Griffiths Emma, Gupta Radhey S
Department of Biochemistry, McMaster University, Hamilton, Ontario, CanadaL8N 3Z51.
Microbiology (Reading). 2001 Sep;147(Pt 9):2611-2622. doi: 10.1099/00221287-147-9-2611.
The phylogenetic placement of the rumen bacterium Fibrobacter succinogenes was determined using a signature sequence approach that allows determination of the relative branching order of the major divisions among Bacteria [Gupta, R. S. (2000) FEMS Microbiol Rev 24, 367-402]. For this purpose, segments of the Hsp60 (groEL), Hsp70 (dnaK), CTP synthase and alanyl-tRNA synthetase genes, which are known to contain signature sequences that are useful for phylogenetic deterministic purposes, were cloned. Using degenerate oligonucleotide primers for highly conserved regions in these proteins, 1.4 kb, 0.75 kb, 401 bp and 171 bp fragments of the Hsp70, Hsp60, CTP synthase and alanyl-tRNA synthetase genes respectively were amplified by PCR, and these fragments were cloned and sequenced. These primers, because of their high degree of conservation, could also be used for cloning these genes from other bacterial species. The Hsp70 homologues from different Gram-negative bacteria contain a 21-23 aa insert that is not found in any Gram-positive bacteria. The presence of this insert in the F. succinogenes Hsp70 supports its placement within the Gram-negative group of bacteria. A conserved insert in F. succinogenes Hsp60 that is commonly present in all bacterial species, except various Gram-positive bacteria, Deinococcus-Thermus groups and green non-sulphur bacteria, provides evidence that F. succinogenes does not belong to these taxa. A particularly useful signature consisting of a 4 aa insert is found in Ala-tRNA synthetase. This insert is present in all proteobacterial homologues as well as in homologues from species belonging to the Chlamydia and Cytophaga-Flavobacterium- Bacteroides (CFB) groups, but it is not found in homologues from any other groups of bacteria. The presence of this insert in F. succinogenes Ala-tRNA synthetase provides evidence that this species is related to these groups. However, two other signatures in CTP synthase and Hsp70 proteins, that are distinctive of the proteobacterial species, are not present in the F. succinogenes homologues. These results provide evidence that F. succinogenes does not belong to the proteobacterial division and thus should be placed in a similar position as the Chlamydia and CFB groups of species.
采用一种特征序列法确定瘤胃细菌琥珀酸丝状杆菌（Fibrobacter succinogenes）的系统发育位置，该方法可确定细菌主要分类群的相对分支顺序[古普塔，R. S.（2000年）《FEMS微生物学评论》24卷，367 - 402页]。为此目的，克隆了热休克蛋白60（Hsp60，即groEL）、热休克蛋白70（Hsp70，即dnaK）、CTP合成酶和丙氨酰 - tRNA合成酶基因的片段，已知这些片段包含对系统发育确定性分析有用的特征序列。使用针对这些蛋白质高度保守区域的简并寡核苷酸引物，通过聚合酶链反应（PCR）分别扩增出Hsp70、Hsp60、CTP合成酶和丙氨酰 - tRNA合成酶基因的1.4 kb、0.75 kb、401 bp和171 bp片段，并将这些片段克隆和测序。由于这些引物具有高度保守性，它们也可用于从其他细菌物种中克隆这些基因。来自不同革兰氏阴性菌的Hsp70同源物含有一段21 - 23个氨基酸的插入序列，在任何革兰氏阳性菌中均未发现。琥珀酸丝状杆菌Hsp70中存在该插入序列，支持其归属于革兰氏阴性菌组。琥珀酸丝状杆菌Hsp60中的一个保守插入序列在所有细菌物种中普遍存在，但各种革兰氏阳性菌、嗜热放线菌 - 嗜热栖热菌组和绿色非硫细菌除外，这表明琥珀酸丝状杆菌不属于这些分类单元。在丙氨酰 - tRNA合成酶中发现了一个特别有用的由4个氨基酸插入组成的特征序列。该插入序列存在于所有变形菌门同源物以及衣原体属和噬纤维菌 - 黄杆菌 - 拟杆菌（CFB）组物种的同源物中，但在任何其他细菌组的同源物中均未发现。琥珀酸丝状杆菌丙氨酰 - tRNA合成酶中存在该插入序列，表明该物种与这些组相关。然而，CTP合成酶和Hsp70蛋白中的另外两个特征序列是变形菌门物种所特有的，在琥珀酸丝状杆菌同源物中不存在。这些结果表明琥珀酸丝状杆菌不属于变形菌门，因此应置于与衣原体属和CFB组物种类似的位置。
