Cyclic Di Gmp Dependent Molecular Mechanisms In E Coli

Download or read book Cyclic Di-GMP-dependent Molecular Mechanisms in E. Coli written by Xin Fang and published by . This book was released on 2014 with total page 175 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacteria are able to undergo the lifestyle switch from the swimming motile single cells to the sedentary multicellular communities called biofilm. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a novel ubiquitous bacterial second messenger, seems to be the key regulator of bacterial lifestyle switch by controlling exopolysaccharide synthesis, cell adhesion, flagella-/pili-based motility, and virulence. So far, little is known about the cellular targets of c-di-GMP and the means by which it exerts its actions. The overall goal of my PhD research is to identify and understand the details of elaborate regulatory mechanisms utilized by c-di-GMP to control the transition between motile and sessile growth in bacteria. In Escherichia coli (E. coli), elevated level of c-di-GMP obtained by mutating glutamate-alanine-leucine (EAL) domain proteins such yhjH represses flagella-based motility. The effect is mediated by a PilZ domain protein YcgR, which blocks cell motility at the post-translational level. Based on co-immunoprecipitation, two-hybrid and mutational analyses, YcgR seems to bind to the flagellum switch complex, FliGMN. The primary interacting partner of YcgR is FliG, and the YcgR-FliG interaction is c-di-GMP-dependent. YcgR binds to a central region of FliG, which also interacts with FliM, the subunit which mediates chemotaxis-induced switching in rotation direction of the flagellum. An increase in counter clockwise bias in flagellum rotation and a decrease in the rotation speed resulting from YcgR-FliG interaction does not impair cell attachment to surfaces. This is consistent with the role of YcgR in the transition from a motile planktonic state to a surface-attached state. During evolution, some c-di-GMP signaling EAL domain proteins have lost their catalytic activity; one such example is the degenerative EAL protein YdiV from E. coli. This protein does not bind or hydrolyze c-di-GMP. Instead, YdiV binds to FlhD and acts as an anti-activator of the master regulator of flagellar gene expression (FlhD4C2). YdiV also functions as an adaptor that brings FlhD4C2 to the ClpXP protease for degradation. Here, we uncovered a new function of YdiV as an inhibitor of colanic acid synthesis associated with the mucoid colony phenotype. Finally, in order to search for novel c-di-GMP receptors, we used a DRaCALA assay to screen the ASKA collection, an E. coli expression gene library, with fluorescently or radioisotope-labeled c-di-GMP. The fluorescently labeled c-di-GMP method produced numerous false-positives and was abandoned. The radioactively-labeled c-di-GMP screen resulted in identification of several c-di-GMP-binding candidate proteins. One of these, BcsE, binds c-di-GMP with submicromolar affinity via a protein domain of unknown function, DUF2819, hereby designated GIL (GGDEF I-site like). A C-terminal fragment of GIL shows similarity to the c-di-GMP-binding I-site present in many GGDEF domain diguanylate cyclases. The RxxD motif involved in c-di-GMP binding in the I-sites is necessary for c-di-GMP binding in the GIL domain, which suggests evolutionary relatedness of the two domains. In S. enterica, the BcsE-c-di-GMP complex affects abundance of the BcsA cellulose synthase protein and therefore, is required for maximal cellulose synthesis. Independent of BcsE, c-di-GMP binds to the PilZ domain of BcsA and activates its glycosyltransferase activity. A two-tier control by c-di-GMP may increase the stringency of regulation of resource-consuming cellulose synthesis. The BcsE homologs from other representatives of enterobacteriaceae also bind c-di-GMP, which indicates that these bacteria use a similar regulatory setup for cellulose synthesis.