RESULTS:

Cyanobacteria modulate intracellular levels of cAMP and cGMP in response to environmental conditions (light nutrients and pH). In an attempt to identify components of the cAMP and cGMP signalling pathways in Synechocystis PCC 6803 the authors screened its complete genome sequence by using bioinformatic tools and data from sequence–function studies performed on both eukaryotic and prokaryotic cAMP/cGMP-dependent proteins. Sll1624 and Slr2100 were tentatively assigned as being two putative cyclic nucleotide phosphodiesterases. Five proteins were identified as having all the determinants required to be cyclic nucleotide receptors two of them being probably more specific for cGMP (an element of two-component regulatory systems – Slr2104 – and a putative cyclic-nucleotide-gated cation channel – Slr1575) the three others being probably more specific for cAMP: (i) a protein of unidentified function (Slr0842); (ii) a putative cyclic-nucleotide-modulated permease (Slr0593) previously annotated as a kinase A regulatory subunit; and (iii) a putative transcription factor (CRP-Syn =Sll1371) which possesses cAMP- and DNA-binding determinants homologous to those of the cAMP receptor protein of Escherichia coli (CRP-Ec). This homology together with the presence in Synechocystis of CRP-Ec-like binding sites upstream of crp cya1 slr1575 and several genes encoding enzymes involved in transport and metabolism strongly suggests that CRP-Syn is a global regulator.

Acid resistance is an important feature of both pathogenic and non-pathogenic Escherichia coli. It enables survival in the acidic regions of mammalian gastrointestinal tracts and is largely responsible for the small number of bacteria required for infection/colonization. Three systems of acid resistance have been identified the most efficient of which requires glutamic acid during pH 2 acid challenge. Three proteins associated with glutamate-dependent acid resistance have been identified. They are glutamate decarboxylase (encompassing two isozymes encoded by gadA and gadB) and a putative glutamate:γ-amino butyric acid antiporter (encoded by gadC). The results confirm that the GadA and GadB proteins increase in response to stationary phase and low environmental pH. The levels of these proteins correspond to concomitant changes in gadA and gadBC mRNA levels. Fusions between lacZ and the gadA and gadBC operons indicate that this control occurs at the transcriptional level. Western blot Northern blot and fusion analyses reveal that regulation of these genes is complex. Expression in rich media is restricted to stationary phase. However in minimal media acid pH alone can trigger induction in exponential or stationary phase cells. Despite this differential control there is only one transcriptional start site for each gene. Expression in rich media is largely dependent on the alternate sigma factor σS and is repressed by the cAMP receptor protein (CRP). In contrast σS has only a minor role in gad transcription in cells grown in minimal media. Deletions of the regulatory region upstream of gadA provided evidence that a 20 bp conserved region located 50 bp from the transcriptional start of both operons is required for expression.

Starvation of Salmonella enterica serovar Typhimurium (S. Typhimurium) for an exogenous source of carbon and energy (C-starvation) induces the starvation-stress response (SSR). The SSR functions to (i) maintain viability during long-term C-starvation and (ii) generate cross-resistance to other environmental stresses. The SSR is at least partially under the control of the alternative sigma factor σS. It is hypothesized that C-starvation causes cell envelope stresses that could induce the σE and/or Cpx regulons both of which control extracytoplasmic functions and thus may play a role in the regulation of the SSR. In support of this hypothesis Western blot analysis showed that the relative levels of σE increased during C-starvation peaking after approximately 72 h of C-starvation; in contrast CpxR levels remained relatively constant from exponential phase up to 72 h of C-starvation. To determine if σE and thus the regulon it controls is an essential component of the SSR several mutant strains were compared for their abilities to survive long-term C-starvation and to develop C-starvation-induced (CSI) cross-resistances. An rpoE mutant strain was significantly impaired in both long-term C-starvation survival (LT-CSS) and in CSI cross-resistance to challenges with 20 mM H2O2 for 40 min 55 °C for 16 min pH 3·1 for 60 min and 870·2 USP U polymyxin B ml−1 (PmB) for 60 min to varying degrees. These results suggest that C-starvation can generate signals that induce the rpoE regulon and that one or more members of the σE regulon are required for maximal SSR function. Furthermore evidence suggests that the σE and σS regulons function through separate mechanisms in the SSR. In contrast C-starvation does not appear to generate signals required for Cpx regulon induction which support the findings that it is not required for LT-CSS or cross-resistance to H2O2 pH 3·1 or PmB challenges. However it was required to achieve maximal cross-resistance to 55 °C. Therefore σE is a key regulatory component of the SSR and represents an additional σ factor required for the SSR of Salmonella.

Group A Streptococcus (GAS) possesses a complex regulatory system enabling the organism to colonize a range of physiologically distinct host sites. Within this network of regulators is the streptococcal regulator of virulence (Srv). Srv is a member of the CRP/FNR family of transcriptional regulators and is most similar to pleiotropic regulatory factor A (PrfA) a positive regulator of virulence in Listeria monocytogenes. Members of this family possess a characteristic C-terminal helix–turn–helix motif (HTH) that facilitates binding to DNA targets. Genome scanning identified four targets in GAS that were similar to the consensus DNA target recognized by PrfA. Furthermore previous amino acid sequence alignments identified conserved residues within the Srv HTH which are necessary for function in PrfA and CRP. Here we investigated the ability of Srv to interact with DNA and evaluated the role of the HTH in this interaction. Purified recombinant Srv (rSrv) was found to co-purify with an untagged form of Srv. Glutaraldehyde cross-linking and gel-filtration chromatography indicated that this co-purification is likely due to the ability of Srv to oligomerize. Electrophoretic mobility shift assays (EMSAs) demonstrated that rSrv retarded the mobility of DNA targets and a supershift analysis confirmed the observation was rSrv-dependent. Competition EMSA indicated that rSrv had a higher relative affinity for the DNA targets studied than non-specific DNA. Site-directed mutagenesis of residues predicted to be in or near the HTH resulted in a decrease or abrogation of DNA binding. Complementation of MGAS5005Δsrv with one of these site-directed mutants failed to restore wild-type SpeB activity. Taken together these data suggest that the Srv HTH is necessary for DNA binding and Srv function.

The ssr3341 locus was previously suggested to encode an orthologue of the RNA chaperone Hfq in the cyanobacterium Synechocystis sp. strain PCC 6803. Insertional inactivation of this gene resulted in a mutant that was not naturally transformable and exhibited a non-phototactic phenotype compared with the wild-type. The loss of motility was complemented by reintroduction of the wild-type gene correlated with the re-establishment of type IV pili on the cell surface. Microarray analyses revealed a small set of genes with drastically reduced transcript levels in the knockout mutant compared with the wild-type cells. Among the most strongly affected genes slr1667 slr1668 slr2015 slr2016 and slr2018 stood out as they belong to two operons that had previously been shown to be involved in motility controlled by the cAMP receptor protein SYCRP1. This suggests a link between cAMP signalling motility and possibly the involvement of RNA-based regulation. This is believed to be the first report demonstrating a functional role of an Hfq orthologue in cyanobacteria establishing a new factor in the control of motility.

Vibrio cholerae is the causative agent of cholera which continues to be a major public health concern in Asia Africa and Latin America. The bacterium can persist outside the human host and alternates between planktonic and biofilm community lifestyles. Transition between the different lifestyles is mediated by multiple signal transduction pathways including quorum sensing. Expression of the Zn-metalloprotease haemagglutinin (HA)/protease is subject to a dual regulation which involves the quorum-sensing regulator HapR and the cAMP receptor protein. In a previous study we observed that a mutant defective in the cAMP-receptor protein (CRP) expressed lower levels of HapR. To further investigate the role of CRP in modulating HapR and other signal transduction pathways we performed global gene expression profiling of a Δcrp mutant of El Tor biotype V. cholerae. Here we show that CRP is required for the biosynthesis of cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected the Δcrp mutant produced more cholera toxin and enhanced biofilm. Expression of flagellar genes reported to be affected in ΔhapR mutants was diminished in the Δcrp mutant. However an epistasis analysis indicated that cAMP–CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. cholerae to colonize the small intestine and cause disease. These genes included ompU ompT and ompW encoding outer-membrane proteins the alternative sigma factor σ E required for intestinal colonization and genes involved in anaerobic energy metabolism. Our results indicate that CRP plays a crucial role in the V. cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. cholerae in the human host and the environment.

The Escherichia coli guaB promoter (P guaB ) regulates transcription of two genes guaB and guaA that are required for the synthesis of guanosine 5′-monophosphate (GMP) a precursor for the synthesis of guanine nucleoside triphosphates. Transcription from P guaB increases as a function of increasing cellular growth rate and this is referred to as growth rate-dependent control (GRDC). Here we investigated the role of the factor for inversion stimulation (FIS) in the regulation of this promoter. The results showed that there are three binding sites for FIS centred near positions −11 +8 and +29 relative to the guaB transcription start site. Binding of FIS to these sites results in repression of P guaB in vitro but not in vivo. Deletion of the fis gene results in increased P guaB activity in vivo but GRDC of P guaB is maintained.

The effect of different carbon sources on the expression of tricarboxylic acid (TCA) cycle genes along with glyoxylate bypass genes in Corynebacterium glutamicum was determined. All TCA cycle genes were coordinately expressed in medium containing acetate. Growth in the presence of acetate gave rise to abundant expression of most TCA cycle genes with the level of gltA transcript being the highest. However when the cells entered the stationary phase triggered by acetate exhaustion all genes were repressed except sucCD and mdhB which were slightly induced. Acetate withdrawal from the growth medium during the exponential phase also led to rapid repression of most TCA cycle genes and a corresponding twofold increase in the expression of sucCD which were strongly induced by citrate and succinate. In addition glucose depletion during the stationary phase led to a corresponding 8–20-fold induction of the sucCD aceA and aceB genes. Addition of glucose to acetate medium resulted in about 10-fold induction of sucCD. The strong dependence of TCA cycle sucCD and glyoxylate bypass aceA and aceB expression on carbon source availability was confirmed and the regulatory system will be studied precisely.

Vfr a global regulator of Pseudomonas aeruginosa virulence factors is a homologue of the Escherichia coli cAMP receptor protein CRP. Vfr is 91 % similar to CRP and maintains many residues important for CRP to bind cAMP bind DNA and interact with RNA polymerase at target promoters. While vfr can complement an E. coli crp mutant in β-galactosidase production tryptophanase production and catabolite repression crp can only complement a subset of Vfr-dependent phenotypes in P. aeruginosa. Using specific CRP binding site mutations it is shown that Vfr requires the same nucleotides as CRP for optimal transcriptional activity from the E. coli lac promoter. In contrast CRP did not bind Vfr target sequences in the promoters of the toxA and regA genes. Footprinting analysis revealed Vfr protected sequences upstream of toxA regA and the quorum sensing regulator lasR that are similar to but significantly divergent from the CRP consensus binding sequence and Vfr causes similar DNA bending to CRP in bound target sequences. Using a preliminary Vfr consensus binding sequence deduced from the Vfr-protected sites Vfr target sequences were identified upstream of the virulence-associated genes plcN plcHR pbpG prpL and algD and in the vfr/orfX argH/fimS pilM/ponA intergenic regions. From these sequences the Vfr consensus binding sequence 5′-ANWWTGNGAWNY : AGWTCACAT-3′ was formulated. This study suggests that Vfr shares many of the same functions as CRP but has specialized functions at least in terms of DNA target sequence binding required for regulation of a subset of genes in its regulon.

The physiological changes induced by indoleacetic acid (IAA) treatment were investigated in the totally sequenced Escherichia coli K-12 MG1655. DNA macroarrays were used to measure the mRNA levels for all the 4290 E. coli protein-coding genes; 50 genes (1.1 %) exhibited significantly different expression profiles. In particular genes involved in the tricarboxylic acid cycle the glyoxylate shunt and amino acid biosynthesis (leucine isoleucine valine and proline) were up-regulated whereas the fermentative adhE gene was down-regulated. To confirm the indications obtained from the macroarray analysis the activity of 34 enzymes involved in central metabolism was measured; this showed an activation of the tricarboxylic acid cycle and the glyoxylate shunt. The malic enzyme involved in the production of pyruvate and pyruvate dehydrogenase required for the channelling of pyruvate into acetyl-CoA were also induced in IAA-treated cells. Moreover it was shown that the enhanced production of acetyl-CoA and the decrease of NADH/NAD+ ratio are connected with the molecular process of the IAA response. The results demonstrate that IAA treatment is a stimulus capable of inducing changes in gene expression enzyme activity and metabolite level involved in central metabolic pathways in E. coli.