枯草芽孢杆菌抗脯氨酸结构类似物突变株的筛选及突变株proBA基因的克隆和序列分析

利用亚硝基胍对枯草芽孢杆菌93151进行诱变处理，获得了耐NaCl浓度达14％的突变株，同时发现该突变株也是一个抗脯氨酸反馈抑制突变菌株，其胞内自由脯氨酸的含量随着盐浓度的提高显著增加，说明其对渗透压的耐受能力与胞内自由脯氨酸的含量紧密相关。利用PCR方法克隆突变株的proBA基因，得到一个约2.3kb的DNA片段，序列分析表明该片段含有一完整的proB基因和部分proA基因，与野生菌株的proB基因相比，突变株proB基因中有3个碱基发生了改变，其中一个碱基的变化（从起始密码子开始第781位由T→A）导致了一个氨基酸发生改变（Ser→Thr)，另外两个碱基变化为沉默位点突变。将该proB基因转入大肠杆菌脯氨酸营养缺陷型菌株，能够与其功能互补。同时对部分proA基因序列分析发现，其与proB基因头尾重叠。在proA基因起始密码子上游第14个碱基处有一个类似于SD的序列，其所编码的氨基酸序列与枯草芽孢杆菌168的同源性为77％。

Cloning and Sequencing of the proBA Gene from the Selected Mutant Resistant to Proline Analogue from Bacillus subtilis

NTG was used to make chemical mutation for Bacillus subtilis 93151. An enhanced osmotolerant mutant was obtained, which could grow in minimal medium containing 14% NaCl (w/v) and was not subject to proline mediated feedback repression. The content of the intracellular free proline from the mutant increased rapidly with the rising of NaCl concentration. A 2.3kb DNA fragment from the mutant was amplified using PCR method. Sequence analysis indicated that three bases changed within the proB gene, compared with the wild type strain. One of the mutations was substitution of an A for a T at nt position 781, leading to a change of a Ser to a Thr at amino acid residue 261 of the deduced protein product, while other two were silent mutations. The recombinant vector pBE2 proB could functionally complement the proline auxotrophy E. coli 1.1252. Sequence analysis of proA showed that proA and proB overlapped by 4 nt, and there was a SD sequence at nt 14 upstream of the start codon of proA . The deduced amino acid of proA gene shared a high similarity with that of Bacillus subtilis 168 (77%).