- Volume 159, Issue Pt_10, 2013

Bacillithiol is the major low molecular mass thiol produced by many firmicutes bacteria, including the model organism Bacillus subtilis and pathogens such as Bacillus anthracis and Staphylococcus aureus. We have previously shown that four genes (bshA, bshB1, bshB2 and bshC) are involved in bacillithiol biosynthesis. Here, we report that these four genes are encoded within three, unlinked operons all expressed from canonical σA-dependent promoters as determined by 5′RACE (rapid amplification of cDNA ends). The bshA and bshB1 genes are embedded within a seven-gene operon additionally including mgsA, encoding methylglyoxal synthase, and the essential genes cca and birA, encoding tRNA nucleotidyltransferase (CCA transferase) and biotin-protein ligase, respectively. The bshB2 gene is co-transcribed with unknown function genes, while bshC is expressed both as part of a two-gene operon (with the upstream putative pantothenate biosynthesis gene ylbQ) and from its own promoter. All three operons are expressed at a reduced level in an spx null mutant, consistent with a direct role of Spx as a transcriptional activator for these operons, and all three operons are induced by the thiol oxidant diamide. In contrast with other Spx-regulated genes characterized to date, the effects of Spx on basal expression and diamide-stimulated expression appear to be independent of Cys10 in the redox centre of Spx. Consistent with the role of Spx as an activator of bacillithiol biosynthetic genes, cellular levels of bacillithiol are reduced several-fold in an spx null mutant.

An E. coli K-12 mutant deficient in S-adenosylmethionine (SAM) synthesis, i.e ΔmetK, but expressing a rickettsial SAM transporter, can grow in glucose minimal medium if provided with both SAM and methionine. It uses the externally provided (R)-enantiomer of SAM as methyl donor to produce most but not all of its methionine, by methylation of homocysteine catalysed by homocysteine methyltransferase (MmuM). The ΔmetK cells are also altered in growth and are twice as long as those of the parent strain. When starved of SAM, the mutant makes a small proportion of very long cells suggesting a role of SAM and of methylation in the onset of crosswall formation.

In response to antibiotics, bacteria activate regulatory systems that control the expression of genes that participate in detoxifying these compounds, like multidrug efflux systems. We previously demonstrated that the BaeSR two-component system from Salmonella enterica serovar Typhimurium (S. Typhimurium) participates in the detection of ciprofloxacin, a bactericidal antibiotic, and in the positive regulation of mdtA, an efflux pump implicated in antibiotic resistance. In the present work, we provide further evidence for a role of the S. Typhimurium BaeSR two-component system in response to ciprofloxacin treatment and show that it regulates sodA expression. We demonstrate that, in the absence of BaeSR, the transcript levels of sodA and the activity of its gene product are lower. Using electrophoretic mobility shift assays and transcriptional fusions, we demonstrate that BaeR regulates sodA by a direct interaction with the promoter region.

The mexCD–oprJ multidrug efflux operon of Pseudomonas aeruginosa is regulated by the NfxB repressor. Two forms of NfxB have been reported [Shiba et al. (1995). J Bacteriol 177, 5872) although mutagenesis studies here confirm that the larger protein (199 amino acids, 22.4 kDa) is the functional repressor. NfxB binds upstream of the mexCD–oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding. Two-hybrid assays confirmed that NfxB is a multimer, with the C-terminal two-thirds of the repressor required for multimerization. Random mutagenesis identified several mutations within the C-terminal region of NfxB required for multimerization, all of which mapped to a three-helix subdomain of the C-terminal region in a structural model of the repressor, which may thus represent the multimerization domain. These mutations compromised NfxB binding to its target DNA in electromobility shift assays, and their introduction into the chromosome of P. aeruginosa enhanced mexCD–oprJ expression and promoted multidrug resistance, consistent with the functional NfxB repressor being a multimer. Site-directed and spontaneous nfxB mutants showing increased mexCD–oprJ expression and multidrug resistance were also recovered, with mutations mapping to the three-helix subdomain again impacting multimerization and DNA binding. Mutations mapping to the N-terminal helix–turn–helix motif implicated in DNA binding did not impact multimerization although they did render the repressor insoluble and unsuitable for mobility shift assays. Size exclusion column chromatography demonstrated that wild-type NfxB forms tetramers in solution, although a mutant form of the repressor carrying a G192D substitution near the C terminus of the protein and compromised for DNA binding and repressor activity forms dimers. These results suggest that NfxB operates as a tetramer (dimer of dimers) and that the C terminus of the protein serves as a tetramerization domain.

Polyphosphate (poly P) metabolism regulates the stress response in mycobacteria. Here we describe the regulatory architecture of a signal transduction system involving the two-component system (TCS) SenX3–RegX3, the extracytoplasmic function sigma factor sigma E (SigE) and the poly P-synthesizing enzyme polyphosphate kinase 1 (PPK1). The ppk1 promoter of Mycobacterium tuberculosis is activated under phosphate starvation. This is attenuated upon deletion of an imperfect palindrome likely representing a binding site for the response regulator RegX3, a component of the two-component system SenX3–RegX3 that responds to phosphate starvation. Binding of phosphorylated RegX3 to this site was confirmed by electrophoretic mobility shift assay. The activity of the ppk1 promoter was abrogated upon deletion of a putative SigE binding site. Pull-down of SigE from M. tuberculosis lysates of phosphate-starved cells with a biotinylated DNA harbouring the SigE binding site confirmed the likely binding of SigE to the ppk1 promoter. In vitro transcription corroborated the involvement of SigE in ppk1 transcription. Finally, the overexpression of RseA (anti-SigE) attenuated ppk1 expression under phosphate starvation, supporting the role of SigE in ppk1 transcription. The regulatory elements identified in ppk1 transcription in this study, combined with our earlier observation that PPK1 is itself capable of regulating sigE expression via the MprAB TCS, suggest the presence of multiple positive-feedback loops in this signalling circuit. In combination with the sequestering effect of RseA, we hypothesize that this architecture could be linked to bistability in the system that, in turn, could be a key element of persistence in M. tuberculosis.

The osmoprotectant glycine betaine can be generated intracellularly from conversion of the exogenous precursor choline by enzymes encoded by the gbsAB operon in Bacillus subtilis. Uptake of choline from outside B. subtilis cells is mediated through two evolutionarily closely related ATP-binding cassette transporters, OpuB and OpuC. Expression of the opuB operon and of the opuC operon is known to be osmoinducible. Here, we show that choline exerts a suppressive effect on opuC expression during normal growth and under osmotic stress. In the absence of the choline-responsive repressor GbsR, opuB expression is also suppressed by choline. We also report that a gene (formerly yvbF, now designated opcR) located immediately upstream of the opuC operon negatively regulates transcription of the opuC operon and, in the absence of GbsR, also that of the opuB operon. An inverted repeat (TTGTAAA-N8-TTTACAA) that overlaps with the −35 hexamer of the promoters of both operons has been identified as the OpcR operator. OpcR belongs to the GbsR-type transcriptional regulators. Its orthologues with unknown function are present in some other Bacillus species. Moreover, deletion analyses revealed that a region located further upstream of the promoters of the opuB operon and the opuC operon is critical for expression of both operons during normal growth and under osmotic stress. Osmotic induction of these two operons appears not to be OpcR mediated. OpcR is not a choline-responsive repressor. The possible biological role of OpcR is discussed.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are major barriers to recombination through recognition of invading nucleic acids, such as phage and plasmids, and promoting their degredation through the action of CRISPR associated (Cas) proteins. The genomic comparison of 17 Corynebacterium diphtheriae strains led to the identification of three novel CRISPR–Cas system variants, based on the Type II (Type II-C) or type I-E systems. The type II-C system was the most common (11/17 isolates) but it lacked the csn2 and cas4 genes that are involved in spacer acquisition. We also identified that this variant type II-C CRISPR–Cas system is present in other bacteria, and the first system was recently characterized in Neisseria meningitidis. In the remaining isolates, the type II-C system was replaced by a variant of type I-E (I-E-a), where the repeat arrays are inserted between the cas3 and cse1 genes. Three isolates with the type II-C system also possess an additional variant of type I-E (I-E-b), elsewhere in the genome, that exhibits a novel divergent gene organization within the cas operon. The nucleotide sequences of the palindromic repeats and the cas1 gene were phylogenetically incongruent to the core genome. The G+C content of the systems is lower (46.0–49.5 mol%) than the overall DNA G+C content (53 mol%), and they are flanked by mobile genetic elements, providing evidence that they were acquired in three independent horizontal gene transfer events. The majority of spacers lack identity with known phage or plasmid sequences, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. These novel CRISPR–Cas systems may represent a unique mechanism for spacer acquisitions and defence against invading DNA.

Previous reports showed that the large linear plasmid SCP1 of Streptomyces coelicolor A3(2) contains a 5.4 kb centrally located replication locus. We report here that SCP1 actually contains three internal replication loci. Subcloning of the 5.4 kb sequence identified a 3.2 kb minimal locus (rep1A/repB/iteron) that determined propagation in Streptomyces lividans. The two newly identified replication genes, rep2A and rep3A, resembled the rep gene of Streptomyces circular plasmid pZL12. Transcription start points of the three replication genes were determined. The three replication loci could independently determine propagation in linear mode in S. lividans. Individual and sequential deletions of the rep1A and rep3A genes were successful. The SCP1-derived linear plasmids with deletions of the rep1A and/or rep3A genes still propagated in similar copy numbers, were inherited largely stable and were transferred efficiently by conjugation in S. coelicolor. Interestingly, SCP1 can be artificially circularized to yield a 280 kb circular plasmid, circular SCP-1 (C-SCP1), which contains the three replication loci. Strikingly, the copy numbers, inheritance and transfer of C-SCP1 resembled that of the linear SCP1 plasmids. Transcripts of the rep1A, rep2A and rep3A genes in linear or artificially circularized SCP1 were detected at all the time points of strain growth.

CspR has been characterized recently as a cold-shock RNA-binding protein in Enterococcus faecalis , a natural member of the gastro-intestinal tract capable of switching from a commensal relationship with the host to an important nosocomial pathogen. In addition to its involvement in the cold-shock response, CspR also plays a role in the long-term survival and virulence of E. faecalis . In the present study, we demonstrated that anti-CspR immune rabbit serum protected larvae of Galleria mellonella against a lethal challenge of the WT strain. These results suggested that CspR might have a surface location. This hypothesis was verified by Western blot that showed detection of CspR in the total as well as in the surface protein fraction. In addition, identification of surface polypeptides by proteolytic shaving of intact bacterial cells followed by liquid chromatography-MS-MS revealed that cold-shock proteins (EF1367, EF2939 and CspR) were present on the cell surface. Lastly, anti-CspR immune rabbit serum was used for immunolabelling and detected with colloidal gold-labelled goat anti-rabbit IgG in order to determine the immunolocalization of CspR on E. faecalis WT strain. Electron microscopy images confirmed that the cold-shock protein RNA-binding protein CspR was present in both cytoplasmic and surface parts of the cell. These data strongly suggest that CspR, in addition to being located intracellularly, is also present in the extracellular protein fraction of the cells and has important functions in the infection process of Galleria larvae.

A putative prenyltransferase gene, NFIA_043650, was amplified from Neosartorya fischeri NRRL 181 and cloned into the expression vector pQE60. The deduced polypeptide consisting of 445 amino acids with a molecular mass of 51 kDa was overproduced in Escherichia coli and purified as His6-tagged protein to near homogeneity. The purified soluble protein was subsequently assayed with potential aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis of the reaction mixtures revealed acceptance of all tested tryptophan-containing cyclic dipeptides. Isolation and structural elucidation of enzyme products of five selected substrates indicated a reverse C2-prenylation on the indole nucleus, proving the enzyme to be a cyclic dipeptide C2-prenyltransferase (CdpC2PT). Differing significantly from two known brevianamide F reverse C2-prenyltransferases NotF and BrePT which use cyclo-l-Trp-l-Pro as their preferred substrate, CdpC2PT showed a clear substrate preference for (S)-benzodiazepinedinone and cyclo-l-Trp-l-Trp with K M values of 84.1 and 165.2 µM and turnover numbers at 0.63 and 0.30 s−1, respectively. A possible role of CdpC2PT in the biosynthesis of fellutanines is discussed.

Aerobic anoxygenic photosynthesis (AAP) is found in an increasing number of proteobacterial strains thriving in ecosystems ranging from extremely oligotrophic to eutrophic. Here, we have investigated whether the fuel oxygenate-degrading betaproteobacterium Aquincola tertiaricarbonis L108 can use AAP to compensate kinetic limitations at low heterotrophic substrate fluxes. In a fermenter experiment with complete biomass retention and also during chemostat cultivation, strain L108 was challenged with extremely low substrate feeding rates of tert-butyl alcohol (TBA), an intermediate of methyl tert-butyl ether (MTBE). Interestingly, formation of photosynthetic pigments, identified as bacteriochlorophyll a and spirilloxanthin, was only induced in growing cells at TBA feeding rates less than or equal to maintenance requirements observed under energy excess conditions. Growth continued at rates between 0.001 and 0.002 h−1 even when the TBA feed was decreased to values close to 30 % of this maintenance rate. Partial sequencing of genomic DNA of strain L108 revealed a bacteriochlorophyll synthesis gene cluster (bchFNBHL) and photosynthesis regulator genes (ppsR and ppaA) typically found in AAP and other photosynthetic proteobacteria. The usage of light as auxiliary energy source enabling evolution of efficient degradation pathways for kinetically limited heterotrophic substrates and for lowering the threshold substrate concentration S min at which growth becomes zero is discussed.

NhaB-like antiporters were the second described class of Na+/H+ antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent K m values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent K m at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of K m in the course of molecular evolution.

A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.