Tamura Haruki, Yamada Arisa, Yoshida Yasuo, Kato Hirohisa
Department of Dental Pharmacology, School of Dentistry, Iwate Medical University, Morioka, Iwate, Japan.
Curr Microbiol. 2009 May;58(5):432-7. doi: 10.1007/s00284-008-9336-0. Epub 2008 Dec 18.
An autolysin gene, atlh, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. Atlh protein encoded by atlh is composed of 879 amino acids, with a molecular weight of 95,902.26. Atlh possesses four 15-amino-acid residue repeats in the putative cell-wall-binding domain and has a catalytic domain in the C-terminus. The deduced amino acid sequence of atlh showed homology to S. mutans autolysin AtlA (68.4% similarity). Inactivation of atlh resulted in elongated chain formation compared to the parent strain. Recombinant proteins Atlh and its derivatives were constructed and analyzed by zymography. Zymographic analysis revealed that the Asp-771 residue of Atlh was essential for lytic activity and that lytic activity was not diminished by the deletion of repetitive regions in the putative cell-wall-binding domain of Atlh. Biofilm assay showed that the wild-type strain formed glucose- and sucrose-dependent biofilms, the atlh mutant diminished this ability. These results suggest that Atlh is associated with cell separation and biofilm formation.
利用简并聚合酶链反应（PCR）和基因步移法，从唐氏链球菌MFe28中鉴定并测序了一个自溶素基因atlh。atlh编码的Atlh蛋白由879个氨基酸组成，分子量为95,902.26。Atlh在假定的细胞壁结合结构域中具有四个15个氨基酸残基的重复序列，并且在C末端具有一个催化结构域。atlh推导的氨基酸序列与变形链球菌自溶素AtlA具有同源性（相似性为68.4%）。与亲本菌株相比，atlh的失活导致链状伸长。构建了重组蛋白Atlh及其衍生物，并通过酶谱分析进行了分析。酶谱分析表明，Atlh的Asp-771残基对裂解活性至关重要，并且Atlh假定的细胞壁结合结构域中重复区域的缺失并未降低裂解活性。生物膜检测表明，野生型菌株形成葡萄糖和蔗糖依赖性生物膜，而atlh突变体则降低了这种能力。这些结果表明，Atlh与细胞分离和生物膜形成有关。
