Yamamoto Y, Sato Y, Takahashi-Abbe S, Abbe K, Yamada T, Kizaki H
Department of Biochemistry, Tokyo Dental College, Chiba City, Japan.
Infect Immun. 1996 Feb;64(2):385-91. doi: 10.1128/iai.64.2.385-391.1996.
We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.
我们利用质粒pVA891克隆文库进行随机诱变后，分离出了变形链球菌GS-5的一个山梨醇阴性突变体。该突变体在厌氧条件下不能代谢山梨醇，但在需氧条件下可以。此突变体中缺失了位于pVA891插入位点两侧的一段10kb的染色体DNA片段。然后通过大肠杆菌的标记拯救方法从亲本菌株GS-5中回收了相应区域。丙酮酸甲酸裂解酶基因pfl在位于染色体缺失区域中部的一个3kb PstI-XbaI片段中被鉴定出来，其在变形链球菌中的失活产生了相同的山梨醇阴性表型。对pfl基因的核苷酸序列分析显示，一个2.3kb的开放阅读框（ORF）之前有潜在的核糖体结合序列和启动子样序列。该ORF编码一个推测的由775个氨基酸残基组成的蛋白质，计算分子量为87,533。从该ORF推导的氨基酸序列与大肠杆菌pfl基因的氨基酸序列有显著相似性。
