- Volume 157, Issue 6, 2011

In the extremely thermophilic bacterium Thermus thermophilus HB8, one of the four TetR-family transcriptional regulators, which we named T. thermophilus FadR, negatively regulated the expression of several genes, including those involved in fatty acid degradation, both in vivo and in vitro. T. thermophilus FadR repressed the expression of the target genes by binding pseudopalindromic sequences covering the predicted −10 hexamers of their promoters, and medium-to-long straight-chain (C10–18) fatty acyl-CoA molecules were effective for transcriptional derepression. An X-ray crystal structure analysis revealed that T. thermophilus FadR bound one lauroyl (C12)-CoA molecule per FadR monomer, with its acyl chain moiety in the centre of the FadR molecule, enclosed within a tunnel-like substrate-binding pocket surrounded by hydrophobic residues, and the CoA moiety interacting with basic residues on the protein surface. The growth of T. thermophilus HB8, with palmitic acid as the sole carbon source, increased the expression of FadR-regulated genes. These results indicate that in T. thermophilus HB8, medium-to-long straight-chain fatty acids can be used for metabolic energy under the control of FadR, although the major fatty acids found in this strain are iso- and anteiso-branched-chain (C15 and 17) fatty acids.

It is becoming increasingly clear that the so-called remnant organelles of microaerophilic unicellular eukaryotes, hydrogenosomes and mitosomes, are significantly reduced versions of mitochondria. They normally lack most of the classic mitochondrial attributes, such as an electron transport chain and a genome. While hydrogenosomes generate energy by substrate-level phosphorylation along a hydrogen-producing fermentation pathway, involving iron–sulfur-cluster-containing enzymes pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, whether mitosomes participate in ATP synthesis is currently unknown. Both enzymes were recently described in the mitosome-bearing diplomonad Giardia intestinalis, also shown to produce molecular hydrogen. As published data show that giardial PFO is a membrane-associated enzyme, it could be suspected that PFO and hydrogenase operate in the mitosome, in which case the latter would by definition be a hydrogenosome. Using antibodies against recombinant enzymes of G. intestinalis, it was shown by Western blot analysis of subcellular fractions and by confocal immunofluorescence microscopy of whole cells that neither PFO nor hydrogenase localize to the mitosome, but are mostly found in the cytosol. The giardial mitosome is known to play a role in iron–sulfur cluster assembly and to contain chaperones Cpn60 and mtHsp70, which assist, in particular, in protein import. In mitochondria, transmembrane potential is essential for this complex process. Using MitoTracker Red and organelle-specific antibodies, transmembrane potential could be detected in the Trichomonas vaginalis hydrogenosome, but not in the G. intestinalis mitosome. These results provide further evidence that the Giardia mitosome is one of the most highly reduced mitochondrial homologues.

Lactococcus lactis is known to take up extracellular peptides via at least three distinct peptide transporters. The well-described oligopeptide transporter Opp alone is able to ensure the growth of L. lactis in milk, while the di- and tripeptide transporter DtpT is involved in a peptide-dependent signalling mechanism. The oligopeptide Opt transporter displays two peptide-binding proteins, OptA and OptS. We previously demonstrated that OptA-dependent transport is dedicated to nutritional peptides, as an optABCDF mutant (of a strain devoid of Opp) has an impaired capacity to grow in milk. Using isogenic peptide transport mutants, this study shows that biosynthesis of the Opt transporter is much less sensitive to downregulation that is dependent on extracellular peptides taken up by DtpT than is Opp biosynthesis; this peptide-dependent regulation relies on the transcriptional repressor CodY. We demonstrate the dual function of the Opt system; while OptA contributes to the bacterial nutrition during growth in milk, OptS is involved in the transport of signalling peptides derived from milk and controlling opp expression. So, these results shed new light on the peptide-dependent regulation relying on two peptide transporters with different specificities: DtpT and Opt (via OptS).

The human pathogen Vibrio cholerae uses quorum sensing to regulate the expression of a number of phenotypes, including virulence factor production, in response to changes in cell density. It produces small molecules called autoinducers that increase in concentration as cell density increases, and these autoinducers bind to membrane sensors once they reach a certain threshold. This binding leads to signalling through a downstream phosphorelay pathway to alter the expression of the transcriptional regulator HapR. Previously, it was shown that the VarS/VarA two-component system acts on a component of the phosphorelay pathway upstream of HapR to regulate HapR expression levels. Here, we show that in addition to this mechanism of regulation, VarS and VarA also indirectly modulate HapR protein activity. This modulation is mediated by the small RNA CsrB but is independent of the known quorum-sensing system that links the autoinducers to HapR. Thus, the VarS/VarA two-component system intersects with the quorum-sensing network at two levels. In both cases, the effect of VarS and VarA on quorum sensing is dependent on the Csr small RNAs, which regulate carbon metabolism, suggesting that V. cholerae may integrate nutrient status and cell density sensory inputs to tailor its gene expression profile more precisely to surrounding conditions.

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz1). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of 14C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R and Rz/Rz1 gene homologues encoded by DLP12 are not merely genetic junk, but rather play an important, though undefined, role in cell wall maintenance.

The bacterial phytochrome of Pseudomonas aeruginosa (PaBphP) is an in vitro-active red/far-red light sensor histidine kinase of a two-component regulatory system. Despite solid biochemical data, its function in this heterotrophic, opportunistic pathogen is still unknown. Previous studies established that the genes encoding the two necessary phytochrome components BphO, a chromophore-producing haem oxygenase, and BphP, the apo-phytochrome, are co-transcribed in a bicistronic operon. Transcription has been shown to be induced in the stationary phase and to be dependent on the alternative sigma factor RpoS. Here we show an additional regulation of bphP expression through the quorum-sensing (QS) regulator LasR. This regulation is also reflected in a combination of expression profile experiments and proteome analyses of wild-type and phytochrome-deficient strains. While PaBphP has a pleiotropic effect on global gene expression, 66 % of the downregulated genes in the phytochrome mutant display a link to the Las QS system. Most of these genes seem to be indirectly regulated by LasR through BphP and the unknown response regulator BphR. A model of phytochrome function within the Las QS network is presented.

Fusarium graminearum, the causal agent of wheat head blight, shows intrinsic resistance to amine fungicides. It is commonly accepted that the amines target sterol C-14 reductase and sterol Δ8–Δ7 isomerase of ergosterol biosynthesis, encoded by the genes ERG24 and ERG2, respectively. Analysis of the genome sequence of F. graminearum revealed that the fungus contains two paralogous FgERG24 genes (FgERG24A and FgERG24B), which are homologous to the ERG24 of Saccharomyces cerevisiae. In this study, we disrupted FgERG24A and FgERG24B in F. graminearum. Compared to the wild-type strain HN9-1, FgERG24A and FgERG24B deletion mutants did not show recognizable phenotypic changes in mycelial growth on potato dextrose agar or in virulence on wheat heads. HPLC analysis showed that the amount of ergosterol in FgERG24A or FgERG24B deletion mutants was not significantly different from that in the wild-type strain. These results indicate that neither of the two genes is essential for growth, pathogenicity or ergosterol biosynthesis in F. graminearum. FgERG24B deletion mutants exhibited significantly increased sensitivity to amine fungicides, including tridemorph, fenpropidin and spiroxamine, but not to non-amine fungicides. In contrast, FgERG24A deletion mutants did not show changed sensitivity to any amine tested. The resistance of the FgERG24B deletion mutant to amines was restored by genetic complementation of the mutant with wild-type FgERG24B. These results indicate that FgERG24B controls the intrinsic resistance of F. graminearum to amines. The finding of this study provides new insights into amine resistance in filamentous fungi.

The alternative sigma factor ComX is a key regulator of natural transformation in members of the genus Streptococcus. ComX controls expression of the late competence genes, which are essential for DNA binding, uptake and recombination. In Streptococcus pneumoniae, it has been demonstrated that ComX is degraded by ClpEP at the end of the competence period. In the present study we show that a different Clp protease complex, ClpCP, contributes to ComX degradation in Streptococcus thermophilus. Mutant strains lacking the ClpC chaperone displayed significantly increased transformability compared with the wild-type strain under conditions where ComX was expressed at relatively low levels. At higher expression levels, ClpCP appears to become saturated and unable to prevent the accumulation of ComX. Together, our results suggest that the role of ClpC is to mediate degradation of ComX when the sigma factor is produced in low amounts, i.e. when the environmental stimulus promoting competence development is weak. This would prevent S. thermophilus from developing the competent state at an inappropriate time and/or place.

Azotobacter vinelandii is a soil bacterium that undergoes differentiation to form cysts that are resistant to desiccation. Upon induction of cyst formation, the bacterium synthesizes alkylresorcinols that are present in cysts but not in vegetative cells. Alternative sigma factors play important roles in differentiation. In A. vinelandii, AlgU (sigma E) is involved in controlling the loss of flagella upon induction of encystment. We investigated the involvement of the sigma factor RpoS in cyst formation in A. vinelandii. We analysed the transcriptional regulation of the rpoS gene by PsrA, the main regulator of rpoS in Pseudomonas species, which are closely related to A. vinelandii. Inactivation of rpoS resulted in the inability to form cysts resistant to desiccation and to produce cyst-specific alkylresorcinols, whereas inactivation of psrA reduced by 50 % both production of alkylresorcinols and formation of cysts resistant to desiccation. Electrophoretic mobility shift assays revealed specific binding of PsrA to the rpoS promoter region and that inactivation of psrA reduced rpoS transcription by 60 %. These results indicate that RpoS and PsrA are involved in regulation of encystment and alkylresorcinol synthesis in A. vinelandii.

The present study investigated the phylogenetic affiliation and physiological characteristics of bacteria responsible for anaerobic ammonium oxidization (anammox); these bacteria were enriched in an anammox reactor with a nitrogen removal rate of 26.0 kg N m−3 day−1. The anammox bacteria were identified as representing ‘Candidatus Brocadia sinica’ on the basis of phylogenetic analysis of rRNA operon sequences. Physiological characteristics examined were growth rate, kinetics of ammonium oxidation and nitrite reduction, temperature, pH and inhibition of anammox. The maximum specific growth rate (μmax) was 0.0041 h−1, corresponding to a doubling time of 7 days. The half-saturation constants (K s) for ammonium and nitrite of ‘Ca. B. sinica’ were 28±4 and 86±4 µM, respectively, higher than those of ‘Candidatus Brocadia anammoxidans’ and ‘Candidatus Kuenenia stuttgartiensis’. The temperature and pH ranges of anammox activity were 25–45 °C and pH 6.5–8.8, respectively. Anammox activity was inhibited in the presence of nitrite (50 % inhibition at 16 mM), ethanol (91 % at 1 mM) and methanol (86 % at 1 mM). Anammox activities were 80 and 70 % of baseline in the presence of 20 mM phosphorus and 3 % salinity, respectively. The yield of biomass and dissolved organic carbon production in the culture supernatant were 0.062 and 0.005 mol C (mol )−1, respectively. This study compared physiological differences between three anammox bacterial enrichment cultures to provide a better understanding of anammox niche specificity in natural and man-made ecosystems.

Bacteria encode multiple protein secretion systems that are crucial for interaction with the environment and with hosts. In recent years, attention has focused on type VI secretion systems (T6SSs), which are specialized transporters widely encoded in Proteobacteria. The myriad of processes associated with these secretion systems could be explained by subclasses of T6SS, each involved in specialized functions. To assess diversity and predict function associated with different T6SSs, comparative genomic analysis of 34 Pseudomonas genomes was performed. This identified 70 T6SSs, with at least one locus in every strain, except for Pseudomonas stutzeri A1501. By comparing 11 core genes of the T6SS, it was possible to identify five main Pseudomonas phylogenetic clusters, with strains typically carrying T6SSs from more than one clade. In addition, most strains encode additional vgrG and hcp genes, which encode extracellular structural components of the secretion apparatus. Using a combination of phylogenetic and meta-analysis of transcriptome datasets it was possible to associate specific subsets of VgrG and Hcp proteins with each Pseudomonas T6SS clade. Moreover, a closer examination of the genomic context of vgrG genes in multiple strains highlights a number of additional genes associated with these regions. It is proposed that these genes may play a role in secretion or alternatively could be new T6S effectors.

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-d-Quip3NAc-(1→3)-α-l-Fucp2OAc-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal ld-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core – namely terminal ld-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.

Nucleolin is a prominent nucleolar protein that is mobilized into the cytoplasm during infection by enteropathogenic Escherichia coli (EPEC). Nucleolin also exists at low levels at the cell surface of eukaryotic cells and here we show that upon infection of an intestinal cell model, EPEC recruits and subsequently sequesters cell-surface EGFP-nucleolin into extracellularly located bacterial microcolonies. The recruitment of nucleolin was evident around bacteria within the centre of the microcolonies that were not directly associated with actin-based pedestals. Incubation of host intestinal cells with different ligands that specifically bind nucleolin impaired the ability of EPEC to disrupt epithelial barrier function but did not inhibit bacterial attachment or other effector-driven processes such as pedestal formation or microvilli effacement. Taken together, this work suggests that EPEC exploits two spatially distinct pools of nucleolin during the infection process.