csrA Mutant Research Articles

CsrA regulates Helicobacter pylori J99 motility and adhesion by controlling flagella formation.

Motility mediated by the flagella of Helicobacter pylori has been shown to be required for normal colonization and is thought to be important for the bacteria to move toward the gastric mucus in niches adjacent to the epithelium. Barnard et al. showed that CsrA appears to be necessary for full motility and the ability to infect mice, but its mechanism of regulation is still unclear. Motility and cell adhesion ability were determined in wild-type, csrA mutant, and revertant J99 strains. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. The expression of two major flagellins, flaA/flaB, and the alternative sigma factor rpoN (σ(54)) were determined by real-time quantitative RT-PCR and Western blot. The csrA mutant showed loss of motility and lower adhesion ability compared with the wild-type and revertant J99 strains. The csrA mutant was not flagellated. Transcription of flaA and flaB mRNA decreased to only 40% and 16%, respectively, in the csrA mutant compared with the wild-type J99 (p = .006 and <.0001, respectively), and Western blot analysis showed dramatically reduced FlaA/FlaB proteins in a csrA mutant. The disruption of csrA also decreased expression of rpoN to 48% in the csrA mutant, but the degradation rate of rpoN mRNA was not changed. These results suggest that CsrA regulates H. pylori J99 flagella formation and thereby affects bacterial motility.

A Novel Endogenous Induction of ColE7 Expression in a csrA Mutant of Escherichia coli

Carbon storage regulator A (CsrA) is an important regulator that controls central metabolic pathways and a variety of physiological functions. We found that disruption of csrA in cells containing the ColE7 operon caused a 12-fold increase in colicin E7 production. Moreover, real-time RT-PCR demonstrated a decrease of around 50 % in the lexA mRNA of the csrA mutant. However, the cellular level of RecA protein and its mRNA were not significantly different from the wild type strain. Our results suggest that a novel induction mechanism might exist in E. coli that allows the expression of ColE7 operon in response to a metabolic shift. Proteomic analysis suggested that csrA deficient mutant may adapt PEP-glyoxylate cycle for energy production. Thus, the physiological changes in the csrA mutant may be similar to carbon source limitation for initiating the expression of ColE7 operon in response to stringent environmental conditions.

Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation

BackgroundAlthough Campylobacter jejuni is consistently ranked as one of the leading causes of bacterial diarrhea worldwide, the mechanisms by which C. jejuni causes disease and how they are regulated have yet to be clearly defined. The global regulator, CsrA, has been well characterized in several bacterial genera and is known to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus Campylobacter. Previously, we reported data illustrating the requirement for CsrA in several virulence related phenotypes of C. jejuni strain 81–176, indicating that the Csr pathway is important for Campylobacter pathogenesis.ResultsWe compared the Escherichia coli and C. jejuni orthologs of CsrA and characterized the ability of the C. jejuni CsrA protein to functionally complement an E. coli csrA mutant. Phylogenetic comparison of E. coli CsrA to orthologs from several pathogenic bacteria demonstrated variability in C. jejuni CsrA relative to the known RNA binding domains of E. coli CsrA and in several amino acids reported to be involved in E. coli CsrA-mediated gene regulation. When expressed in an E. coli csrA mutant, C. jejuni CsrA succeeded in recovering defects in motility, biofilm formation, and cellular morphology; however, it failed to return excess glycogen accumulation to wild type levels.ConclusionsThese findings suggest that C. jejuni CsrA is capable of efficiently binding some E. coli CsrA binding sites, but not others, and provide insight into the biochemistry of C. jejuni CsrA.

Circuitry linking the Csr and stringent response global regulatory systems

CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.

Metabolic engineering of Escherichia coli to enhance phenylalanine production

The global regulatory system of Escherichia coli, carbon storage regulator (Csr), was engineered to increase the intracellular concentration of phosphoenolpyruvate. We examined the effects of csrA and csrD mutations and csrB overexpression on phenylalanine production in E. coli NST37 (NST). Overexpression of csrB led to significantly greater phenylalanine production than csrA and csrD mutations (2.33 vs 1.67 and 1.61 g l(-1), respectively; P < 0.01). Furthermore, the overexpression of csrB was confirmed by the observed increase in csrB transcription level. We also determined the effect of overexpressing transketolase A (TktA) or glucose-6-phosphate dehydrogenase (Zwf) in NST and the csrA mutant of NST (NSTCSRA) on phenylalanine production. The NSTCSRA strain overexpressing TktA (NSTCSRA [pTktA]) produced significantly more phenylalanine than that of Zwf (2.39 vs 1.61 g l(-1); P > 0.01). Furthermore, we examined the effect of overexpressing TktA, 3-deoxy-D: -arabino-heptulosonate-7-phosphate synthase (AroF(FR)), and chorismate mutase/prephenate dehydratase (PheA(FR)) together in NSTCSRA (NSTCSRA [pTkaFpA]). It is interesting to note that NSTCSRA [pTkaFpA] produced significantly less phenylalanine than both NSTCSRA [pTktA] and NST overexpressing csrB (NST [pCsrB]) (1.84 vs 2.39 and 2.33 g l(-1), respectively; P < 0.01). Thus, csrB overexpression or csrA mutation in combination with tktA overexpression was more effective than previous approaches that targeted the glycolytic or aromatic pathway enzymes for enhancing phenylalanine production.

Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects virulence-associated regulators and growth in Acanthamoeba castellanii

Legionella bacteria have a developmental cycle in which they go from existing in the aquatic environment to replicating inside eukaryotic host cells. The adaptation to the new environment requires an efficient regulatory system. Overexpression of CsrA, a global regulatory protein found in a variety of Gram-negative bacteria has been shown to suppress virulence-associated traits in Legionella pneumophila. Since evidence resulting only from overproduction may not be sufficient to validate the role of a regulatory protein, a csrA mutant strain, CsrA(−), with a drastically reduced production of CsrA, was created. Using RNA slot blots and Western blotting it was shown that fliA and flaA, genes which contribute to flagellation, were expressed early in the mutant. Additionally, in CsrA(−) the levels of the stationary-phase sigma factor, RpoS, and a recently described regulator of virulence traits, LetE, were increased. Growth curves of CsrA(−) bacteria were delayed with pigment production occurring at the same OD 578 but at reduced levels in the mutant. Replication ability of the CsrA(−) mutant in amoebae was also affected. Based on these results, we could show that CsrA is involved in the regulation of the bacterial switch from the replicative to the transmissible form.

Global regulation of virulence and the stress response by CsrA in the highly adapted human gastric pathogen Helicobacter pylori

Although successful and persistent colonization of the gastric mucosa depends on the ability to respond to changing environmental conditions and co-ordinate the expression of virulence factors during the course of infection, Helicobacter pylori possesses relatively few transcriptional regulators. We therefore investigated the contribution of the regulatory protein CsrA to global gene regulation in this important human pathogen. CsrA was necessary for full motility and survival of H. pylori under conditions of oxidative stress. Loss of csrA expression deregulated the oxidant-induced transcriptional responses of napA and ahpC, the acid induction of napA, cagA, vacA, the urease operon, and fur, as well as the heat shock responses of napA, groESL and hspR. Although the level of napA transcript was higher in the csrA mutant, its stability was similar in the wild-type and mutant strains, and less NapA protein was produced in the mutant strain. Finally, H. pylori strains deficient in the production of CsrA were significantly attenuated for virulence in a mouse model of infection. This work provides evidence that CsrA has a broad role in regulating the physiology of H. pylori in response to environmental stimuli, and may be important in facilitating adaptation to the different environments encountered during colonization of the gastric mucosa. Furthermore, CsrA appears to mediate its effects in H. pylori at the post-transcriptional level by influencing the processing and translation of target transcripts, with minimal effect on the stability of the target mRNAs.

CsrA regulates translation of the Escherichia coli carbon starvation gene, cstA, by blocking ribosome access to the cstA transcript.

CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia coli genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA'-'lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by Esigma(70). We investigated the influence of sigma(S) on cstA expression and found that a sigma(S) deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA Shine-Dalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.

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.

Role of RsmA in the regulation of swarming motility and virulence factor expression in Proteus mirabilis.

Swarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper-flagellated swarm cells that undergo cycles of rapid and co-ordinated population migration across surfaces and exhibit high levels of virulence gene expression. RsmA (repressor of secondary metabolites) and CsrA, its homologue in Escherichia coli, control many phenotypic traits, such as motility and pathogenesis in Erwinia species, glycogen biosynthesis, cell size and biofilm formation in Escherichia coli and swarming motility in Serratia marcescens. To investigate the role of RsmA in Proteus mirabilis, the rsmA gene from Proteus mirabilis (hereafter referred to as rsmA(Pm)) was cloned. RsmA(Pm) showed high sequence similarity to Escherichia coli CsrA and RsmA cloned from Erwinia carotovora subsp. carotovora, Serratia marcescens, Haemophilus influenzae and Bacillus subtilis and could complement an Escherichia coli csrA mutant in glycogen synthesis. A low-copy-number plasmid carrying rsmA(Pm) expressed from its native promoter caused suppression of swarming motility and expression of virulence factors in Proteus mirabilis. mRNA stability assays suggested that RsmA(Pm) inhibited virulence factor expression through promoting mRNA degradation. RsmA homologues cloned from Serratia marcescens and Erwinia carotovora subsp. carotovora could also inhibit swarming and virulence factor expression in Proteus mirabilis.

Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli.

Many species of bacteria devote considerable metabolic resources and genetic information to the ability to sense the environment and move towards or away from specific stimuli using flagella. In Escherichia coli and related species, motility is regulated by several global regulatory circuits, which converge to modulate the overall expression of the master operon for flagellum biosynthesis, flhDC. We now show that the global regulator CsrA of E. coli K-12 is necessary for motility under a variety of growth conditions, as a result of its role as an activator of flhDC expression. A chromosomally encoded flhDC'-'lacZ translational fusion was expressed at three- to fourfold higher levels in csrA wild-type strains than in isogenic csrA mutants. Purified recombinant CsrA protein stimulated the coupled transcription-translation of flhDC'-' lacZ in S-30 extracts and bound to the 5' segment of flhDC mRNA in RNA mobility shift assays. The steady-state level of flhDC mRNA was higher and its half-life was approximately threefold greater in a csrA wild-type versus a csrA mutant strain. Thus, CsrA stimulates flhDC gene expression by a post-transcriptional mechanism reminiscent of its function in the repression of glycogen biosynthesis.

Regulation of Salmonella enterica serovar typhimurium invasion genes by csrA.

Penetration of intestinal epithelial cells by Salmonella enterica serovar Typhimurium requires the expression of invasion genes, found in Salmonella pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of Escherichia coli csrA, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of csrA was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes, hilA, invF, sipC, and prgH. Overexpression of csrA from an induced araBAD promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected hilA, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate hilA. We also found that both the loss and the overexpression of csrA reduced the expression of two regulators of hilA, hilC and hilD, suggesting that csrA exerts its control of hilA through one or both of these regulators. We further found, however, that CsrA could affect the expression of both invF and sipC independent of its effects on hilA. One additional striking phenotype of the csrA mutant, not observed in a comparable E. coli mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the csrA mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the csrA-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the csrA mutant arise from effects of CsrA on different targets.