sequence or concatenated-gene sequence comparisons.MLSA data also suggested that strain JSM 104105 could represent a different phylogenic subline separated from recognized Halomonas species.The results of comparative genomics analysis supported the conclusion from MLSA data.Either ANI(78.9%-91.6%)or dDDH values(22.1%-43.7%)were all well below the standard criteria(ANI,95%-96%;dDDH≥70%)for delineation of prokaryotic species.The results of phylogenomic analysis also showed that strain JSM 104105 clearly represented an independent subclade in Halomonas.[Conclusion]From the perspective of molecular phylogeny,the combination of MLSA and comparative genomics analysis demonstrated definitely that strain JSM 104105 belongs to Halomonas,being phylogenetically closely related to H.gudaonensis,H.azerbaijanica and H.lysinitropha,and that strain JSM 104105 cannot be assigned to any recognized species but represents a novel genospecies of the genus Halomonas.