Bandekar Divya, Chouhan Om Prakash, Mohapatra Swati, Hazra Mousumi, Hazra Saugata, Biswas Sumit
VISTA Lab, BITS, Pilani - K K Birla Goa Campus, Zuarinagar, Goa, India.
Department of Biotechnology, Indian Institute of Technology Roorkee, Uttarakhand, India.
Microbiol Res. 2017 Sep;202:61-70. doi: 10.1016/j.micres.2017.05.003. Epub 2017 May 23.
The hallmark of the lifecycle of Vibrio cholerae is its ability to switch between two lifestyles - the sessile, non-pathogenic form and the motile, infectious form in human hosts. One of these changes is in the formation of surface biofilms, when in sessile aquatic habitats. The cell-cell interactions within a V. cholerae biofilm are stabilized by the production of an exopolysachharide (EPS) matrix, which in turn is regulated by the ubiquitous secondary messenger, cyclic di-GMP (c-di-GMP), synthesized by proteins containing GGD(/E)EF domains in all prokaryotic systems. Here, we report the functional role of the VC0395_0300 protein (Sebox3) encoded by the chromosome I of V. cholerae, with a GGEEF signature sequence, in the formation of surface biofilms. In our study, we have shown that Escherichia coli containing the full-length Sebox3 displays enhanced biofilm forming ability with cellulose production as quantified and visualized by multiple assays, most notably using FEG-SEM. This has also been corroborated with the lack of motility of host containing Sebox3 in semi-solid media. Searching for the reasons for this biofilm formation, we have demonstrated in vitro that Sebox3 can synthesize c-di-GMP from GTP. The homology derived model of Sebox3 displayed significant conservation of the GGD(/E)EF architecture as well. Hence, we propose that the putative protein VC0395_0300 from V. cholerae is a diguanylate cyclase which has an active role in biofilm formation.
霍乱弧菌生命周期的标志是其能够在两种生活方式之间转换——在人类宿主中，一种是固着的、无致病性的形式，另一种是游动的、具有传染性的形式。其中一个变化发生在固着于水生栖息地时表面生物膜的形成过程中。霍乱弧菌生物膜内的细胞间相互作用通过胞外多糖（EPS）基质的产生得以稳定，而EPS基质又受普遍存在的第二信使环二鸟苷酸（c-di-GMP）调控，c-di-GMP由所有原核系统中含GGD(/E)EF结构域的蛋白质合成。在此，我们报道了霍乱弧菌染色体I编码的具有GGEEF特征序列的VC0395_0300蛋白（Sebox3）在表面生物膜形成中的功能作用。在我们的研究中，我们发现含有全长Sebox3的大肠杆菌表现出增强的生物膜形成能力，通过多种检测方法（最显著的是使用场发射枪扫描电子显微镜（FEG-SEM））对纤维素产量进行定量和可视化分析得以证实。这一点也通过含有Sebox3的宿主在半固体培养基中缺乏运动性得到了佐证。为探究这种生物膜形成的原因，我们在体外证明Sebox3能够从鸟苷三磷酸（GTP）合成c-di-GMP。Sebox3的同源衍生模型也显示出GGD(/E)EF结构具有显著的保守性。因此，我们提出霍乱弧菌的推定蛋白VC0395_0300是一种二鸟苷酸环化酶，在生物膜形成中发挥着积极作用。
