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Volume 158,
Issue 5,
2012
Volume 158, Issue 5, 2012
- Review
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Multiple leptospiral sphingomyelinases (or are there?)
More LessCulture supernatants of leptospiral pathogens have long been known to haemolyse erythrocytes. This property is due, at least in part, to sphingomyelinase activity. Indeed, genome sequencing reveals that pathogenic Leptospira species are richly endowed with sphingomyelinase homologues: five genes have been annotated to encode sphingomyelinases in Leptospira interrogans. Such redundancy suggests that this class of genes is likely to benefit leptospiral pathogens in their interactions with the mammalian host. Surprisingly, sequence comparison with bacterial sphingomyelinases for which the crystal structures are known reveals that only one of the leptospiral homologues has the active site amino acid residues required for enzymic activity. Based on studies of other bacterial toxins, we propose that leptospiral sphingomyelinase homologues, irrespective of their catalytic activity, may possess additional molecular functions that benefit the spirochaete. Potential secretion pathways and roles in pathogenesis are discussed, including nutrient acquisition, dissemination, haemorrhage and immune evasion. Although leptospiral sphingomyelinase-like proteins are best known for their cytolytic properties, we believe that a better understanding of their biological role requires the examination of their sublytic properties as well.
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Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors
More LessSalmonella enterica serovars cause severe disease in humans, such as gastroenteritis and typhoid fever. The bacteria are able to invade and replicate within host cells, including epithelial cells and macrophages. Pathogenesis of Salmonella is facilitated by a type III secretion system (T3SS) encoded by genes of Salmonella pathogenicity island 2 (SPI-2). Intracellular replication occurs in a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and depends on translocation of approximately 30 effector proteins via the SPI-2 T3SS into the host endomembrane system and cytoplasm. In this review we discuss the many different functions of these effectors, which range from maintaining the integrity of the SCV and its juxtanuclear location, to interference with the host cytoskeleton and immune signalling.
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- Cell and Molecular Biology of Microbes
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Multiple deletions reveal the essentiality of the DedA membrane protein family in Escherichia coli
More LessThe DedA family is a highly conserved, ancient family of membrane proteins with representatives in most sequenced genomes. A characteristic of prokaryotic DedA family genes is extensive gene duplication, with most bacterial genomes carrying two or more homologues. The Escherichia coli genome carries eight DedA genes, each individually nonessential. We previously described an E. coli mutant (BC202; ΔyghB : : kanR , ΔyqjA : : tetR ) with in-frame deletions of two DedA genes encoding proteins with 61 % amino acid identity. BC202 fails to complete cell division or grow at elevated temperatures. Here, we report that restoration of normal growth and cell division of BC202 is possible by overexpression of a subset of the eight E. coli DedA genes (yabI, yohD, yqjA and yghB) but not others (dedA, ydjX, ydjZ and yqaA), suggesting the existence of two functional groups within the family. We have constructed individual E. coli strains in which all eight DedA genes are deleted in a nonpolar manner, and growth is supported by a single DedA family gene under control of an inducible promoter. Strain BAL801 (with growth supported by cloned dedA) and BAL802 (with growth supported by cloned yqjA) exhibit slow growth that is absolutely dependent upon the presence of the arabinose inducer. Growth in the presence of glucose results in cell death. These results indicate that while not individually essential, the E. coli DedA family proteins are collectively essential. These observations suggest important functions for the E. coli DedA protein family.
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A novel GlnR target gene, nnaR, is involved in nitrate/nitrite assimilation in Streptomyces coelicolor
More LessGlnR is the global transcriptional regulator of nitrogen assimilation in Streptomyces coelicolor. Under nitrogen starvation, GlnR controls the transcription of at least nine genes associated with nitrogen metabolism. In this study, we identified a new GlnR target gene, SCO2958, named nnaR (nitrate/nitrite assimilation regulator). In silico analysis of NnaR revealed the presence of two distinct domains: an N-terminal uroporphyrinogen-III synthase (HemD)-like enzymatic domain and a C-terminal DNA binding domain. Complementation experiments with a haemin auxotroph Escherichia coli ΔhemD mutant strain revealed that NnaR has no HemD activity. Physiological studies of an S. coelicolor nnaR : : Tn5062 mutant showed that NnaR is involved in regulating nitrite reduction. By electrophoretic mobility shift assays the functionality of the NnaR DNA binding domain was confirmed, and it was found that NnaR binds in front of the genes narK (putative nitrate extrusion protein), nirB (nitrite reductase), nirA (putative nitrite/sulphite reductase) and nasA (putative nitrate reductase), which are associated with nitrate/nitrite assimilation. Furthermore, a cooperative binding of NnaR together with GlnR to the nirB promoter was observed, suggesting that NnaR may act as a GlnR co-activator.
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Identification of C-terminal hydrophobic residues important for dimerization and all known functions of ParB of Pseudomonas aeruginosa
More LessThe ParB protein of Pseudomonas aeruginosa is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParB P.a. . By in silico modelling of the C-terminal domain (amino acids 242–290) the hydrophobic residues L282, V285 and I289 (but not L286) are engaged in leucine-zipper-like structure formation, whereas the charged residues R290 and Q266 are implicated in forming a salt bridge involved in protein stabilization. Five parB mutant alleles were constructed and their functionality was defined in vivo and in vitro. In agreement with model predictions, the substitution L286A had no effect on mutant protein activities. Two ParBs with single substitutions L282A or V285A and deletions of two or seven C-terminal amino acids were impaired in both dimerization and DNA binding and were not able to silence genes adjacent to parS, suggesting that dimerization through the C terminus is a prerequisite for spreading on DNA. The defect in dimerization also correlated with loss of ability to interact with partner protein ParA. Reverse genetics demonstrated that a parB mutant producing ParB lacking the two C-terminal amino acids as well as mutants producing ParB with single substitution L282A or V285A had defects similar to those of a parB null mutant. Thus so far all the properties of ParB seem to depend on dimerization.
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Involvement of pnp in survival of UV radiation in Escherichia coli K-12
More LessPolynucleotide phosphorylase (PNPase), a multifunctional protein, is a 3′→5′ exoribonuclease or exoDNase in the presence of inorganic phosphate (Pi), and extends a 3′-OH of RNA or ssDNA in the presence of ADP or dADP. In Escherichia coli, PNPase is known to protect against H2O2- and mitomycin C-induced damage. Recent reports show that Bacillus subtilis PNPase is required for repair of H2O2-induced double-strand breaks. Here we show that absence of PNPase makes E. coli cells sensitive to UV, indicating that PNPase has a role in survival of UV radiation damage. Analyses of various DNA repair pathways show that in the absence of nucleotide excision repair, survival of UV radiation depends critically on PNPase function. Consequently, uvrA pnp, uvrB pnp and uvrC pnp strains show hypersensitivity to UV radiation. Whereas the pnp mutation is non-epistatic to recJ, recQ and recG mutations with respect to the UV-sensitivity phenotype, it is epistatic to uvrD, recB and ruvA mutations, implicating it in the recombinational repair process.
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Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23
More LessLactobacillus casei is a lactic acid bacterium commonly found in the gastrointestinal tract of animals, and some strains are used as probiotics. The ability of probiotic strains to survive the passage through the gastrointestinal tract is considered a key factor for their probiotic action. Therefore, tolerance to bile salts is a desirable feature for probiotic strains. In this study we have characterized the response of L. casei BL23 to bile by a transcriptomic and proteomic approach. The analysis revealed that exposure to bile induced changes in the abundance of 52 proteins and the transcript levels of 67 genes. The observed changes affected genes and proteins involved in the stress response, fatty acid and cell wall biosynthesis, metabolism of carbohydrates, transport of peptides, coenzyme levels, membrane H+-ATPase, and a number of uncharacterized genes and proteins. These data provide new insights into the mechanisms that enable L. casei BL23 to cope with bile stress.
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Expression of budding yeast IPT1 produces mannosyldiinositol phosphorylceramide in fission yeast and inhibits cell growth
More LessIn Saccharomyces (Sacc.) cerevisiae, the final step of the complex sphingolipid biosynthetic pathway requires Ipt1p for synthesis of mannosyldiinositol phosphorylceramide [M(IP)2C]. No fission yeast equivalent to Ipt1p has been found in the Schizosaccharomyces (Schiz.) pombe genome, and the most abundant complex sphingolipid is mannosylinositol phosphorylceramide. To examine the effect of expressing Sacc. cerevisiae IPT1 (ScIPT1) in Schiz. pombe, the ScIPT1 gene was cloned into an inducible fission yeast integrative vector and expressed in wild-type Schiz. pombe. In the Schiz. pombe ScIPT1-expressing cells, M(IP)2C was detected, indicating that ScIpt1p functions in M(IP)2C synthesis in Schiz. pombe. Expression of ScIPT1 caused pleiotropic phenotypes, including aberrant morphology and mislocalization of ergosterols in the plasma membrane. Furthermore, growth of Schiz. pombe was severely impaired. We analysed the sphingolipid composition of ScIPT1-expressing cells following a prolonged lag phase, and found that M(IP)2C was not synthesized, indicating that Ipt1p had been inactivated. GFP-tagged ScIpt1 localized primarily in the Golgi apparatus in wild-type Schiz. pombe. Over time, ScIpt1p was eventually transported to the vacuolar lumen through the multivesicular body pathway. These results indicate that M(IP)2C is toxic to Schiz. pombe and that fission yeast possesses an unknown mechanism to effectively extrude toxic sphingolipids from cells.
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In vivo features of signal transduction by the essential response regulator RpaB from Synechococcus elongatus PCC 7942
More LessThe NblS-RpaB signalling pathway, the most conserved two-component system in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions and is involved in negative regulation of high-light-induced genes. However, relevant regulatory details of the NblS-RpaB signalling pathway remain to be elucidated. We recently showed that the response regulator RpaB is regulated by specific (de)phosphorylation from the histidine kinase NblS and that RpaB and its phosphorylatable residue Asp56 are both required for viability of Synechococcus elongatus PCC 7942. We show here that the phosphorylated form of RpaB is present in cells growing under standard laboratory conditions and that high light stress affected the ratio of phosphorylated to non-phosphorylated RpaB. It also decreased the amount of rpaB transcripts without appreciably changing the total levels of RpaB. Quantitative Western blotting and confocal microscopy analyses were consistent with RpaB being a very abundant regulator, with nucleoid localization. A genetically engineered RpaB-GFP (green fluorescent protein) fusion protein rescued lethality of the rpaB null mutant, indicating that it was functional. This is, to our knowledge, the first study demonstrating in a cyanobacterium, and for a two-component response regulator, that the in vivo ratio of phosphorylated to non-phosphorylated protein changes in response to environmental conditions.
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Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli
The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids phosphatidylglycerol and cardiolipin. We found that the Rcs activation, and consequent thermosensitivity, were suppressed by overexpression of the lgt gene, encoding diacylglyceryltransferase, which catalyses the modification of prolipoproteins that is the first step in the maturation and localization process of lipoproteins, and is a prerequisite for the later steps. The outer-membrane lipoprotein RcsF is an essential component of Rcs signalling. This lipoprotein was poorly localized to the outer membrane in the pgsA null mutant, probably because of the absence of phosphatidylglycerol, the major donor of diacylglycerol in the Lgt reaction. Even in a pgsA + background, the Rcs system was activated when RcsF was mislocalized to the inner membrane by alteration of the residues at positions 2 and 3 of its mature form to inner-membrane retention signals, or when it was mislocalized to the periplasm by fusing the mature form to maltose-binding protein. These results suggest that RcsF functions as a ligand for RcsC in activating Rcs signalling. Mislocalized versions of RcsF still responded to mutations pgsA, mdoH and tolB, further activating the Rcs system, although the rfaP mutation barely caused activation. It seems that RcsF must be localized in the outer membrane to respond effectively to stimuli from outside the cell.
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The phosphatidylcholine synthase of Pseudomonas putida A ATCC 12633 is responsible for the synthesis of phosphatidylcholine, which acts as a temporary reservoir for Al3+
More LessIn Pseudomonas putida A ATCC 12633 cells grown with tetradecyltrimethylammonium bromide and exposed to Al3Cl, phosphatidylcholine (PC) levels increased, which alleviated stress caused by the Al3+. Here we cloned and sequenced a gene from this strain that encodes a phosphatidylcholine synthase (PCS) and characterized a pcs-deficient mutant. In the pcs-deficient mutant, PC could not be detected, whereas the mutant could be successfully complemented and expressed the enzyme, indicating that PC synthesis occurs exclusively via the PCS pathway in this organism. Although under non-stressing growth conditions the pcs-deficient mutant showed growth like that of the wild-type strain, the mutant was much more sensitive when challenged with Al3+, which strongly supports the supposition that PC is involved in the response of P. putida to Al3+ and acts as a temporary reservoir of available ions through the formation of Al3+ : PC complexes.
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Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans
The opportunistic pathogen Candida albicans has a single protein phosphatase Z (PPZ) candidate gene termed CaPPZ1, which shows significant allele variability. We demonstrate here that bacterially expressed CaPpz1 protein exhibits phosphatase activity which can be inhibited by recombinant Hal3, a known inhibitor of Saccharomyces cerevisiae Ppz1. Site-directed mutagenesis experiments based on natural polymorphisms allowed the identification of three amino acid residues that affect enzyme activity or stability. The expression of CaPPZ1 in ppz1 S. cerevisiae and pzh1 Schizosaccharomyces pombe cells partially rescued the salt and caffeine phenotypes of the deletion mutants. CaPpz1 also complemented the slt2 S. cerevisiae mutant, which is crippled in the mitogen-activated protein (MAP) kinase that mediates the cell wall integrity signalling pathway. Collectively, our results suggest that the orthologous PPZ enzymes have similar but not identical functions in different fungi. The deletion of the CaPPZ1 gene in C. albicans resulted in a mutant that was sensitive to salts such as LiCl and KCl, to caffeine, and to agents that affect cell wall biogenesis such as Calcofluor White and Congo red, but was tolerant to spermine and hygromycin B. Reintegration of the CaPPZ1 gene into the deletion mutant alleviated all of the mutant phenotypes tested. Thus CaPpz1 is involved in cation homeostasis, cell wall integrity and the regulation of the membrane potential of C. albicans. In addition, the germ tube growth rate, and virulence in the BALB/c mouse model, were reduced in the null mutant, suggesting a novel function for CaPpz1 in the yeast to hypha transition that may have medical relevance.
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Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP
More LessProteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His–Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His–Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant SpxDD (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxΔC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx–ClpX interaction, resulting in rapid degradation.
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Reduction of the monounsaturated fatty acid content of Escherichia coli results in increased resistance to oxidative damage
Reactive oxygen species (ROSs) affect several macromolecules and cellular components in eukaryotic and prokaryotic cells. In this work, the effect of various ROS-generating compounds on the Escherichia coli membrane was studied. Membrane fatty acid profiles, oxidative damage levels and bacterial resistance to these toxicants were determined. Studies included wild-type cells as well as a strain exhibiting a modified monounsaturated fatty acid (MUFA) profile (accomplished by overexpressing the β-hydroxyacyl acyl carrier protein dehydratase-encoding gene, fabA). Levels of membrane MUFAs and oxidative damage markers decreased slightly upon toxicant exposure with a concomitant increase in cell resistance to these ROS-generating compounds. A direct relationship between MUFAs and lipid peroxidation was observed. The lower the MUFA the lower the peroxide levels, suggesting that MUFAs are targets for membrane lipid oxidation.
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Suppression of pleiotropic phenotypes of a Burkholderia multivorans fur mutant by oxyR mutation
More LessFur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes, such as an inability to efficiently use several carbon sources, as well as high sensitivity to hydrogen peroxide (H2O2), paraquat (a superoxide-producing compound) and nitric oxide (NO). To gain more insight into the pleiotropic role of the Fur protein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 fur mutant that restored tolerance to NO were isolated and characterized in this study. The microarray-based comparative genomic analysis and subsequent sequencing analysis indicated that such suppressor mutants had a 2 bp deletion in the oxyR gene, whose orthologues encode H2O2-responsive transcriptional regulators in other bacterial species. The suppressor mutants and the reconstructed fur–oxyR double-deletion mutant showed indistinguishable phenotypes in that they were all (i) more resistant than the fur mutant to H2O2, superoxide, NO and streptonigrin (an iron-activated antibiotic) and (ii) able to use carbon sources that cannot efficiently support the growth of the fur mutant. These results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant. The fur–oxyR double mutants were found to overexpress the KatG (catalase/peroxidase) and AhpC1 and AhpD (alkyl hydroperoxide reductase subunits C and D) proteins, and their enzymic activities to remove reactive oxygen and nitrogen species were suggested to be responsible for the suppression of phenotypes caused by the fur mutation.
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Expression of Vibrio vulnificus insulin-degrading enzyme is regulated by the cAMP–CRP complex
More LessComponents of the bacterial phosphoenolpyruvate (PEP) : carbohydrate phosphortransferase system (PTS) have multiple regulatory roles in addition to PEP-dependent transport/phosphorylation of numerous carbohydrates. We have recently shown that, in an opportunistic human pathogen, Vibrio vulnificus, enzyme IIAGlc (EIIAGlc) interacts with a peptidase that has high sequence similarity to mammalian insulin-degrading enzymes, called Vibrio insulin-degrading enzyme (vIDE). Although the vIDE–EIIAGlc interaction is independent of the phosphorylation state of EIIAGlc, vIDE shows no peptidase activity unless complexed with the unphosphorylated form of EIIAGlc. A deletion mutant of ideV, the gene encoding vIDE, shows remarkably lower degrees of survival and virulence than the wild-type strain in mice, implying that vIDE is a virulence factor. In this study, we investigated regulation of ideV expression at the transcriptional level. Primer extension analysis identified two different transcriptional start sites of ideV: PL for the longer transcript and PS for the shorter transcript. We performed ligand fishing experiments by using the promoter region of ideV and found that the cAMP receptor protein (CRP) specifically binds to the promoter. DNase I footprinting experiments revealed that CRP binds to a region between the two promoters. In vitro transcription assays showed that CRP activates ideV PS transcription in the presence of cAMP whose concentration is regulated by EIIAGlc. These results suggest that EIIAGlc regulates the expression level of vIDE as well as its activity.
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- Environmental and Evolutionary Microbiology
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Passage of Campylobacter jejuni through the chicken reservoir or mice promotes phase variation in contingency genes Cj0045 and Cj0170 that strongly associates with colonization and disease in a mouse model
Human illness due to Camplyobacter jejuni infection is closely associated with consumption of poultry products. We previously demonstrated a 50 % shift in allele frequency (phase variation) in contingency gene Cj1139 (wlaN) during passage of C. jejuni NCTC11168 populations through Ross 308 broiler chickens. We hypothesized that phase variation in contingency genes during chicken passage could promote subsequent colonization and disease in humans. To test this hypothesis, we passaged C. jejuni strains NCTC11168, 33292, 81-176, KanR4 and CamR2 through broiler chickens and analysed the ability of passaged and non-passaged populations to colonize C57BL6 IL-10-deficient mice, our model for human colonization and disease. We utilized fragment analysis and nucleotide sequence analysis to measure phase variation in contingency genes. Passage through the chicken reservoir promoted phase variation in five specific contingency genes, and these ‘successful’ populations colonized mice. When phase variation did not occur in these same five contingency genes during chicken passage, these ‘unsuccessful’ populations failed to colonize mice. Phase variation during chicken passage generated small insertions or deletions (indels) in the homopolymeric tract (HT) in contingency genes. Single-colony isolates of C. jejuni strain KanR4 carrying an allele of contingency gene Cj0170 with a10G HT colonized mice at high frequency and caused disease symptoms, whereas single-colony isolates carrying the 9G allele failed to colonize mice. Supporting results were observed for the successful 9G allele of Cj0045 in strain 33292. These data suggest that phase variation in Cj0170 and Cj0045 is strongly associated with mouse colonization and disease, and that the chicken reservoir can play an active role in natural selection, phase variation and disease.
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- Genes and Genomes
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Inner-membrane transporters for the siderophores pyochelin in Pseudomonas aeruginosa and enantio-pyochelin in Pseudomonas fluorescens display different enantioselectivities
More LessIron uptake and transcriptional regulation by the enantiomeric siderophores pyochelin (Pch) and enantio-pyochelin (EPch) of Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, are stereospecific processes. The iron-loaded forms of Pch (ferriPch) and of EPch (ferriEPch) are recognized stereospecifically (i) at the outer membrane by the siderophore receptors FptA in P. aeruginosa and FetA in P. fluorescens and (ii) in the cytoplasm by the two AraC-type regulators PchR, which are activated by their cognate siderophore. Here, stereospecific siderophore recognition is shown to occur at the inner membrane also. In P. aeruginosa, translocation of ferriPch across the inner membrane is carried out by the single-subunit siderophore transporter FptX. In contrast, the uptake of ferriEPch into the cytoplasm of P. fluorescens was found to involve a classical periplasmic binding protein-dependent ABC transporter (FetCDE), which is encoded by the fetABCDEF operon. Expression of a translational fetA–gfp fusion was repressed by ferric ions, and activated by the cognate siderophore bound to PchR, thus resembling the analogous regulation of the P. aeruginosa ferriPch transport operon fptABCX. The inner-membrane transporters FetCDE and FptX were expressed in combination with either of the two siderophore receptors FetA and FptA in a siderophore-negative P. aeruginosa mutant deleted for the fptABCX operon. Growth tests conducted under iron limitation with ferriPch or ferriEPch as the iron source revealed that FptX was able to transport ferriPch as well as ferriEPch, whereas FetCDE specifically transported ferriEPch. Thus, stereospecific siderophore recognition occurs at the inner membrane by the FetCDE transporter.
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- Microbial Pathogenicity
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Interaction of environmental Burkholderia cenocepacia strains with cystic fibrosis and non-cystic fibrosis bronchial epithelial cells in vitro
Burkholderia cenocepacia is an important human pathogen in patients with cystic fibrosis (CF). Non-clinical reservoirs may play a role in the acquisition of infection, so it is important to evaluate the pathogenic potential of environmental B. cenocepacia isolates. In this study, we investigated the interactions of two environmental B. cenocepacia strains (Mex1 and MCII-168) with two bronchial epithelial cell lines, 16HBE14o− and CFBE41o−, which have a non-CF and a CF phenotype, respectively. The environmental strains showed a significantly lower level of invasion into both CF and non-CF cells in comparison with the clinical B. cenocepacia LMG16656T strain. Exposure of polarized CFBE41o− or 16HBE14o− cells to the environmental strains resulted in a significant reduction in transepithelial resistance (TER), comparable with that observed following exposure to the clinical strain. A different mechanism of tight junction disruption in CF versus non-CF epithelia was found. In the 16HBE41o− cells, the environmental strains resulted in a drop in TER without any apparent effect on tight junction proteins such as zonula occludens-1 (ZO-1). In contrast, in CF cells, the amount of ZO-1 and its localization were clearly altered by the presence of both the environmental strains, comparable with the effect of LMG16656. This study demonstrates that even if the environmental strains are significantly less invasive than the clinical strain, they have an effect on epithelial integrity comparable with that of the clinical strain. Finally, the tight junction regulatory protein ZO-1 appears to be more susceptible to the presence of environmental strains in CF cells than in cells which express a functional cystic fibrosis transmembrane regulator (CFTR).
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Characterization of HrpB2 from Xanthomonas campestris pv. vesicatoria identifies protein regions that are essential for type III secretion pilus formation
More LessThe Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. T3S depends on HrpB2, which is essential for assembly of the extracellular T3S pilus and is itself weakly secreted. To characterize the role of HrpB2, we used a transposon mutagenesis approach, which led to the insertion of pentapeptide-encoding sequences into hrpB2. Complementation studies with HrpB2 mutant derivatives revealed that the N-terminal region of HrpB2 tolerates pentapeptide insertions, whereas insertions in the regions spanning amino acids 60–74 and 93–130, respectively, resulted in a loss of bacterial pathogenicity and T3S, including secretion of HrpB2 itself. The C-terminal region (amino acids 93–130) of HrpB2 contains a conserved VxTLxK amino acid motif that is also present in predicted inner rod proteins from animal-pathogenic bacteria and is required for the contribution of HrpB2 to pilus assembly and T3S. Electron microscopy and fractionation studies revealed that HrpB2 is not a component of the extracellular pilus structure but localizes to the bacterial periplasm and the outer membrane. We therefore propose that the essential contribution of HrpB2 to T3S and pilus assembly is linked to its possible function as a periplasmic component of the T3S system at the base of the pilus.
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Volumes and issues
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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)
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