Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella

The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.     

Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of Escherichia coli

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA'-'lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.     

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused ∼5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem–loop distal to cei, and the putative CsrA binding site overlapping the Shine–Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions.     

Comprehensive alanine-scanning mutagenesis of Escherichia coli CsrA defines two subdomains of critical functional importance

The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA'-'lacZ, flhDC'-'lacZ, and pgaA'-'lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (beta1, residues 2-7) and containing the last (beta5, residues 40-47) beta-strands of CsrA. The beta1 and beta5 strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.     

Global regulation by CsrA in Salmonella typhimurium

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.     

细菌全局性调控因子CsrA的结构与功能

碳存储调控因子A (carbon storage regulator, CsrA) 是一种RNA结合蛋白,在细菌的碳代谢、生物被膜形成、运动性、病原菌毒力、群体感应、环二鸟苷酸信号合成、应激感应等多种生理过程中具有重要调节功能,是全局性调控蛋白.它通过与靶标mRNA的特异结合,抑制其翻译或增强其稳定性来调控下游基因的表达,属于转录后调控因子的范畴.CsrA蛋白的表达与活性受碳存储调控(Csr)系统本身多个自主调节回路的精密控制：　一些小的非编码RNA (snmRNAs,如CsrB/C）作为拮抗因子与CsrA二聚体结合并抑制其活性;而这些snmRNAs在体内又可在CsrD的辅助下被核糖核酸内切酶E和多核苷酸磷酸化酶降解,释放CsrA的活性.当前,对于Csr系统的调节作用、调控通路与机制的研究是细菌学研究的热点,本文综述了该蛋白及Csr系统的结构、功能和作用机制的最新研究进展.