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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.

Campylobacter jejuni is a gastrointestinal pathogen of humans but can asymptomatically colonize the avian gut. C. jejuni therefore grows at both 37 degrees C and 42 degrees C, the internal temperatures of humans and birds respectively. Microarray and proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 42 degrees C of two proteins, Cj0414 and Cj0415, orthologous to gluconate dehydrogenase (GADH) from Pectobacterium cypripedii. 81-176 demonstrated GADH activity, converting d-gluconate to 2-keto-d-gluconate, that was higher at 42 degrees C than at 37 degrees C. In contrast, cj0414 and cj0415 mutants lacked GADH activity. Wild-type but not cj0415 mutant bacteria exhibited gluconate-dependent respiration. Neither strain grew in defined media with d-gluconate or 2-keto-d-gluconate as a sole carbon source, revealing that gluconate was used as an electron donor rather than as a carbon source. When administered to chicks individually or in competition with wild-type, the cj0415 mutant was impaired in establishing colonization. In contrast, there were few significant differences in colonization of BALB/c-ByJ mice in single or mixed infections. These results suggest that the ability of C. jejuni to use gluconate as an electron donor via GADH activity is an important metabolic characteristic that is required for full colonization of avian but not mammalian hosts.

Summary The Campylobacter fetus surface layer proteins (SLPs), encoded by five to nine sapA homologues, are major virulence factors. To characterize the sapA homologues further, a 65.9 kb C. fetus genomic region encompassing the sap locus from wild-type strain 23D was completely sequenced and analysed; 44 predicted open reading frames (ORFs) were recognized. The 53.8 kb sap locus contained eight complete and one partial sapA homologues, varying from 2769 to 3879 bp, sharing conserved 553–2622 bp 5′ regions, with partial sharing of 5′ and 3′ non-coding regions. All eight sapA homologues were expressed in Escherichia coli as antigenic proteins and reattached to the surface of SLP– strain 23B, indicating their conserved function. Analysis of the sap homologues indicated three phylogenetic groups. Promoter-specific polymerase chain reactions (PCRs) and sapA homologue-specific reverse transcription (RT)-PCRs showed that the unique sapA promoter can potentially express all eight sapA homologues. Reciprocal DNA recombination based on the 5′ conserved regions can involve each of the eight sapA homologues, with frequencies from 10−1 to 10−3. Intragenic recombination between sapA7 and sapAp8, mediated by their conserved regions with a 10−1−10−2 frequency, allows the formation of new sap homologues. As divergent SLP C-termini possess multiple antigenic sites, their reciprocal recombination behind the unique sap promoter leads to continuing antigenic variation.

A collection of 20 strains of Helicobacter pylori from several regions of the world was studied to better understand the population genetic structure and diversity of this species. Sequences of fragments from seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, yphC ) and two virulence-associated genes (cagA, vacA) showed high levels of synonymous sequence variation (mean percentage Ks of 10-27%) and lower levels of non-synonymous variation (mean percentage Ka of 0.2-5.6%). Cluster analysis of pairwise differences between alleles revealed the existence of two weakly clonal groupings, which included half of the strains investigated. All six strains isolated from Japanese and coastal Chinese were assigned to the 'Asian' clonal grouping, probably reflecting descent from a distinct common ancestor. The clonal groupings were not totally uniform; recombination, as measured by the homoplasy test and compatibility matrices, was extremely common within all genes tested, except cagA. The fact that clonal descent could still be discerned despite such frequent recombination possibly reflects founder effects and geographical separation and/or selection for particular alleles of these genes.

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.

Neisseria meningitidis FAM20 has recently been shown to produce two Fe-regulated proteins (FrpA and FrpC) related to the RTX family of cytotoxins. Here we report the cloning and DNA sequence of the locus containing the gene encoding the larger meningococcal RTX protein FrpC. FrpC was highly similar to FrpA throughout much of the predicted protein, with two main differences. Whereas the FrpA protein had 13 copies of the nine-amino-acid repeat units typical of RTX proteins, FrpC had 43 copies. The additional copies in FrpC apparently arose from a threefold tandem amplification of a 600bp DNA fragment encoding the repeats. In addition, the frpC gene lacked good promoter consensus sequences. An open reading frame (ORF1) of unknown function was found immediately upstream of frpC, suggesting the possibility that frpC was cotranscribed with ORF1. A probable promoter was found 300bp upstream of ORF1, and it contained a Fur protein-binding sequence found in the promoters of Fe-regulated Escherichia coli genes. DNA upstream of the ORF1/frpC promoter was homologous to IS1016-like elements surrounding capsulation loci of strains of Haemophilus influenzae. A FrpC-like protein (reactive in immunoblots with monoclonal antibody 9D4; multiple reactive bands of about 200 to 120 kDa) was found in five out of eight meningococcal strains but only in one out of 14 other Neisseria, suggesting that FrpC may participate in the pathogenesis of meningococcal disease.

Campylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS (Campylobacter planktonic growth regulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Delta cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Delta cprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in Delta cprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.

Summary To investigate urease-independent mechanisms by which Helicobacter pylori resists acid stress, subtractive RNA hybridization was used to identify H. pylori genes whose expression is induced after exposure to acid pH. This approach led to the isolation of a gene that encoded a predicted 34.8 kDa protein (WbcJ), which was homologous to known bacterial O-antigen biosynthesis proteins involved in the conversion of GDP-mannose to GDP-fucose. An isogenic wbcJ null mutant strain failed to express O-antigen and Lewis X or Lewis Y determinants and was more sensitive to acid stress than was the wild-type strain. Qualitative differences in LPS profiles were observed in H. pylori cells grown at pH 5 compared with pH 7, which suggests that H. pylori may alter its LPS structure in response to acidic pH. This may be an important adaptation facilitating H. pylori colonization of the acidic gastric environment.