- Volume 157, Issue 4, 2011

The soxRS regulon protects Escherichia coli cells against superoxide and nitric oxide. Oxidation of the SoxR sensor, a [2Fe–2S]-containing transcriptional regulator, triggers the response, but the nature of the cellular signal sensed by SoxR is still a matter of debate. In vivo, the sensor is maintained in a reduced, inactive state by the activities of SoxR reductases, which employ NADPH as an electron donor. The hypothesis that NADPH levels affect deployment of the soxRS response was tested by transforming E. coli cells with genes encoding enzymes and proteins that lead to either build-up or depletion of the cellular NADPH pool. Introduction of NADP+-reducing enzymes, such as wheat non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase or E. coli malic enzyme, led to NADPH accumulation, inhibition of the soxRS regulon and enhanced sensitivity to the superoxide propagator methyl viologen (MV). Conversely, expression of pea ferredoxin (Fd), a redox shuttle that can oxidize NADPH via ferredoxin-NADP(H) reductase, resulted in execution of the soxRS response in the absence of oxidative stress, and in higher tolerance to MV. Processes that caused NADPH decline, including oxidative stress and Fd activity, correlated with an increase in total (NADP++NADPH) stocks. SoxS expression can be induced by Fd expression or by MV in anaerobiosis, under conditions in which NADPH is oxidized but no superoxide can be formed. The results indicate that activation of the soxRS regulon in E. coli cells exposed to superoxide-propagating compounds can be triggered by depletion of the NADPH stock rather than accumulation of superoxide itself. They also suggest that bacteria need to finely regulate homeostasis of the NADP(H) pool to enable proper deployment of this defensive response.

Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni2+ or Zn2+, and roughly halved in the presence of Co2+ or Fe3+. The limB gene was maximally expressed at 3–7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.

Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ–ProA–ProH route is responsible for the production of proline as an osmoprotectant, and the ProB–ProA–ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB–treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.

Azospirillum brasilense, a plant-growth-promoting rhizobacterium, is exposed to changes in its abiotic environment, including fluctuations in temperature, salinity, osmolarity, oxygen concentration and nutrient concentration, in the rhizosphere and in the soil. Since extra-cytoplasmic function (ECF) sigma factors play an important role in stress adaptation, we analysed the role of ECF sigma factor (also known as RpoE or σ E) in abiotic stress tolerance in A. brasilense. An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slow-growing, and was sensitive to salt, ethanol and methylene blue stress. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis and sensitivity to different stresses. Based on data from reverse transcriptase-PCR, a two-hybrid assay and a pull-down assay, we present evidence that rpoE is cotranscribed with chrR and the proteins synthesized from these two overlapping genes interact with each other. Identification of the transcription start site by 5′ rapid amplification of cDNA ends showed that the rpoE–chrR operon was transcribed by two promoters. The proximal promoter was less active than the distal promoter, whose consensus sequence was characteristic of RpoE-dependent promoters found in alphaproteobacteria. Whereas the proximal promoter was RpoE-independent and constitutively expressed, the distal promoter was RpoE-dependent and strongly induced in response to stationary phase and elevated levels of ethanol, salt, heat and methylene blue. This study shows the involvement of RpoE in controlling carotenoid synthesis as well as in tolerance to some abiotic stresses in A. brasilense, which might be critical in the adaptation, survival and proliferation of this rhizobacterium in the soil and rhizosphere under stressful conditions.

CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10- to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR–operator complexes are 18-fold less stable than RNA polymerase (RNAP)–pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO4 footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.

Formation of the segrosome, a nucleoprotein complex crucial for proper functioning of plasmid partition systems, involves interactions between specific partition proteins (ParA-like and ParB-like), ATP and specific DNA sequences (the centromeric sites). Although partition systems have been studied for many years, details of the segrosome formation are not yet clear. Organization of the pSM19035-encoded partition system is unique; in contrast with other known par systems, here, the δ and ω genes do not constitute an operon. Moreover, Omega [a ParB-like protein which has a Ribbon-Helix-Helix (RHH) structure] recognizes multiple centromeric sequences located in the promoters of δ, ω and copS (copy-number control gene). The ParA-like protein Delta is a Walker-type ATPase. In this work, we identify the interaction domains and requirements for dimerization and hetero-interactions of the Delta and Omega proteins of pSM19035 plasmid. The RHH structures are involved in Omega dimerization in vivo and its N-terminal unstructured part is indispensable for association with Delta, both in vivo and in vitro. Omega does not need to form dimers to interact with Delta. ATP binding is not required for Delta dimerization but is important for interaction with Omega in vivo. The in vitro interaction between Delta and Omega depends on ATP but does not require the presence of specific DNA segments (the centromere) recognized by Omega. The C-terminal part of the Delta protein (aa 198–284) is indispensable for interaction with Omega. Delta most probably interacts with Omega as a dimer since two amino acid substitutions in a conserved region between the A′ and B motifs abolish both the dimerization of Delta and its interaction with Omega.

The Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Saccharomyces cerevisiae Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4–Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used S. cerevisiae mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X3-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4–Ctr5 complex, whereas the Met-X3-Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of Sch. pombe cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface.

There are six essential genes in the Saccharomyces cerevisiae genome which encode proteins bearing the tetratricopeptide repeat (TPR) domain that mediates protein–protein interaction. Thus far, the function of one of them, YNL313c, remains unknown. Our conditional mutants of YNL313c display osmoremedial temperature sensitivity, hypersensitivity to both Calcofluor White and low concentrations of SDS, and osmoremedial caffeine sensitivity. These are hallmarks of mutants that display cell wall defects. Accordingly we rename the gene as EMW1 (essential for maintenance of the cell wall). Loss of Emw1p function is not associated with abrogation of the cell wall integrity (CWI) MAP kinase cascade. Instead, emw1ts mutants activate this cascade even at permissive temperature, indicating that loss of Emw1p function does not cause a defect in sensors and effectors of cell wall signalling, but leads to a cell wall defect directly. Constitutive activation of the CWI cascade is reflected by the overproduction of chitin by emw1ts mutants, a compensatory response frequently displayed by cell wall mutants. Growth is restored to emw1ts mutants incubated at otherwise non-permissive temperature when GFA1 is overexpressed. GFA1 encodes the hexosephosphate aminotransferase that catalyses the rate-limiting step in the pathway that synthesizes the chitin precursor UDP-GlcNAc. The possibility that Emw1p is required for function of Gfa1p was ruled out, because the emw1ts phenotype persists when the requirement for Gfa1p is bypassed. Furthermore, if loss of Emw1p function leads to loss of function of Gfa1p, then chitin synthesis would be diminished. Instead, a stimulation of the synthesis of this polymer is detected. Consequently, the defect associated with emw1ts mutants may be associated with compromise in one of the remaining processes that depend on UDP-GlcNAc, namely N-glycosylation or glycosylphosphatidylinositol (GPI)-anchor synthesis.

Biofilm production by staphylococci is an important virulence determinant mediated by the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or by surface and extracellular proteins. Deletion of the Staphylococcus accessory regulator sarX significantly reduced biofilm-forming capacity in Staphylococcus epidermidis CSF41498, whereas multicopy sarX complemented the sarX mutant and increased wild-type biofilm production. In Staphylococcus aureus, SarX negatively regulates the accessory gene regulator (Agr) system, which in turn has strain-specific effects on biofilm regulation. Here we found that purified S. epidermidis SarX protein bound specifically to the agr P3 promoter. However RT-PCR analysis revealed that both mutation of sarX and multicopy sarX activated RNAIII transcription, making it difficult to correlate sarX-mediated biofilm regulation with altered agr activity. In contrast, RT-PCR and immunoblot analysis revealed that icaA transcription and PIA expression were decreased in the sarX mutant, whereas multicopy sarX increased ica and PIA expression. Furthermore, multicopy sarX did not promote biofilms in an icaC mutant. Finally, purified SarX protein bound specifically to the ica operon promoter. Taken together, these data reveal that the S. epidermidis SarX protein regulates the transcriptional activity of the agr and ica loci and controls the biofilm phenotype, primarily by regulating icaADBC transcription and PIA production.

Horizontal genetic exchange strongly influences the evolution of many bacteria, substantially contributing to difficulties in defining their position in taxonomic groups. In particular, how clusters of related bacterial genotypes – currently classified as microbiological species – evolve and are maintained remains controversial. The nature and magnitude of gene exchange between two closely related (approx. 15 % nucleotide divergence) microbiologically defined species, Campylobacter jejuni and Campylobacter coli, was investigated by the examination of mosaic alleles, those with some ancestry from each population. A total of 1738 alleles from 2953 seven-locus housekeeping gene sequence types (STs) were probabilistically assigned to each species group with the model-based clustering algorithm structure. Alleles with less than 75 % assignment probability to one of the populations were confirmed as mosaics using the structure linkage model. For each of these, the putative source of the recombinant region was determined and the allele was mapped onto a clonalframe genealogy derived from concatenated ST sequences. This enabled the direction and frequency of introgression between the two populations to be established, with 8.3 % of C. coli clade 1 alleles having acquired C. jejuni sequence, compared to 0.5 % for the reciprocal process. Once generated, mosaic genes spread within C. coli clade 1 by a combination of clonal expansion and lateral gene transfer, with some evidence of erosion of the mosaics by reacquisition of C. coli sequence. These observations confirm previous analyses of the exchange of complete housekeeping alleles and extend this work by describing the processes of horizontal gene transfer and subsequent spread within recipient species.

The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and β subunits. Transcriptomic analysis was performed with cultures growing at 37 °C at 1, 4 and 6 h after inoculation with mutants that lack combinations of HU α and HU β. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants, revealing not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and SPI2 pathogenicity islands. The hupA hupB double mutant was defective in invasion of epithelial cell lines and in its ability to survive in macrophages. The double mutant also had defective swarming activity and a competitive fitness disadvantage compared with the wild-type. In contrast, inactivation of just the hupB gene resulted in increased fitness and correlated with the upregulation of members of the RpoS regulon in exponential-phase cultures. Our data show that HU coordinates the expression of genes involved in central metabolism and virulence and contributes to the success of S. enterica as a pathogen.

The replacement of the bladder with a neobladder made from ileal tissue is the prescribed treatment in some cases of bladder cancer or trauma. Studies have demonstrated that individuals with an ileal neobladder have recurrent colonization by Escherichia coli and other species that are commonly associated with urinary tract infections; however, pyelonephritis and complicated symptomatic infections with ileal neobladders are relatively rare. This study examines the genomic content of two E. coli isolates from individuals with neobladders using comparative genomic hybridization (CGH) with a pan-E. coli/Shigella microarray. Comparisons of the neobladder genome hybridization patterns with reference genomes demonstrate that the neobladder isolates are more similar to the commensal, laboratory-adapted E. coli and a subset of enteroaggregative E. coli than they are to uropathogenic E. coli isolates. Genes identified by CGH as exclusively present in the neobladder isolates among the 30 examined isolates were primarily from large enteric isolate plasmids. Isolations identified a large plasmid in each isolate, and sequencing confirmed similarity to previously identified plasmids of enteric species. Screening, via PCR, of more than 100 isolates of E. coli from environmental, diarrhoeagenic and urinary tract sources did not identify neobladder-specific genes that were widely distributed in these populations. These results taken together demonstrate that the neobladder isolates, while distinct, are genomically more similar to gastrointestinal or commensal E. coli, suggesting why they can colonize the transplanted intestinal tissue but rarely progress to acute pyelonephritis or more severe disease.

Manganese has an important yet undefined role in the virulence of many bacterial pathogens. In this study we confirm that a null mutation in Yersinia pseudotuberculosis mntH reduces intracellular manganese accumulation. An mntH mutant was susceptible to killing by reactive oxygen species when grown under manganese-limited conditions. The mntH mutant was defective in survival and growth in macrophages expressing functional Nramp1, but in macrophages deficient in Nramp the bacteria were able to survive and replicate. In Galleria mellonella, the mntH mutant was attenuated. Taken together, these data suggest a role for manganese in Y. pseudotuberculosis during macrophage intracellular survival, protecting the bacteria from the antimicrobial products released during the respiratory burst.

Enteropathogenic and enterohaemorrhagic Escherichia coli are related pathotypes of bacteria that cause acute watery diarrhoea and haemorrhagic colitis, respectively, and enterohaemorrhagic E. coli can lead to a serious complication known as haemolytic uraemic syndrome. In both bacteria the global regulatory protein Ler controls virulence. The ler gene is found within the locus of enterocyte effacement, or LEE, encoding a type III secretion system necessary for injecting effector proteins into intestinal epithelial cells and causing net secretory diarrhoea. The nucleoid-associated protein H-NS silences, whereas Ler serves as an anti-silencer of, multiple LEE operons. Although Ler has a higher affinity for DNA than does H-NS, the precise molecular mechanism by which Ler increases LEE transcription remains to be determined. In this report we investigate the oligomerization activity of Ler. In solution, Ler forms dimers and soluble aggregates of up to 5000 kDa molecular mass, and appears to oligomerize more readily than the related protein H-NS. An insertional mutation into the Ler linker region diminished oligomerization activity. Despite being proteins of similar mass and having homologous DNA-binding domains, Ler and H-NS complexed to DNA migrated to distinct locations, as determined by an electrophoretic mobility shift assay, implying that the related proteins form different 3D shapes in the presence of DNA. Lastly, we present electron microscopy images of toroidal Ler–DNA structures that are predicted to be involved in stimulating gene expression.