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Volume 151,
Issue 9,
2005
Volume 151, Issue 9, 2005
- Pathogens And Pathogenicity
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The MAP kinase Hog1p differentially regulates stress-induced production and accumulation of glycerol and d-arabitol in Candida albicans
More LessCandida albicans produces and accumulates large amounts of the polyols d-arabitol and glycerol in culture, and/or in infected mammalian tissues. However, the effects of environmental stresses on production and accumulation of these polyols, and the means by which polyol production and accumulation are regulated have not been studied. C. albicans grown in glucose at 30 °C (i) produced maximal amounts of glycerol within 6 h, (ii) produced maximal amounts of d-arabitol and ribitol within 12 h, and (iii) released most of these polyols into the extracellular environment. C. albicans responded to osmotic and citric acid stress by producing and accumulating more glycerol, and to temperature and oxidative stresses by producing more d-arabitol. The increase in intracellular glycerol was proportional to extracellular osmolarity, suggesting that glycerol functions as an osmolyte. The MAP kinase Hog1p is required for wild-type glycerol production in several fungal species subjected to osmotic stress, but it is not known if Hog1p plays a role in regulating d-arabitol production. Therefore, two C. albicans hog1 null mutants were constructed and tested for the ability to produce glycerol and d-arabitol in response to environmental stresses. The ability to grow and produce glycerol when exposed to osmotic or citric acid stresses, and to produce d-arabitol when exposed to oxidative stress, was partially dependent on Hog1p, but the ability to produce d-arabitol when exposed to temperature stress was Hog1p independent. These results imply that multiple pathways regulate glycerol and d-arabitol synthesis in C. albicans.
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A novel bacteriocin-like substance (BLIS) from a pathogenic strain of Vibrio harveyi
More LessInter-strain and inter-species inhibition mediated by a bacteriocin-like inhibitory substance (BLIS) from a pathogenic Vibrio harveyi strain VIB 571 was demonstrated against four isolates of the same species, and one culture each of a Vibrio sp., Vibrio fischeri, Vibrio gazogenes and Vibrio parahaemolyticus. The crude BLIS, which was obtained by ammonium-sulphate precipitation of the cell-free supernatant of a 72 h broth culture of strain VIB 571, was inactivated by lipase, proteinase K, pepsin, trypsin, pronase E, SDS and incubation at ≥60 °C for 10 min. The activity was stable between pH 2–11 for at least 5 h. Anion-exchange chromatography, gel filtration, SDS-PAGE and two-dimensional gel electrophoresis revealed the presence of a single major peak, comprising a protein with a pI of ∼5·4 and a molecular mass of ∼32 kDa. The N-terminal amino acid sequence of the protein comprised Asp-Glu-Tyr-Ile-Ser-X-Asn-Lys-X-Ser-Ser-Ala-Asp-Ile (with X representing cysteine or modified amino acid residues). A similarity search based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) generated peptide masses and the N-terminal sequence did not yield any significant matches.
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Subinhibitory cerulenin inhibits staphylococcal exoprotein production by blocking transcription rather than by blocking secretion
More LessCerulenin is an antibiotic that inhibits fatty acid synthesis by covalent modification of the active thiol of the chain-elongation subtypes of β-ketoacyl-acyl carrier protein synthase. It also inhibits other processes that utilize essential thiols. Cerulenin has been widely reported to block protein secretion at sub-MIC levels, an effect that has been postulated to represent interference with membrane function through interference with normal fatty acid synthesis. This study confirms the profound reduction in extracellular proteins caused by low concentrations of the antibiotic, and shows by Northern blot hybridization that this reduction is due to interference with transcription. By exchanging promoters between entB, a gene that is inhibited by cerulenin, and entA, a gene that is not, it was also shown that the antibiotic does not block secretion. Subinhibitory concentrations of cerulenin were also found to block transcriptional activation of at least two regulatory determinants, agr and sae, that function by signal transduction. Interference with the activation of these and other regulatory determinants probably accounts for much of the inhibitory effect on exoprotein production of sub-MIC concentrations of cerulenin.
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Characterization of the role of LtgB, a putative lytic transglycosylase in Neisseria gonorrhoeae
More LessNeisseria gonorrhoeae releases monomeric peptidoglycan (PG) fragments during growth. These PG fragments affect pathogenesis-related phenotypes including induction of inflammatory cytokines and killing of ciliated fallopian tube cells. Although the biological activities of these molecules have been established in multiple systems, the genes and gene products responsible for their production in N. gonorrhoeae have not been determined. The authors previously identified genes for three lytic transglycosylase homologues (ltgA, ltgB and ltgC) in the N. gonorrhoeae genome sequence. Mutation of ltgA was found to affect PG fragment release, and mutation of ltgC affected cell separation. In this study the effects of complete deletion or point mutations in ltgB were characterized. Point mutations were introduced by a combination of insertion-duplication mutagenesis and positive and negative selection, thereby generating selectable marker-less mutations. The ltgB deletion mutant had normal growth characteristics and was not affected in PG fragment release. When expressed in Escherichia coli, gonococcal LtgB was able to substitute for lambda endolysin to cause cell lysis. Mutation of the predicted catalytic-site glutamic acid residue did not decrease lysis in this system. However, mutation of a nearby glutamic acid residue eliminated lysis activity.
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Ca2+ release from host intracellular stores and related signal transduction during Campylobacter jejuni 81-176 internalization into human intestinal cells
More LessCampylobacter jejuni is the leading bacterial cause of human diarrhoeal disease in many parts of the world, including the USA. The ability of C. jejuni to invade the host intestinal epithelium is an important determinant of virulence. A common theme among pathogenic invasive micro-organisms is their ability to usurp the eukaryotic cell-signalling systems both to allow for invasion and to trigger disease pathogenesis. Ca2+ is very important in a great variety of eukaryotic cell-signalling processes (e.g. calmodulin-activated enzymes, nuclear transcriptional upregulation, and cytoskeletal rearrangements). This study analyses the effects of Ca2+ availability on invasion of human INT407 intestinal epithelial cells by C. jejuni strain 81-176. The ability of C. jejuni to invade INT407 cells was not blocked by chelation of any remaining extracellular Ca2+ from host cells incubated in Ca2+-free, serum-free media. In contrast, C. jejuni invasion was markedly reduced either by chelating host intracellular Ca2+ with 1,2-bis-(2-)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA, AM) or by blocking the release of Ca2+ from intracellular stores with dantrolene or U73122. Moreover, Bay K8644, a plasma-membrane Ca2+-channel agonist, was observed to stimulate C. jejuni invasion, presumably by increasing host intracellular free Ca2+ levels. Measurement of host-cell cytosolic Ca2+ via spectrofluorimetry and fluorescence microscopy revealed an increase in Ca2+ from 10 min post-infection. Monolayer pretreatment with either a calmodulin antagonist or a specific inhibitor of protein kinase C was found to cause a marked reduction in C. jejuni invasion, suggesting roles for these Ca2+-activated modulators in signal-transduction events involved in C. jejuni invasion. These results demonstrate that C. jejuni induces the mobilization of Ca2+ from host intracellular stores, which is an essential step in the invasion of intestinal cells by this pathogen.
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Molecular and serological evidence of Pneumocystis circulation in a social organization of healthy macaques (Macaca fascicularis)
Simian populations represent valuable models for understanding the epidemiology of human pneumocystosis. The present study aims to describe the circulation of Pneumocystis organisms within a social organization of healthy crab-eating macaques (Macaca fascicularis) living in a natural setting in France. Animals were followed for up to 2 years. Deep nasal swab and blood samples were collected monthly from each animal under general anaesthesia. Environmental air was sampled for a 1 week period every month in the park where the macaques dwelt. Pneumocystis DNA was detected by nested-PCR of mitochondrial large subunit rRNA (mtLSU) gene in nasal swab and air samples. Anti-Pneumocystis IgG antibodies were detected in serum samples by indirect immuno-fluorescence assay. Pneumocystis DNA was detected in 168 of 500 swab samples examined (33·6 %). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. Positive detection of Pneumocystis DNA was not related to the detection of serum anti-Pneumocystis antibody. During the second year of the study, Pneumocystis DNA was amplified more frequently from unweaned macaques than from adults or subadults. The mtLSU sequence showed marked polymorphism with eight Pneumocystis sequence types representing two distinct groups. On the whole, a constant and intensive circulation of Pneumocystis organisms within the community was observed. However, the implication of the various members of the colony was probably different and several levels of colonization by Pneumocystis may occur in immunocompetent macaques.
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Identification and expression of a host-recognized antigen, FspA, from Flavobacterium psychrophilum
More LessFlavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome, an economically important disease of immature salmonid fish for which there is no vaccine. Convalescent serum from the host, rainbow trout (Oncorhynchus mykiss), reacted strongly with a ∼20 kDa, Flavobacterium-specific protein antigen (subsequently named FspA) from F. psychrophilum. Protein-enriched, detergent-partitioned samples were separated by two-dimensional gel electrophoresis and the protein target was excised, proteolytically cleaved and the resulting peptides analysed by MS. Quadrupole-time-of-flight MS was used to generate a fragmented peptide spectrum. The resulting peptide sequences were then used to design degenerate PCR primers to amplify the gene (fspA) of interest: 612 bp encoding 203 aa, including a putative 19 aa N-terminal signal sequence which predicted a processed 19 303·6 Da protein. FspA proved to be unique and only homologous to two unspecified sequences reported from Flavobacterium johnsoniae, although weakly homologous to a Yersinia pseudotuberculosis adhesin. An amplified gene fragment (537 bp, encoding 179 aa) was further cloned into an expression vector, expressed as a ∼30 kDa N-terminal fusion protein and found to retain its strong reactivity with host serum antibodies. These results suggest that the surface-localized FspA may be an important subunit vaccine candidate antigen against F. psychrophilum.
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- Physiology
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Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp.
More LessBacteria undergo a variety of physiological changes following a switch from planktonic growth to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular proteomes. Calcium influences these proteome responses, and affects the amount of surface-associated biomass and extracellular matrix material produced by Pseudoalteromonas sp. 1398. Four extracellular proteins, characterized by N-terminal sequencing, showed increased abundances, while one protein, flagellin, showed reduced abundance at higher [Ca2+]. Immunoblotting and transmission-electron-microscopy analysis confirmed that higher [Ca2+] and surface-associated growth results in the repression of flagella production. Two-dimensional gel electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses demonstrated that Ca2+ had a greater regulatory influence on Pseudoalteromonas sp. growing in biofilms than on planktonic cultures. Approximately 22 % of the total cytosolic proteins resolved by 2DGE had differing abundances in response to a switch from planktonic growth to surface-associated growth when the cells were cultivated in 1 mM Ca2+. At higher [Ca2+] this number increased to 38 %. Fifteen cellular proteins that were differentially expressed in response to biofilm growth and/or Ca2+ were analysed by N-terminal sequencing and/or MS/MS. These proteins were identified as factors involved in cellular metabolic functions, putative proteases and transport proteins, although there were several proteins that had not been previously characterized. These results indicate that Ca2+ causes global changes in matrix material, as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These changes are more pronounced when the bacterium grows in biofilms than when it grows in planktonic culture.
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Desferrioxamine E produced by Streptomyces griseus stimulates growth and development of Streptomyces tanashiensis
The authors previously reported that interspecific stimulatory events between Streptomyces species for antibiotic production and/or morphological differentiation mediated by putative diffusible metabolites take place at a high frequency. This paper reports the isolation and characterization of a substance produced by Streptomyces griseus that stimulates the growth and development of Streptomyces tanashiensis. The substance was purified from the culture supernatant of S. griseus by using anion-exchange chromatography, gel filtration chromatography and reverse-phase HPLC. FAB-MS and NMR analyses of the purified preparation indicated the substance to be desferrioxamine E (synonym: nocardamine), a siderophore that is widely produced by Streptomyces species and related organisms. Similar stimulatory effects on the growth and development of S. tanashiensis were exerted by desferrioxamine E produced by another actinomycete strain, but not by other siderophores tested, including ferrichrome and nocobactin and free ferric ion. An exogenous supply of desferrioxamine E stimulated secondary metabolite formation and/or morphological differentiation in various actinomycete strains. Disruption of the desferrioxamine biosynthesis gene cluster in Streptomyces coelicolor A3(2) abolished the production of desferrioxamine E and the activity to stimulate the growth and differentiation of S. tanashiensis. The S. coelicolor mutant showed impaired growth and development on Bennett's/glucose agar medium, but it was rescued by the exogenous supply of desferrioxamine E. These results indicate that desferrioxamines play an important role in streptomycete physiology. Similar to several pathogenic bacteria and fungi, S. tanashiensis may be defective in the production of siderophores; however, it can utilize the siderophores excreted by other organisms.
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High-level resistance to oxidative stress in Lactococcus lactis conferred by Bacillus subtilis catalase KatE
More LessLactococcus lactis, a lactic acid bacterium widely used for food fermentations, is often exposed to damaging stress conditions. In particular, oxidative stress leads to DNA, protein and membrane damages that can be lethal. As L. lactis has no catalase, the impact of production of the Bacillus subtilis haem catalase KatE on its oxidative stress resistance was tested. This cytoplasmic catalase was engineered for extracellular expression in L. lactis with an optimization strategy based on fusion to the nisin-inducible promoter and a lactococcal signal peptide (SPUsp45). The production of KatE by L. lactis conferred an 800-fold increase in survival after 1 h exposure to 4 mM hydrogen peroxide, and a 160-fold greater survival in long-term (3 days) survival of aerated cultures in a cydA mutant, which is unable to respire. The presence of KatE protected DNA from oxidative damage and limited its degradation after long-term aeration in a cydA/recA mutant, defective in DNA repair. L. lactis is thus able to produce active catalase that can provide efficient antioxidant activity.
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Analysis of teichoic acid biosynthesis regulation reveals that the extracytoplasmic function sigma factor σ M is induced by phosphate depletion in Bacillus subtilis W23
More LessThe expression of the Bacillus subtilis W23 tar genes specifying the biosynthesis of the major wall teichoic acid, the poly(ribitol phosphate), was studied under phosphate limitation using lacZ reporter fusions. Three different regulation patterns can be deduced from these β-galactosidase activity data: (i) tarD and tarL gene expression is downregulated under phosphate starvation; (ii) tarA and, to a minor extent, tarB expression after an initial decrease unexpectedly increases; and (iii) tarO is not influenced by phosphate concentration. To dissect the tarA regulatory pattern, its two promoters were analysed under phosphate limitation: The P tarA -ext promoter is repressed under phosphate starvation by the PhoPR two-component system, whereas, under the same conditions, the P tarA -int promoter is upregulated by the action of an extracytoplasmic function (ECF) σ factor, σ M. In contrast to strain 168, σ M is activated in strain W23 in phosphate-depleted conditions, a phenomenon indirectly dependent on PhoPR, the two-component regulatory system responsible for the adaptation to phosphate starvation. These results provide further evidence for the role of σ M in cell-wall stress response, and suggest that impairment of cell-wall structure is the signal activating this ECF σ factor.
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- Plant-Microbe Interactions
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Isolation of Ralstonia solanacearum hrpB constitutive mutants and secretion analysis of hrpB-regulated gene products that share homology with known type III effectors and enzymes
More LessThe Hrp type III secretion system (TTSS) is essential for the pathogenicity of the Gram-negative plant pathogen Ralstonia solanacearum. To examine the secretion of type III effector proteins via the Hrp TTSS, a screen was done of mutants constitutively expressing the hrpB gene, which encodes an AraC-type transcriptional activator for the hrp regulon. A mutant was isolated that in an hrp-inducing medium expresses several hrpB-regulated genes 4·9–83-fold higher than the wild-type. R. solanacearum Hrp-secreted outer proteins PopA and PopC were secreted at high levels into the culture supernatants of the hrpB constitutive (hrpB c) mutant. Using hrpB c mutants, the extracellular secretion of several hrpB-regulated (hpx) gene products that share homology with known type III effectors and enzymes was examined. Hpx23, Hpx24 and Hpx25, which are similar in sequence to Pseudomonas syringae pv. tomato effector proteins HopPtoA1, HolPtoR and HopPtoD1, are also secreted via the Hrp TTSS in R. solanacearum. The secretion of two hpx gene products that share homology with known enzymes, glyoxalase I (Hpx19) and Nudix hydrolase (Hpx26), was also examined. Hpx19 is accumulated inside the cell, but interestingly, Hpx26 is secreted outside the cell as an Hrp-secreted outer protein, suggesting that Hpx19 functions intracellularly but Hpx26 is a novel effector protein of R. solanacearum.
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The sigma factor RpoS is required for stress tolerance and environmental fitness of Pseudomonas fluorescens Pf-5
More LessMany micro-organisms exist in natural habitats that are subject to severe or dramatically fluctuating environmental conditions. Such is the case for bacteria inhabiting plant surfaces, where they are exposed to UV irradiation, oxygen radicals, and large fluctuations in temperature and moisture. This study focuses on the role of RpoS, a central regulator of stationary-phase gene expression in bacterial cells, in stress response and environmental fitness of Pseudomonas fluorescens Pf-5. Strain Pf-5 is a rhizosphere-inhabiting bacterium that suppresses plant diseases caused by several plant-pathogenic fungi and oomycetes. Previous studies demonstrated that rpoS was required for osmotic and oxidative stress resistance of Pf-5. The results of this study demonstrate a role for rpoS in tolerance of Pf-5 to freezing, starvation, UV irradiation and desiccation stress. In field studies, an rpoS mutant was compromised in rhizosphere colonization of plants in dry soil, whereas similar rhizosphere populations were established by Pf-5 and an rpoS mutant in well-irrigated soils. RpoS is a key determinant in stress response and environmental fitness of the rhizosphere bacterium P. fluorescens Pf-5.
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