Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in <em>Bacillus</em>

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed.芽孢杆菌属（Bacillus）微生物在经济、医学及生物防御领域均具有极高的研究与应用价值。基于16S rRNA序列分析的芽孢杆菌菌株鉴定，通常仅能将其鉴定至物种水平。此外，枯草芽孢杆菌（Bacillus subtilis）菌株的分类划分存在一定偏差。在核糖体数据库项目（Ribosomal Database Project, RDP）与美国国家生物技术信息中心（National Center for Biotechnology Information, NCBI）的数据库中，隶属于芽孢杆菌属175个不同物种的共计2611条16S rDNA序列里，仅1586条可鉴定至物种水平。本研究选取10种芽孢杆菌的16S rRNA序列用于构建物种特异性分类框架与鉴定探针以实现快速鉴定，这10种芽孢杆菌分别为：炭疽芽孢杆菌（Bacillus anthracis，153株）、蜡样芽孢杆菌（Bacillus cereus，211株）、苏云金芽孢杆菌（Bacillus thuringiensis，108株）、枯草芽孢杆菌（Bacillus subtilis，271株）、地衣芽孢杆菌（Bacillus licheniformis，131株）、短小芽孢杆菌（Bacillus pumilus，83株）、巨大芽孢杆菌（Bacillus megaterium，47株）、球形芽孢杆菌（Bacillus sphaericus，42株）、克劳氏芽孢杆菌（Bacillus clausii，39株）以及嗜碱芽孢杆菌（Bacillus halodurans，36株）。研究人员对10个芽孢杆菌物种的1121条16S rDNA序列进行系统发育聚类，共得到89个簇，进而基于34条代表性序列构建了系统发育分类框架。利用该框架，可将当前归类为芽孢杆菌未定种（Bacillus sp.）的1025条16S rDNA序列中的305条鉴定至物种水平。该鉴定结果可通过针对这10种芽孢杆菌的20~30个核苷酸长度的特征序列以及计算机模拟限制性酶切分析得到验证。本整合策略可将约30%的芽孢杆菌未定种鉴定至物种水平，同时研究发现枯草芽孢杆菌菌株可被划分为两个系统发育上截然不同的类群，其中一个类群或许需要重新命名。