- Volume 159, Issue Pt_1, 2013

Lipoteichoic acid (LTA) is an important cell envelope component of Gram-positive bacteria. Bacillus subtilis has four homologous genes for LTA synthesis: ltaS (yflE), yfnI, yqgS and yvgJ. The products LtaS (YflE), YfnI and YqgS are bona fide LTA synthetases, whereas YvgJ functions only as an LTA primase. To clarify whether defects in LTA on the cell envelope trigger extracytoplasmic function (ECF) sigma factors, mRNA levels of the autoregulated ECF sigma factors in cells with singly and multiply deleted alleles of the ltaS homologues were examined by real-time RT-PCR. This revealed that sigM and sigX were induced in cells with a null allele of ΔltaS and ΔyfnI, respectively, and that no ECF sigma factor was induced in cells with a single null allele of ΔyqgS or ΔyvgJ. In cells with double null alleles (ΔltaS and ΔyfnI), sigW and ylaC were induced in addition to sigM and sigX. Cells with triple null alleles (ΔltaS ΔyfnI and ΔyqgS) showed a pattern of induction similar to that of the double null. In cells with quadruple null alleles, sigV and sigY were newly induced. Cells with ΔltaS had approximatley 1/4 the diglucosyldiacylglycerol and over 10 times the CDP-diacylglycerol of wild-type cells. Compensatory elevation of the mRNA level of other homologues was observed (in ΔltaS cells the level of yfnI was elevated; in ΔyfnI cells that of yqgS and yvgJ was elevated; both were even higher in ΔltaS ΔyfnI cells). In ΔltaS cells, the mRNA level of yfnI was corroborated to be regulated by σM, which is activated in the null mutant cells. In ΔyfnI cells, the mRNA levels of yqgS and yvgJ reverted to less than those of wild-type when a defective sigX allele was introduced. Since sigX was activated in cells with ΔyfnI, this suggests that the induction of yqgS and yvgJ is dependent on σX. The LTAs produced by the four ltaS homologues seem to play distinct physiological roles to maintain the full function of LTA on the B. subtilis cell envelope.

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.

Directed motility, or chemotaxis, is required for Helicobacter pylori to establish infection in the stomach, although the full repertoire of this bacterium’s chemotactic responses is not yet known. Here we report that H. pylori responds to zinc as an attractant and nickel as a repellent. To reach this conclusion, we employed both a temporal chemotaxis assay based on bacterial reversals and a supplemented soft agar spatial assay. We refined the temporal assay using a previously described chemorepellent, acid, and found that H. pylori requires rich media with serum to maintain optimal swimming motility. Surprisingly, we found that some strains respond to acid as an attractant, and that the TlpC chemoreceptor correlated with whether acid was sensed as an attractant or repellent. Using this same assay, we detected weak repellent responses to nickel and copper, and a varied response to zinc. We thus developed an alternative spatial chemotactic assay called the supplemented soft agar assay, which utilizes soft agar medium supplemented with the test compound. With Escherichia coli, the attractant serine slowed overall bacterial migration, while the repellent nickel increased the speed of overall migration. In H. pylori we detected slowed migration with doubled tryptone media, as well as zinc, consistent with an attractant response. In contrast, nickel increased migration, consistent with repulsion.

Flagellar biogenesis in the gastric pathogen Helicobacter pylori involves a transcriptional hierarchy that utilizes all three sigma factors found in this bacterium (RpoD, RpoN and FliA). Transcription of the RpoN-dependent genes requires the sensor kinase FlgS and response regulator FlgR. It is thought that FlgS senses some cellular cue to regulate transcription of the RpoN-dependent flagellar genes, but this signal has yet to be identified. Previous studies showed that transcription of the RpoN-dependent genes is inhibited by mutations in flhA, which encodes a membrane-bound component of the flagellar protein export apparatus. We found that depending on the H. pylori strain used, insertion mutations in flhA had different effects on expression of RpoN-dependent genes. Mutations in flhA in H. pylori strains B128 and ATCC 43504 (the type strain) were generated by inserting a chloramphenicol resistance cassette so as to effectively eliminate expression of the gene (ΔflhA), or within the gene following codon 77 (designated flhA77) or codon 454 (designated flhA454), which could allow expression of truncated FlhA proteins. All three flhA mutations severely inhibited transcription of the RpoN-dependent genes flaB and flgE in H. pylori B128. In contrast, levels of flaB and flgE transcripts in H. pylori ATCC 43504 bearing either flhA77 or flhA454, but not ΔflhA, were ~60 % of wild-type levels. The FlhA454 variant was detected in membrane fractions prepared from H. pylori ATCC 43504 but not H. pylori B128, which may account for the phenotypic differences in the flhA mutations of the two strains. Taken together, these findings suggest that only the N-terminal region of FlhA is needed for transcription of the RpoN regulon. Interestingly, expression of an flaB′-′xylE reporter gene in H. pylori ATCC 43504 bearing the flhA77 allele was about eightfold higher than that of a strain with the wild-type allele, suggesting that expression of flaB is not only regulated at the level of transcription but also regulated post-transcriptionally.

The role of the CcpC regulatory protein as a repressor of the genes encoding the tricarboxylic acid branch enzymes of the Krebs cycle (citrate synthase, citZ; aconitase, citB; and isocitrate dehydrogenase, citC) has been established for both Bacillus subtilis and Listeria monocytogenes. In addition, hyperexpression of citB–lacZ reporter constructs in an aconitase null mutant strain has been reported for B. subtilis. We show here that such hyperexpression of citB occurs in L. monocytogenes as well as in B. subtilis and that in both species the hyperexpression is unexpectedly dependent on CcpC. We propose a revision of the existing CcpC–citB regulatory scheme and suggest a mechanism of regulation in which CcpC represses citB expression at low citrate levels and activates citB expression when citrate levels are high.

The eukaryotic basic leucine zipper (bZIP) transcription factors play critical roles in the organismal response to the environment. Recently, a novel YAP-like bZIP, restorer of secondary metabolism A (RsmA), was found in a suppressor screen of an Aspergillus nidulans secondary metabolism (SM) mutant in which overexpression of rsmA was found to partially remediate loss of SM in Velvet Complex mutants. The Velvet Complex is a conserved fungal transcriptional heteromer that couples SM with sexual development in fungi. Here we characterized and contrasted SM in mutants of RsmA and four other A. nidulans bZIP proteins (NapA, ZipA, ZipB and ZipC) with predicted DNA binding motifs similar to RsmA. Only two overexpression mutants exhibited both SM and sexual abnormalities that were noteworthy: OE : : rsmA resulted in a 100-fold increase in sterigmatocystin and a near loss of meiotic spore production. OE : : napA displayed decreased production of sterigmatocystin, emericellin, asperthecin, shamixanthone and epishamixanthone, coupled with a shift from sexual to asexual development. Quantification of bZIP homodimer and heterodimer formation using fluorescence resonance energy transfer (FRET) suggested that these proteins preferentially self-associate.

Bifidobacterium asteroides, originally isolated from honeybee intestine, was found to grow under 20 % O2 conditions in liquid shaking culture using MRS broth. Catalase activity was detected only in cells that were exposed to O2 and grown in medium containing a haem source, and these cells showed higher viability on exposure to H2O2. Passage through multiple column chromatography steps enabled purification of the active protein, which was identified as a homologue of haem catalase on the basis of its N-terminal sequence. The enzyme is a homodimer composed of a subunit with a molecular mass of 55 kDa, and the absorption spectrum shows the typical profile of bacterial haem catalase. A gene encoding haem catalase, which has an amino acid sequence coinciding with the N-terminal amino acid sequence of the purified protein, was found in the draft genome sequence data of B. asteroides. Expression of the katA gene was induced in response to O2 exposure. The haem catalase from B. asteroides shows about 70–80 % identity with those from lactobacilli and other lactic acid bacteria, and no homologues were found in other bifidobacterial genomes.

The Rhodobacter capsulatus response regulator CtrA controls the expression of 227 genes, some of which are upregulated by both the phosphorylated and unphosphorylated forms of CtrA. Therefore, CtrA concentration alone, regardless of phosphorylation state, may determine expression of downstream genes, yet little is known about the regulation of ctrA in R. capsulatus. In this study we used a ctrA : : lacZ fusion plasmid to study the effects of medium composition, growth conditions and growth phase on R. capsulatus ctrA gene expression. These experiments indicate that ctrA expression is higher when cultures are grown in phototrophic (anaerobic) conditions compared with chemotrophic (aerobic) conditions, and is higher when grown in a minimal medium compared with a rich medium. We used several mutants to investigate possible regulatory pathways, and found that in R. capsulatus ctrA is not autoregulated but is regulated by a quorum-sensing system. The expression of ctrA increased as cell cultures moved through exponential phase and into stationary phase, with high levels of expression persisting long after culture turbidity plateaued. Although this growth phase-dependent pattern of expression was also observed in a quorum-sensing mutant, the magnitude of ctrA expression was about 50 % of the wild-type strain at all phases. Furthermore, reduction of phosphate concentration in the growth medium decreased ctrA expression in a culture density-independent manner, whereas reduction of malic acid (carbon source) or ammonium (nitrogen source) concentration had no effect. The regulation of ctrA expression in R. capsulatus appears to require the coordination of multiple pathways involved in detecting a variety of environmental conditions.

Extraordinary claims have been made for the biological properties of the red rain cells of Kerala, including a suggestion that they lack DNA. We have investigated the fluorescence properties of red rain cells, and the solubility of the red pigment in a variety of solvents. Extraction of the pigment with DMSO allowed successful demonstration of DNA using DAPI staining. Cellular impermeability to staining reagents due to the red pigment is the likely explanation for the failure of previous efforts to demonstrate DNA in red rain cells.

Helicobacter hepaticus open reading frame HH0352 was identified as a nickel-responsive regulator NikR. The gene was disrupted by insertion of an erythromycin resistance cassette. The H. hepaticus nikR mutant had five- to sixfold higher urease activity and at least twofold greater hydrogenase activity than the wild-type strain. However, the urease apo-protein levels were similar in both the wild-type and the mutant, suggesting the increase in urease activity in the mutant was due to enhanced Ni-maturation of the urease. Compared with the wild-type strain, the nikR strain had increased cytoplasmic nickel levels. Transcription of nikABDE (putative inner membrane Ni transport system) and hh0418 (putative outer membrane Ni transporter) was nickel- and NikR-repressed. Electrophoretic mobility shift assays (EMSAs) revealed that purified HhNikR could bind to the nikABDE promoter (P nikA ), but not to the urease or the hydrogenase promoter; NikR-P nikA binding was enhanced in the presence of nickel. Also, qRT-PCR and EMSAs indicated that neither nikR nor the exbB-exbD-tonB were under the control of the NikR regulator, in contrast with their Helicobacter pylori homologues. Taken together, our results suggest that HhNikR modulates urease and hydrogenase activities by repressing the nickel transport/nickel internalization systems in H. hepaticus, without direct regulation of the Ni-enzyme genes (the latter is the case for H. pylori). Finally, the nikR strain had a two- to threefold lower growth yield than the parent, suggesting that the regulatory protein might play additional roles in the mouse liver pathogen.

Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NFκB). In turn, NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NFκB activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1–SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients.

Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.

Carbon starvation is a common stress for micro-organisms both in nature and in industry. The carbon starvation stress response (CSSR) involves the regulation of several important processes including programmed cell death and reproduction of fungi, secondary metabolite production and extracellular hydrolase formation. To gain insight into the physiological events of CSSR, DNA microarray analyses supplemented with real-time RT-PCR (rRT-PCR) experiments on 99 selected genes were performed. These data demonstrated that carbon starvation induced very complex changes in the transcriptome. Several genes contributing to protein synthesis were upregulated together with genes involved in the unfolded protein stress response. The balance between biosynthesis and degradation moved towards degradation in the case of cell wall, carbohydrate, lipid and nitrogen metabolism, which was accompanied by the production of several hydrolytic enzymes and the induction of macroautophagy. These processes provide the cultures with long-term survival by liberating nutrients through degradation of the cell constituents. The induced synthesis of secondary metabolites, antifungal enzymes and proteins as well as bacterial cell wall-degrading enzymes demonstrated that carbon-starving fungi should have marked effects on the micro-organisms in their surroundings. Due to the increased production of extracellular and vacuolar enzymes during carbon starvation, the importance of the endoplasmic reticulum increased considerably.

Mycolic acids, very long-chain α-alkyl, β-hydroxylated fatty acids, occur in the members of the order Corynebacteriales where their chain lengths (C26–C88) and structural features (oxygen functions, cis or trans double bonds, cyclopropane rings and methyl branches) are genus- and species-specific. The molecular composition and structures of the mycolic acids of two species belonging to the genus Segniliparus were determined by a combination of modern analytical chemical techniques, which include MS and NMR. They consist of mono-ethylenic C62–C64 (α′), di-ethylenic C77–C79 (α) and extremely long-chain mycolic acids (α+) ranging from 92 to 98 carbon atoms and containing three unsaturations, cis and/or trans double bonds and/or cyclopropanes. The double bonds in each class of mycolic acids were positioned by oxidative cleavage and exhibit locations similar to those of α- and α′-mycolic acids of mycobacteria. For the ultralong chain α-mycolic acids, the three double bonds were located at equally spaced carbon intervals (C13–C16), with the methyl branches adjacent to the proximal and distal trans double bonds. Examination of the Segniliparus rotundus genome compared with those of other members of the Corynebacteriales indicated two obvious differences in genes encoding the elongation fatty acid (FAS-II) enzymes involved in the biosynthesis of mycolic acids: the organization of 3-ketoacyl-ACP synthases (KasA and KasB) and (3R)-hydroxyacyl-ACP dehydratases (HadAB/BC), on one hand, and the presence of two copies of the hadB gene encoding the catalytic domain of the latter enzyme type, on the other. This observation is discussed in light of the most recent data accumulated on the biosynthesis of this hallmark of Corynebacteriales.