Wang Xiaoyu, Luo Chuping, Liu Yongfeng, Liu Youzhou, Nie Yafeng, Chen Zhiyi
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
Wei Sheng Wu Xue Bao. 2009 Oct;49(10):1295-300.
Bacillus subtilis Bs-916 has obvious effects against Rhizoctonia solani. We demonstrated that the mutant strain, M49 obtained by means of low energy ion implantation in strain Bs-916, which produces significantly lower levels of surfactin, had no obvious effects against R. solani.
In order to identify the influencing factors of surfactin-decrease mutant strain M49, its phenotype and related gene expression levels were studied.
Strains to be tested for sporulation were grown for 24 h in sporulation medium. Plasmid pDG1728 (1 microg/mL) was used for DNA transformation to test competence development of M49 and Bs-916 strains. RT-PCR (Semi-quantitative reverse transcriptase-polymerase chain-reaction) was used to determine the expression levels of selected genes involved in competence and sporulation in both the wildtype Bs-916 and mutant M49 strains, such as comS, rapA, rapC gene.
Our data showed that mutant strain M49 confers a leaky competence phenotype, typified by a ten-fold reduction. This indicated a fact that DNA fragments are more easily transformed to wildtype strain than M49 mutant. The M49 strain also appeared to exhibit a sporulation-deficient phenotype, compared with the wild-type Bs-916, its spore's number declined by about 75%. RT-PCR results showed that both the comS and rapC genes were expressed in the Bs-916 strain but not in the M49 strain.
Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter, PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild type Bs-916 and mutant M49 strains is significantly different. When we integrated comA orf into the chromosome of M49 at amyE locus, M49 restored antifungal activity. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis.
枯草芽孢杆菌Bs - 916对茄丝核菌有明显的抑制作用。我们证明，通过低能离子注入菌株Bs - 916获得的突变菌株M49，其表面活性素的产生水平显著降低，对茄丝核菌没有明显的抑制作用。
为了确定表面活性素减少突变菌株M49的影响因素，研究了其表型和相关基因表达水平。
将用于产孢测试的菌株在产孢培养基中培养24小时。使用质粒pDG1728（1微克/毫升）进行DNA转化，以测试M49和Bs - 916菌株的感受态发育情况。使用RT - PCR（半定量逆转录聚合酶链反应）来确定野生型Bs - 916和突变型M49菌株中参与感受态和产孢的选定基因的表达水平，如comS、rapA、rapC基因。
我们的数据表明，突变菌株M49表现出渗漏性感受态表型，其典型特征是降低了10倍。这表明一个事实，即DNA片段更容易转化到野生型菌株而不是M49突变体中。与野生型Bs - 916相比，M49菌株似乎也表现出产孢缺陷表型，其孢子数量下降了约75%。RT - PCR结果表明，comS和rapC基因在Bs - 916菌株中表达，但在M49菌株中不表达。
srfA启动子和ComP - ComA信号转导系统都是导致表面活性素产生的因素的重要组成部分。对Bs - 916和M49的序列进行Blast比较表明，srfA操纵子启动子PsrfA没有差异，但comA基因的编码序列存在差异。这些差异导致M49 ComA蛋白内出现三个错义突变。RT - PCR分析结果表明，野生型Bs - 916和突变型M49菌株中参与感受态和产孢的选定基因的表达水平存在显著差异。当我们将comA orf整合到M49的amyE位点染色体上时，M49恢复了抗真菌活性。这些数据表明，ComA中三个关键氨基酸的突变极大地影响了枯草芽孢杆菌的生物活性。
