Yam Hokchai, Rahim Ainihayati Abdul, Mohamad Suriani, Mahadi Nor Muhammad, Manaf Uyub Abdul, Shu-Chien Alexander Chong, Najimudin Nazalan
School of Biological Sciences, Universiti Sains Malaysia, Minden, Pulau Pinang, Malaysia.
School of Biological Sciences, Universiti Sains Malaysia, Minden, Pulau Pinang, Malaysia; Faculty of Agro Based Industry, Universiti Malaysia Kelantan, Jeli, Kelantan, Malaysia.
PLoS One. 2014 Jun 13;9(6):e99218. doi: 10.1371/journal.pone.0099218. eCollection 2014.
Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β' subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.
类鼻疽伯克霍尔德菌是一种机会致病菌，也是类鼻疽的病原体。它能够适应恶劣环境，并能在受感染宿主的细胞内生存。在本研究中，对与RNA聚合酶的β'亚基（RpoC）相关的转录因子进行了鉴定。已知该亚基的N端区域与σ因子结合时会引发启动子解链。使用组氨酸标记的类鼻疽伯克霍尔德菌RpoC N端区域作为诱饵进行的下拉试验表明，一种假定蛋白BPSS1356是与之结合的蛋白之一。这种假定蛋白在所有类鼻疽伯克霍尔德菌菌株中都保守，且仅存在于伯克霍尔德菌属中。构建了BPSS1356缺失突变体以研究其生物学功能。该突变菌株在LB培养基中生长的稳定期表现出生物膜形成减少和细胞密度降低。电子显微镜分析显示，与野生型相比，ΔBPSS1356突变体细胞的细胞质收缩，表明细胞发生质壁分离，且表面更粗糙。RNA微阵列结果显示，共有63个基因在转录水平上受到BPSS1356缺失的影响，其倍数变化值高于4。在ΔBPSS1356突变体中，一组编码膜定位转运蛋白的基因表达同时下调。在受影响的基因中，假定的离子转运基因受到的抑制最为严重。缺失BPSS1356也下调了精氨酸脱亚氨酶系统、甘油代谢、III型分泌系统簇2、细胞色素bd氧化酶和抗砷相关基因的转录。因此，很明显BPSS1356对许多基因发挥多种调节作用。
