- Volume 158, Issue 10, 2012
Volume 158, Issue 10, 2012
- Review
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Copper-responsive gene regulation in bacteria
More LessCopper is an essential cofactor of various enzymes, but free copper is highly toxic to living cells. To maintain cellular metabolism at different ambient copper concentrations, bacteria have evolved specific copper homeostasis systems that mostly act as defence mechanisms. As well as under free-living conditions, copper defence is critical for virulence in pathogenic bacteria. Most bacteria synthesize P-type copper export ATPases as principal defence determinants when copper concentrations exceed favourable levels. In addition, many bacteria utilize resistance-nodulation-cell division (RND)-type efflux systems and multicopper oxidases to cope with excess copper. This review summarizes our current knowledge on copper-sensing transcriptional regulators, which we assign to nine different classes. Widespread one-component regulators are CueR, CopY and CsoR, which were initially identified in Escherichia coli, Enterococcus hirae and Mycobacterium tuberculosis, respectively. CueR activates homeostasis gene expression at elevated copper concentrations, while CopY and CsoR repress their target genes under copper-limiting conditions. Besides these one-component systems, which sense the cytoplasmic copper status, many Gram-negative bacteria utilize two-component systems, which sense periplasmic copper concentrations. In addition to these well-studied transcriptional factors, copper control mechanisms acting at the post-transcriptional and the post-translational levels will be discussed.
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- Cell and Molecular Biology of Microbes
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Differential response of Porphyromonas gingivalis to varying levels and duration of hydrogen peroxide-induced oxidative stress
Porphyromonas gingivalis, an anaerobic oral pathogen implicated in adult periodontitis, can exist in an environment of oxidative stress. To evaluate its adaptation to this environment, we have assessed the response of P. gingivalis W83 to varying levels and durations of hydrogen peroxide (H2O2)-induced stress. When P. gingivalis was initially exposed to a subinhibitory concentration of H2O2 (0.1 mM), an adaptive response to higher concentrations could be induced. Transcriptome analysis demonstrated that oxidative stress can modulate several functional classes of genes depending on the severity and duration of the exposure. A 10 min exposure to H2O2 revealed increased expression of genes involved in DNA damage and repair, while after 15 min, genes involved in protein fate, protein folding and stabilization were upregulated. Approximately 9 and 2.8 % of the P. gingivalis genome displayed altered expression in response to H2O2 exposure at 10 and 15 min, respectively. Substantially more genes were upregulated (109 at 10 min; 47 at 15 min) than downregulated (76 at 10 min; 11 at 15 min) by twofold or higher in response to H2O2 exposure. The majority of these modulated genes were hypothetical or of unknown function. One of those genes (pg1372) with DNA-binding properties that was upregulated during prolonged oxidative stress was inactivated by allelic exchange mutagenesis. The isogenic mutant P. gingivalis FLL363 (pg1372 : : ermF) showed increased sensitivity to H2O2 compared with the parent strain. Collectively, our data indicate the adaptive ability of P. gingivalis to oxidative stress and further underscore the complex nature of its resistance strategy under those conditions.
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6S RNA – an old issue became blue-green
More Less6S RNA from Escherichia coli acts as a versatile transcriptional regulator by binding to the RNA polymerase and changing promoter selectivity. Although homologous 6S RNA structures exist in a wide range of bacteria, including cyanobacteria, our knowledge of 6S RNA function results almost exclusively from studies with E. coli. To test for potential structural and functional conservation, we selected four predicted cyanobacterial 6S RNAs (Synechocystis, Synechococcus, Prochlorococcus and Nostoc), which we compared with their E. coli counterpart. Temperature-gradient gel electrophoresis revealed similar thermodynamic transition profiles for all 6S RNAs, indicating basically similar secondary structures. Subtle differences in melting behaviour of the different RNAs point to minor structural variations possibly linked to differences in optimal growth temperature. Secondary structural analysis of three cyanobacterial 6S RNAs employing limited enzymic hydrolysis and in-line probing supported the predicted high degree of secondary structure conservation. Testing for functional homology we found that all cyanobacterial 6S RNAs were active in binding E. coli RNA polymerase and transcriptional inhibition, and had the ability to act as template for transcription of product RNAs (pRNAs). Deletion of the 6S RNA gene in Synechocystis did not significantly affect cell growth in liquid media but reduced fitness during growth on solid agar. While our study shows that basic 6S RNA functions are conserved in species as distantly related as E. coli and cyanobacteria, we also noted a subtle degree of divergence, which might reflect fundamental differences in transcriptional regulation and lifestyle, thus providing the first evidence for a possible physiological role in cyanobacteria.
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The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication
More LessThe master regulator CtrA oscillates during the Caulobacter cell cycle due to temporally regulated proteolysis and transcription. It is proteolysed during the G1–S transition and reaccumulates in predivisional cells as a result of transcription from two sequentially activated promoters, P1 and P2. CtrA reinforces its own synthesis by directly mediating the activation of P2 concurrently with repression of P1. To explore the role of P1 in cell cycle control, we engineered a mutation into the native ctrA locus that prevents transcription from P1 but not P2. As expected, the ctrA P1 mutant exhibits striking growth, morphological and DNA replication defects. Unexpectedly, we found CtrA and its antagonist SciP, but not DnaA, GcrA or CcrM accumulation to be dramatically reduced in the ctrA P1 mutant. SciP levels closely paralleled CtrA accumulation, suggesting that CtrA acts as a rheostat to modulate SciP abundance. Furthermore, the reappearance of CtrA and CcrM in predivisional cells was delayed in the P1 mutant by 0.125 cell cycle unit in synchronized cultures. High levels of ccrM transcription despite low levels of CtrA and increased transcription of ctrA P2 in the ctrA P1 mutant are two examples of robustness in the cell cycle. Thus, Caulobacter can adjust regulatory pathways to partially compensate for reduced and delayed CtrA accumulation in the ctrA P1 mutant.
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Three pathway-specific regulators control streptolydigin biosynthesis in Streptomyces lydicus
More LessThe streptolydigin biosynthetic gene cluster from Streptomyces lydicus NRRL 2433 contains three putative regulatory genes, slgR1, slgR2 and slgY, encoding proteins belonging to TetR and LuxR transcriptional regulator families and ATP/GTP-binding proteins of DNA and RNA helicase superfamily I, respectively. Inactivation of slgR1 or slgR2 resulted in the abolition of streptolydigin production, suggesting that these proteins are pathway-specific positive regulators. In the case of the slgR1 mutant, low amounts of streptolydigin C were produced instead of streptolydigin. RT-PCR transcription analysis of streptolydigin biosynthesis genes revealed a hierarchical regulation process. SlgY was found to control the expression of the regulator slgR2. SlgR2 regulates the expression of structural genes involved in the formation of the streptolydigin bicyclic ketal moiety, incorporation and processing of 3-methylaspartate, and the regulator slgR1. On the other hand, SlgR1 controls the expression of slgE1–E2, involved in the conversion of glutamate to 3-methylaspartate, and putative glycoside hydrolases slgC1 and slgC2. Ectopic expression of slgR1, slgR2 and slgY regulatory genes in S. lydicus led to considerable increases in streptolydigin yields, 18-, 11- and 8.5-fold, respectively. Ectopic expression of slgY in an slgR1 mutant led to a 14-fold increase of streptolydigin C yields, while no effect was observed to result from expression of slgR2.
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BfpL is essential for type IV bundle-forming pilus biogenesis and interacts with the periplasmic face of BfpC
More LessEnteropathogenic Escherichia coli (EPEC) causes diarrhoea among infants in developing countries. The bundle-forming pilus (BFP), a type IV pilus found on the surface of EPEC, is essential for full virulence of typical EPEC strains. The machinery for BFP assembly and function is encoded by an operon of 14 genes. Here we investigate the role in pilus biogenesis of BfpL, a small protein with a single N-terminal predicted transmembrane domain reminiscent of pilin-like proteins. We confirmed that a bfpL mutant lacks BFP, and associated auto-aggregation and localized adherence phenotypes. Furthermore, we found that a double mutant unable to express both the putative retraction ATPase BfpF and BfpL also lacks BFP and associated phenotypes, distinguishing BfpL from pilin-like proteins. Western blots of sheared pilus preparations did not suggest that BfpL is a component of BFP. Topology studies using C-terminal truncations and a dual reporter revealed that most of the BfpL protein resides in the periplasm. Further, we demonstrated through yeast two-hybrid assays and confirmed by fluorescence anisotropy that BfpL interacts with the periplasmic face of BfpC. Thus, BfpL has a function distinct from those of pilin-like proteins and is instead part of an inner-membrane subassembly complex that is believed to extract bundlin, the main pilus subunit, from the inner membrane to be incorporated into BFP.
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The all0458/lti46.2 gene encodes a low temperature-induced Dps protein homologue in the cyanobacteria Anabaena sp. PCC 7120 and Anabaena variabilis M3
More LessDNA-binding proteins from starved cells (Dps), which are encoded by many bacterial genomes, protect genomic DNA via non-specific DNA binding, as well as inhibition of free radical formation by chelating Fe(II). In the filamentous cyanobacterium Anabaena, the second gene (lti46.2) in the low temperature-induced gene operon lti46 in strain M3 was found to encode a homologue of Dps, but for a long time this gene remained poorly characterized. A gene cluster, all0459–all0458–all0457, was found later to be 100 % identical to the lti46 gene cluster in a closely related strain, PCC 7120. In the present study, we detected ferroxidase activity of the Lti46.2/All0458 protein, which formed a dodecamer, as found in other Dps proteins. In addition, three homologues of all0458 were found in strain PCC 7120, namely, all1173, alr3808 and all4145. We analysed expression of the lti46 or all0459-8-7 gene cluster in both strains, M3 and PCC 7120, and confirmed its induction by low temperature. We found that the All0458–GFP fusion protein and the All1173–GFP fusion protein were localized to the nucleoids. In the all0458 null mutant, the transcript of the alr3808 gene accumulated. These results suggest that there might be complex cooperation of various members of the dps family in protecting the genome from environmental stresses such as changing temperature.
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All0809/8/7 is a DevBCA-like ABC-type efflux pump required for diazotrophic growth in Anabaena sp. PCC 7120
More LessEfflux pumps export a wide variety of proteinaceous and non-proteinaceous substrates across the Gram-negative cell wall. For the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the ATP-driven glycolipid efflux pump DevBCA–TolC has been shown to be crucial for the differentiation of N2-fixing heterocysts from photosynthetically active vegetative cells. In this study, a homologous system was described. All0809/8/7–TolC form a typical ATP-driven efflux pump as shown by surface plasmon resonance. This putative exporter is also involved in diazotrophic growth of Anabaena sp. PCC 7120. A mutant in all0809 encoding the periplasmic membrane fusion protein of the pump was not able to grow without combined nitrogen. Although heterocysts of this mutant were not distinguishable from those of the wild-type in light and electron micrographs, they were impaired in providing the microoxic environment necessary for N2 fixation. RT-PCR of all0809 transcripts and localization studies on All0807–GFP revealed that All0809/8/7 was initially downregulated during heterocyst maturation and upregulated at later stages of heterocyst formation in all cells of the filament. A substrate of the efflux pump could not be identified in ATP hydrolysis assays. We discuss a role for All0809/8/7–TolC in maintaining the continuous periplasm and how this would be of special importance for heterocyst differentiation.
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PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide
More LessProtein-tyrosine phosphorylation plays a significant role in multiple cellular functions in bacteria. Bacterial tyrosine phosphatases catalyse the dephosphorylation of tyrosyl-phosphorylated proteins. Myxococcus xanthus PhpA shares homology with DNA polymerase and histidinol phosphatase family members. Recombinant His-tagged PhpA requires Mn2+ or Co2+ for phosphatase activity, and shows strict specificity for phosphorylated tyrosine residues. The k m values of PhpA for p-nitrophenyl phosphate (pNPP) and phosphotyrosine peptide, RRLIEDAEpYAARG, were 803 and 139 µM, respectively. The phosphatase activity of PhpA was inhibited by sodium orthovanadate with a k i of 33 µM. phpA gene expression was observed under both vegetative and developmental conditions, but peaked during late fruiting body formation. A phpA mutant exhibited an elevated level of tyrosine phosphorylation of a 79 kDa protein and cytoplasmic tyrosine kinase, BtkA. In M. xanthus, exopolysaccharide (EPS) is essential for cell–cell adhesion and fruiting body formation. phpA mutant cells exhibited enhanced capacity for cell–cell agglutination in agglutination buffer. Under starvation conditions, phpA mutation caused early aggregation and sporulation. The EPS production assay showed that the phpA mutant produced an increased amount of EPS in comparison with the wild-type. These results indicate that PhpA may negatively regulate the production of EPS in M. xanthus.
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The hydrophobic core of FliG domain II is the stabilizer in the Salmonella flagellar motor
More LessThe flagellar protein FliG is the major component of the flagellar torque generator, and consists of two separate domains, I and II. Domain I is essential for flagellar assembly, while domain II in the C-terminal region is not essential for flagellar assembly but is dedicated to torque generation. Previously, we found that some fliG mutants were temperature-hypersensitive (hyper-TS) and identified three residues (F236V, D244Y and K273E) on domain II responsible for the temperature-sensitive (TS) phenotype. In this study, we substituted the three residues with all 20 amino acids (X) and analysed the behaviour of the variants at various temperatures. Each group of F236X, D244X and K273X variants gave rise to several hyper-TS mutants. In F236X, only substitution with F and W gave rise to wild-type, while other hydrophobic residues resulted in hyper-TS mutants and hydrophilic residues resulted in non-motile variants. The atomic arrangement around the F236 residue indicated that F236 together with neighbouring residues forms a hydrophobic core in the centre of domain II, which is well conserved among many species. These data suggest that the hydrophobic core may play an essential role in stabilizing the whole structure of domain II, so that changes of physiological conditions in the microenvironment of domain II do not perturb torque generation.
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The mcsB gene of the clpC operon is required for stress tolerance and virulence in Staphylococcus aureus
More LessThe clpC operon in Staphylococcus aureus comprises four genes, denoted ctsR, mcsA, mcsB and clpC. A mutation within the mcsB gene resulted in hypersensitivity to heavy metal stress, temperature stress, osmotic pressure stress and oxidative stress. This mutation also resulted in sensitivity to variations in pH and lowered expression of the clpC operon under adverse extracellular conditions, as determined by quantitative real-time PCR (qRT-PCR). Additionally, virulence traits such as haemolytic activity, proteolysis, biofilm formation, and evasion from peritoneal fluid killing were substantially reduced in the ΔmcsB strain. Interestingly, mutated mcsB also caused a significant reduction in expression of virulence determinants hla and saeS. To be a successful pathogen, S. aureus must effectively overcome these types of stresses that are encountered within the host. These data show that an S. aureus strain lacking functional mcsB is stress hypersensitive and therefore less viable when introduced into hostile environments. For the first time, these studies have identified mcsB as a crucial and necessary component of stress and pathogenicity mechanisms.
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- Environmental and Evolutionary Microbiology
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Lipid diversity among botulinum neurotoxin-producing clostridia
More LessClostridium botulinum has been classified into four groupings (groups I to IV) based on physiological characteristics and 16S rRNA sequencing. We have examined the lipid compositions of 11 representative strains of C. botulinum and a strain of Clostridium sporogenes by 2D-TLC and by MS. All strains contained phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylethanolamine (PE) in both the all-acyl and the alk-1′-enyl (plasmalogen) forms. Five strains in proteolytic group I, which are related to C. sporogenes, contained varying amounts of an ethanolamine-phosphate derivative of N-acetylglucosaminyl-diradylglycerol, which is also present in C. sporogenes. Three strains in group II, which are related to Clostridium butyricum, Clostridium beijerinckii and Clostridium acetobutylicum, contained lipids characteristic of these saccharolytic species: a glycerol acetal and a PG acetal of the plasmalogen form of PE. Two group III strains, which are related to Clostridium novyi, contained amino-acyl derivatives of PG, which are also found in C. novyi. A strain in group IV had PE, PG and CL, but none of the distinguishing lipids. This work shows that the lipidome of C. botulinum is consistent with its classification by other methods.
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- Genes and Genomes
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Genomics of alternative sulfur utilization in ascomycetous yeasts
More LessThirteen ascomycetous yeast strains with sequenced genomes were assayed for their ability to grow on chemically defined medium with 16 different sulfur compounds as the only significant source of sulfur. These compounds included sulfoxides, sulfones, sulfonates, sulfamates and sulfate esters. Broad utilization of alternative sulfur sources was observed in Komagataella pastoris (syn. Pichia pastoris), Lodderomyces elongisporus, Millerozyma farinosa (syn. Pichia sorbitophila), Pachysolen tannophilus, Scheffersomyces stipitis (syn. Pichia stipitis), Spathaspora passalidarum, Yamadazyma tenuis (syn. Candida tenuis) and Yarrowia lipolytica. Kluyveromyces lactis, Saccharomyces cerevisiae and Zygosaccharomyces rouxii were mainly able to utilize sulfonates and sulfate esters, while Lachancea thermotolerans and Schizosaccharomyces pombe were limited to aromatic sulfate esters. Genome analysis identified several candidate genes with bacterial homologues that had been previously shown to be involved in the utilization of alternative sulfur sources. Analysis of candidate gene promoter sequences revealed a significant overrepresentation of DNA motifs that have been shown to regulate sulfur metabolism in Sacc. cerevisiae.
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- Microbial Pathogenicity
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The non-classical ArsR-family repressor PyeR (PA4354) modulates biofilm formation in Pseudomonas aeruginosa
PyeR (PA4354) is a novel member of the ArsR family of transcriptional regulators and modulates biofilm formation in Pseudomonas aeruginosa. Characterization of this regulator showed that it has negative autoregulatory properties and binds to a palindromic motif conserved among PyeR orthologues. These characteristics are in line with classical ArsR-family regulators, as is the fact that PyeR is part of an operon structure (pyeR-pyeM-xenB). However, PyeR also exhibits some atypical features in comparison with classical members of the ArsR family, as it does not harbour metal-binding motifs and does not appear to be involved in metal perception or resistance. Hence, PyeR belongs to a subgroup of non-classical ArsR-family regulators and is the second ArsR regulator shown to be involved in biofilm formation.
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A Brucella abortus cstA mutant is defective for association with endoplasmic reticulum exit sites and displays altered trafficking in HeLa cells
More LessMembers of the genus Brucella are facultative intracellular pathogenic bacteria able to control maturation of their vacuoles. In several cell types, Brucella is able to reach a proliferation compartment derived from the endoplasmic reticulum (ER). Since ER exit site (ERES) functions are required for Brucella proliferation, we performed a yeast two-hybrid screen between human ERES-associated proteins and the predicted brucella proteome. This screening led to the identification of CstA, a conserved protein that specifically interacts with Sec24A, a component of the ERES. We found that a tagged CstA is secreted in Brucella abortus culture medium. This secretion is independent of the type IV secretion system VirB and the flagellum, suggesting that CstA is secreted through another system. We also discovered that a B. abortus cstA mutant is impaired for its association with the Sec23 ERES marker. The B. abortus cstA mutant displayed peculiar trafficking, with reduced association with LAMP1 and Calnexin 12 h post-infection in HeLa cells. However, its intracellular proliferation kinetics was not affected. The data reported here suggest that CstA could be directly or indirectly involved in the control of B. abortus intracellular trafficking in HeLa cells.
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Haemin represses the haemolytic activity of Staphylococcus aureus in an Sae-dependent manner
More LessStaphylococcus aureus is a major human pathogen and a common cause of nosocomial infections. This facultative pathogen produces a large arsenal of virulence factors, including the haemolysins, which allow the bacterium to lyse erythrocytes and thereby release large amounts of the haem-containing haemoglobin. The released haem is thought to be the main iron source of this organism during the course of infection, and is considered to be crucial for bacterial proliferation in vivo. High concentrations of haem and its degradation products, on the other hand, are known to be toxic for S. aureus, making it essential for the pathogen to tightly control haem release from red blood cells. Here we show that S. aureus responds to haemin by downregulating the expression of haemolysins. Subinhibitory concentrations of haemin were found to significantly reduce transcription of the haemolysin genes hlb (encoding β-haemolysin) and hlgA (encoding the S-class component of γ-haemolysin), while hla (encoding α-haemolysin) and RNAIII (encoding δ-haemolysin) transcription did not appear to be affected. The presence of haemin also reduced the haemolytic potential of the supernatants of S. aureus LS1 cultures. Inactivation of the sae locus in LS1 abolished the haemin effect on the transcription of haemolysin genes, indicating that the two-component regulatory system is required for this regulatory effect. Iron limitation, on the other hand, was found to induce the expression of haemolysins, and this effect was again abolished in the sae mutant, indicating that S. aureus modulates its haemolysin production in response to iron and haem availability in an Sae-dependent manner.
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Antigen variability in Anaplasma phagocytophilum during chronic infection of a reservoir host
More LessAnaplasma phagocytophilum is an obligately intracellular, tick-transmitted, bacterial pathogen of humans and other animals. In order to evade host immunity during the course of infection, A. phagocytophilum utilizes gene conversion to shuffle approximately 100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which encodes the major surface antigen, major surface protein 2 (Msp2). The role and extent of msp2(p44) recombination in a reservoir host for A. phagocytophilum have not been evaluated. In the current study, we explored patterns of recombination and expression site variability of the msp2(p44) gene in three chronically infected woodrats, a reservoir for the disease in the Western USA. All three woodrats developed persistent infection of at least 6 months duration; two of them maintained active infection for at least 8 months. In total, we detected the emergence of 60 unique msp2(p44) expression site variants with no common temporal patterns of expression site recombination among the three A. phagocytophilum populations. Both the strength of infection (i.e. pathogen load) and the genetic diversity of pseudogenes detected at the msp2(p44) expression site fluctuated periodically during the course of infection. An analysis of the genomic pseudogene exhaustion rate showed that the repertoire of pseudogenes available to the A. phagocytophilum population could in theory become depleted within a year. However, the apparent emergence of variant pseudogenes suggests that the pathogen could potentially evade host immunity indefinitely. Our findings suggest a tightly co-evolved relationship between A. phagocytophilum and woodrats in which the pathogen perpetually evades host immunity yet causes no detectable disease.
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The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength
Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor–regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na+/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.
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Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri
Species in the genus Naegleria are free-living amoebae of the soil and warm fresh water. Although around 30 species have been recognized, Naegleria fowleri is the only one that causes primary amoebic meningoencephalitis (PAM) in humans. PAM is an acute and fast progressing disease affecting the central nervous system. Most of the patients die within 1–2 weeks of exposure to the infectious water source. The fact that N. fowleri causes such fast progressing and highly lethal infections has opened many questions regarding the relevant pathogenicity factors of the amoeba. In order to investigate the pathogenesis of N. fowleri under defined experimental conditions, we developed a novel high- versus low-pathogenicity model for this pathogen. We showed that the composition of the axenic growth media influenced growth behaviour and morphology, as well as in vitro cytotoxicity and in vivo pathogenicity of N. fowleri. Trophozoites maintained in Nelson's medium were highly pathogenic for mice, demonstrated rapid in vitro proliferation, characteristic expression of surface membrane vesicles and a small cell diameter, and killed target mouse fibroblasts by both contact-dependent and -independent destruction. In contrast, N. fowleri cultured in PYNFH medium exhibited a low pathogenicity, slower growth, increased cell size and contact-dependent target cell destruction. However, cultivation of the amoeba in PYNFH medium supplemented with liver hydrolysate (LH) resulted in trophozoites that were highly pathogenic in mice, and demonstrated an intermediate proliferation rate in vitro, diminished cell diameter and contact-dependent target cell destruction. Thus, in this model, the presence of LH resulted in increased proliferation of trophozoites in vitro and enhanced pathogenicity of N. fowleri in mice. However, neither in vitro cytotoxicity mechanisms nor the presence of membrane vesicles on the surface correlated with the pathologic potential of the amoeba. This indicated that the pathogenicity of N. fowleri remains a complex interaction between as-yet-unidentified cellular mechanisms.
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- Physiology and Biochemistry
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Aerobic glycerol dissimilation via the Enterococcus faecalis DhaK pathway depends on NADH oxidase and a phosphotransfer reaction from PEP to DhaK via EIIADha
More LessTwo pathways for glycerol dissimilation are present in Enterococcus faecalis. Either glycerol is first phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase with molecular oxygen as the electron acceptor (GlpO/GlpK pathway), or it is first oxidized by glycerol dehydrogenase with NAD+ as the acceptor of the reduction equivalents and then phosphorylated by dihydroxyacetone kinase (GldA/DhaK pathway). The final end product in both cases is dihydroxyacetone phosphate (DHAP). The genes of the GldA/DhaK pathway are present in a four-gene operon structure encoding GldA, a small hypothetical protein (EF1359), and two subunits of dihydroxyacetone kinase (DhaK and DhaL). We demonstrate in this study that protein EF1359 is part of a phosphorylation cascade which phosphorylates dihydroxyacetone in a phosphoenolpyruvate (PEP)-dependent reaction via EI, HPr, EF1359 and DhaLK. Furthermore we show that aerobic dissimilation of glycerol via the GldA/DhaK pathway is dependent on active NADH oxidase to regenerate NADH in Ent. faecalis. A refined model of the aerobic metabolism of glycerol via the GldA/DhaK pathway is presented.
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Role of Hog1, Tps1 and Sod1 in boric acid tolerance of Saccharomyces cerevisiae
More LessIn order to identify genetic contributions to boric acid (BA) resistance, a yeast knockout collection was screened for BA-sensitive mutants. Prominent among the BA-sensitive mutants were strains with defects in the cytoplasmic part of the high osmolarity/glycerol (HOG) signalling pathway, the trehalose-synthesis pathway (TPS1/TPS2) and the copper–zinc superoxide dismutase SOD1. An analysis of HOG-pathway mutants and fluorescence microscopy of Hog1–GFP fusions showed that the non-redundant cytoplasmic components of the pathway, Pbs2p and Hog1p, are required to maintain BA resistance, but that import of the activated Hog1p kinase into the nucleus neither occurs during BA stress nor is necessary for wild-type-like BA tolerance. Pbs2p and Hog1p are also required to support normal morphogenesis during BA stress as their absence leads to BA-induced hyperpolarized growth. An analysis of Sod1p and Tps1p expression revealed that BA stress induces superoxide dismutase and increases trehalose synthesis activity, albeit only after a 7 h delay. We conclude that normal BA resistance of Saccharomyces cerevisiae depends on the functioning of HOG signalling, the trehalose synthesis pathway and superoxide dismutase activity.
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)