Bandara Aloka B, Schurig Gerhardt G, Sriranganathan Nammalwar, Prasad Rajeev, Boyle Stephen M
Department of Biomedical Sciences & Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0910, USA.
Vet Microbiol. 2009 Feb 2;133(4):387-93. doi: 10.1016/j.vetmic.2008.07.019. Epub 2008 Aug 5.
The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16M possesses seven pbp genes: three in pbp-1 family (designated as 1A, 1B, and 1C); one in pbp-2 family; and three in pbp-6 family (designated as 6A, 6B, and 6C). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis. A recombinant B. melitensis strain (designated 16MDeltapbp1C) was generated by disrupting the pbp-1C of strain 16M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16M. Electron microscopy revealed that strain 16M exhibited native cocco-bacillus morphology, while 16MDeltapbp1C possessed a spherical morphology. Strain 16MDeltapbp1C did not differ from strain 16M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis. Expression of mRNA of fixR, the gene downstream of pbp-1C was similar between the strains 16M and 16MDeltapbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A, pbp-1B, or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis. Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A, pbp-1B, and pbp-2 are essential for viability of B. melitensis.
青霉素结合蛋白（PBPs）是调节细菌细胞壁肽聚糖层组装的酶。羊种布鲁氏菌16M菌株的基因组拥有7个pbp基因：pbp - 1家族中有3个（命名为1A、1B和1C）；pbp - 2家族中有1个；pbp - 6家族中有3个（命名为6A、6B和6C）。我们研究了pbp - 1和pbp - 2基因对羊种布鲁氏菌的生存能力、细胞形态和感染性的重要性。通过等位基因交换破坏16M菌株的pbp - 1C基因，构建了一株重组羊种布鲁氏菌菌株（命名为16MDeltapbp1C）。该菌株在胰蛋白胨大豆琼脂平板上形成的菌落比16M菌株小近20%，并且在胰蛋白胨大豆肉汤中的生长速度比16M菌株慢。电子显微镜观察显示，16M菌株呈现天然的球杆菌形态，而16MDeltapbp1C具有球形形态。16MDeltapbp1C菌株在从感染的小鼠巨噬细胞系J774.1中恢复，或从感染的BALB/c小鼠脾脏中恢复方面，与16M菌株没有差异，这表明pbp - 1C对于羊种布鲁氏菌在细胞内的持续存在是可有可无的。pbp - 1C下游基因fixR的mRNA表达在16M和16MDeltapbp1C菌株之间相似，这表明pbp - 1C的破坏没有诱导任何极性效应。多次尝试突变pbp - 1A、pbp - 1B或pbp - 2基因均失败，很可能是因为这些基因对于羊种布鲁氏菌的生存能力是不可或缺的。我们的研究结果表明，pbp - 1C调节体外生长和细胞形态，而pbp - 1A、pbp - 1B和pbp - 2对于羊种布鲁氏菌的生存能力至关重要。
