- Volume 146, Issue 10, 2000
Volume 146, Issue 10, 2000
- Microbiology Comment
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- Biochemistry
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The carboxyl terminus of the Bacillus subtilis SecA is dispensable for protein secretion and viability
More LessThe Escherichia coli secretion-dedicated chaperone SecB targets a subset of proteins to the translocase by interacting with the carboxyl (C-) terminus of SecA. This region of SecA is highly conserved in Eubacteria, but despite its presence in the Bacillus subtilis SecA, the B. subtilis genome does not appear to contain a gene for a clear homologue of SecB. Deletion of the C-terminus of the B. subtilis SecA yields cells that have normal viability, but that exhibit a response reminiscent of oxidative stress and the loss of a number of secretory proteins from the culture supernatant. Semi-quantitative RT-PCR demonstrates that these proteins are expressed at lower levels. The C-terminus of SecA fused to glutathione S-transferase (GST) specifically binds a cytosolic protein, termed MrgA. This protein has been reported to function in relation to oxidative stress, but deletion of the mrgA gene does not result in a secretion defect nor does it cause an oxidative stress response. It is concluded that the C-terminus of the B. subtilis SecA is not essential for secretion and viability.
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Dependence of Trichomonas vaginalis upon polyamine backconversion
More LessTrichomonas vaginalis grown for 16 h in the presence of [14C]spermine formed a high intracellular pool of [14C]spermidine and a small but detectable pool of [14C]putrescine. When [3H]putrescine was added to the growth medium, a large intracellular pool of [3H]putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T. vaginalis. Spermidine:spermineN 1-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine. Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction. In the presence of saturating substrate, the trichomonad SSAT had an activity of 0·39±0·09 nmol min−1 (mg protein)−1 (the mean of five analyses) and an apparent K m for spermine of 1·7 μM. The enzyme was competitively inhibited by di(ethyl)norspermine with a K i of 28 μM. Growth studies indicated that 50 μM di(ethyl)norspermine caused a 68% and 84% reduction in the intracellular concentrations of spermidine and spermine, respectively. The trichomonad polyamine oxidase required FAD as a cofactor and had an apparent K m of 6·0 μM forN 1-acetylspermine. The potential of bis(alkyl) polyamine analogues as antitrichomonad agents is discussed.
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- Biotechnology
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Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system
More LessA series of chimeric α-amylase genes derived from amyL, which encodes the liquefying α-amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques. The gene constructs were cloned in Bacillus subtilis, where their ability to direct the synthesis and secretion of active α-amylase was determined. Detectable α-amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wild-type AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzymes were degraded to a greater extent than the native enzyme. These findings suggest that cell-associated proteolysis is a significant problem affecting the use of B. subtilis as host bacterium for the production of heterologous proteins.
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Intracellular pH determination of pristinamycin-producing Streptomyces pristinaespiralis by image analysis
More LessIntracellular pH (pHi) is an essential parameter in the regulation of intracellular processes. Thus, its measurement might provide clues regarding the physiological state of cells cultivated in vitro. pHi of the filamentous, pristinamycin-producing Streptomyces pristinaespiralis was determined by epifluorescence microscopy and image analysis using the pH-sensitive fluorescent probe BCECF-AM [2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. Staining cell culture samples (OD660=1) of S. pristinaespiralis with 20 μM BCECF at 28 °C for 30 min yielded a green/red fluorescence ratio (R 527/600) that correlated with the pHi of the cells for values ranging from 6·5 to 8·5. When S. pristinaespiralis was cultivated in pristinamycin-producing conditions (in batch mode, with a constant external pH of 6·8), the measured pHi varied between 6·3 and 8·7. In fact, pHi correlated with the excretion of pristinamycins and glucose consumption during the production process.
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- Genetics And Molecular Biology
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Expression of the ftsY gene, encoding a homologue of the α subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis
More LessBacillus subtilis FtsY (Srb) is a homologue of the α subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth. The ftsY gene is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor σA. During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of σK and gerE mutants. Therefore, ftsY is solely expressed during sporulation from a σK- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene. To examine the role of FtsY during sporulation, the B. subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state. Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.
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The Vibrio cholerae O1 chromosomal integron
More LessThe GenBank accession numbers for the sequences determined in this work are AF025662, AF055586, X64097, AF179593 and AF179596.
Until the discovery of the V ibrio c holerae repeat (VCR), the gene capture and expression systems termed integrons had been typically associated with antibiotic-resistance gene cassettes with usually less than five genes in an array. A method is described for the cloning of the ends of large cassette arrays. Conserved restriction sites within VCRs facilitated the mapping by Southern hybridization and cloning of the 5’ end of the VCR array, and using appropriate fragments it was possible to develop a physical map of the region of the V. cholerae chromosome. Sequence determination of the predicted beginning of this region revealed intI4, a member of the integron family of integrases. Comparison of these sequences from El Tor, Classical and serotype O134 V. cholerae strains identified the 3’ end of the attI site, thereby defining the class 4 integron in one of the V. cholerae chromosomes, and providing the first evidence for integron-like site-specific recombination within V. cholerae. Conduction assays demonstrated IntI1-mediated recombination between VCRs. Restriction mapping places the sequences of intI4 and 26 VCR gene cassettes in arrays within a 120 kb region of the V. cholerae O1 strain 569B genome. This region contains an estimated 150 VCR gene cassettes, dwarfing previously described arrays. Southern analysis of genomic DNA from strains of Vibrio anguillarum, Vibrio mimicus and a number of V. cholerae serotypes revealed fragments that hybridized with VCR-specific probes but showed a high degree of restriction fragment length polymorphism. These data facilitate the identification of part of a new class 5 integron from V. mimicus.
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The nitric oxide regulated nor promoter of Paracoccus denitrificans
More LessThe promoter of the Paracoccus denitrificans nitric oxide reductase operon (norCBQDEF) has been characterized by primer extension and deletion analysis. A major transcript that is detectable only in anaerobically grown cells initiates 43·5 bp downstream of the centre of a putative binding site for the transcription factor NNR (nitrite and nitric oxide reductase regulator, which is known to regulate nor expression). A minor transcript initiates 121 bp upstream of the major transcript and is detectable in cells grown aerobically or anaerobically. Deletion derivatives of the nor promoter region were constructed and analysed in vivo using transcriptional fusions to the reporter gene lacZ. Expression patterns from promoter deletions in a wild-type strain and an nnr mutant confirmed that the minor transcript is NNR independent, and makes a small contribution to nor expression under both aerobic and anaerobic growth conditions. A deletion derivative truncated to within 7 bp of the putative NNR-binding site showed a near wild-type response to anaerobic growth, showing that no upstream DNA sequences are required for activation of the major promoter. Site-directed mutagenesis of the putative NNR-binding site confirmed that this is the major cis-acting sequence mediating the anaerobic inducibility of nor expression.
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T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis
More LessThe consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli σ70 promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The −10, −16, −42 and −52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the −10, −16 and −35 regions, but also that of position −33, and even more importantly, maintenance of the upstream promoter element at position −42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.
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A mutation which affects both the specificity of PtsG sugar transport and the regulation of ptsG expression by Mlc in Escherichia coli
More LessNormally glucosamine (GlcN) is not a substrate for EIICBGlc of the glucose phosphotransferase system (PTS), encoded by ptsG, but it is transported by the mannose (Man) PTS, encoded by manXYZ. A mutation, umgC, has been described in Escherichia coli which allows a strain mutated in the Man PTS to grow on GlcN. The umgC mutation was mapped to the ptsG region and was proposed to make ptsG expression constitutive. Transcription of ptsG is regulated by the repressor Mlc so that mutations in mlc enhance the expression of ptsG. An mlc mutation, however, is not sufficient to allow good growth on GlcN, unlike the umgC mutation. The umgC mutation is shown to enhance expression of ptsG even in the absence of any PTS sugar transport, but the increase is greater in the presence of GlcN or Man. The umgC mutation also increases expression of the ptsHI and manXYZ operons, which are both regulated by Mlc. The umgC mutation was sequenced and two mutations were found: one, G176D, within the IIC membrane domain and the second, E472K, within the soluble IIB domain of PtsG. The cloned UmgC allele shows the enhanced transport and regulatory characteristics of the chromosomal mutation. Analysis of the two mutations present individually on plasmids shows that the IIC mutation is responsible for both the effect on sugar specificity and regulation.
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Antisense PNA effects in Escherichia coli are limited by the outer-membrane LPS layer
More LessAntisense peptide nucleic acids (PNAs) can inhibit Escherichia coli gene expression and cell growth through sequence-specific RNA binding, and this opens possibilities for novel anti-infective agents and tools for microbial functional genomics. However, the cellular effects of PNAs are limited relative to effects in cell extracts, presumably because of cell barrier components such as the outer-membrane lipopolysaccharide (LPS) layer or drug efflux pumps, both of which function to exclude antibiotics and other foreign molecules. To evaluate the importance of such cellular factors on PNA effects, the authors developed a positive assay for antisense inhibition by targeting the lac operon repressor and compared PNA susceptibilities in mutant and wild-type E. coli by assessing lacZ induction. Strains with defective LPS (AS19 and D22) were more permeable to the antibiotic nitrocefin and more susceptible to PNA than the wild-type. Also, PNA potency was improved in wild-type cells grown in the presence of certain cell-wall-permeabilizing agents. In contrast, the activities of the Acr and Emr drug efflux pumps were not found to affect PNA susceptibility. The results show that the LPS layer is a major barrier against cell entry, but PNAs that can enter E. coli are likely to remain active inside cells.
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Dioctyl phthalate increases the percentage of unsaturated fatty acids with a concomitant decrease in cellular heat shock sensitivity in the yeast Saccharomyces cerevisiae
More LessIn the past it has been reproducibly demonstrated that 37 °C-grown DBY747 yeast cells have 29% more unsaturated fatty acids and a 3 °C higher maximal heat shock response (HSR) than their 25 °C counterparts. Suddenly the HSR and lipid profiles of cells grown at 25 °C and 37 °C became indistinguishable from one another. This paper reports an aberrantly high level of unsaturated fatty acids and an abnormally insensitive HSR in cells grown at 25 °C in yeast nitrogen base (YNB) that has been reconstituted from dehydrated medium packaged in ’new’ plastic containers. Effective even at a 1:600 dilution of reconstituted medium in laboratory-made YNB, the ’active ingredient’ was identified using a combination of HPLC and mass spectroscopy as dioctyl phthalate (a plasticising agent). Furthermore, the same levels of increase in the percentage of unsaturated fatty acids and decrease in the sensitivity of HSR were found in cells grown in laboratory-made YNB that contained as little as 36 μM pure dioctyl phthalate. This compound nevertheless failed to elicit an observable effect on cellular growth rate at levels up to and including 144 μM. These results suggest that dioctyl phthalate causes yeast cells to accumulate high levels of unsaturated fatty acids with a concomitant decrease in the sensitivity of the HSR, without compromising overall cellular function. They also support earlier work that suggested that the HSR is exquisitely sensitive to the level of unsaturated fatty acids present in yeast cells.
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The Aspergillus nidulans cysA gene encodes a novel type of serine O-acetyltransferase which is homologous to homoserine O-acetyltransferases
More LessThe GenBank accession number for the sequence reported in this paper is AF029885.
The Aspergillus nidulans cysA gene was cloned by functional complementation of the cysA1 mutation that impairs the synthesis of O-acetylserine. The molecular nature of cysA1 and cysA103 alleles was characterized; a nucleotide substitution and a frame shift were found in the former and a deletion mutation in the latter. The CYSA protein is 525 amino acids long and is encoded by an uninterrupted open reading frame. Expression of the cysA gene appears not to be regulated by sulfur, carbon and nitrogen sources. Protein sequence analysis reveals extensive similarity to homoserine O-acetyltransferases, particularly the bacterial ones, and no homology with known serine O-acetyltransferases. The authors propose that the CYSA protein is analogous to serine O-acetyltransferases, i.e. it catalyses the same reaction but has an independent evolutionary origin.
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- Genomics
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Genome size and genetic map of Cowdria ruminantium
More LessCowdria ruminantium is the cause of a serious tick-borne disease of domestic ruminants, known as heartwater or cowdriosis. The organism belongs to the tribe Ehrlichieae, which contains obligate intracellular pathogens, causing several important animal and human diseases. Although a few C. ruminantium genes have been cloned and sequenced, very little is known about the size, gross structure and organization of the genome. This paper presents a complete physical map and a preliminary genetic map for C. ruminantium. Chromosomal C. ruminantium DNA was examined by PFGE and Southern hybridization. PFGE analysis revealed that C. ruminantium has a circular chromosome approximately 1576 kb in size. A physical map was derived by combining the results of PFGE analysis of DNA fragments resulting from digestion of the whole genome with KspI, RsrII and SmaI and Southern hybridization analysis with a series of gene probes and isolated macrorestriction fragments. A genetic map for C. ruminantium with a mean resolution of 290 kb was established, the first for a member of the Ehrlichieae. A total of nine genes or cloned C. ruminantium DNA fragments were mapped to specific KspI, RsrII and SmaI fragments, including the major antigenic protein gene, map-1.
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- Pathogenicity And Medical Microbiology
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Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase
More LessThe GenBank accession numbers for the nucleotide sequences for the C. neoformans mannitol-1-phosphate dehydrogenase cDNA and gene are AF175685 and AF186474, respectively.
Cryptococcus neoformans, the causative agent of cryptococcosis, produces large amounts of mannitol in culture and in infected mammalian hosts. Although there is considerable indirect evidence that mannitol synthesis may be required for wild-type stress tolerance and virulence in C. neoformans, this hypothesis has not been tested directly. It has been proposed that mannitol-1-phosphate dehydrogenase (MPD) is required for fungal mannitol synthesis, but no MPD-deficient fungal mutants or cDNAs or genes encoding fungal MPDs have been described. Therefore, C. neoformans was purified from a 148 kDa homotetramer of 36 kDa subunits that catalysed the reaction mannitol1-phosphate+NAD→←fructose 6-phosphate+NADH. Partial peptide sequences were used to isolate the corresponding cDNA and gene, and the deduced MPD protein was found to be homologous to the zinc-containing long-chain alcohol/polyol dehydrogenases. Lysates of Saccharomyces cerevisiae transformed with the cDNA of interest (but not vector-transformed controls) contained MPD catalytic activity. Lastly, Northern analyses demonstrated MPD mRNA in glucose- and mannitol-grown C. neoformans cells. Thus, MPD has been purified and characterized from C. neoformans, and the corresponding cDNA and gene (MPD1) cloned and sequenced. Availability of C. neoformans MPD1 should permit direct testing of the hypotheses that (i) MPD is required for mannitol biosynthesis and (ii) the ability to synthesize mannitol is essential for wild-type stress tolerance and virulence.
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- Physiology And Growth
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Mitochondrial respiratory mutants of Saccharomyces cerevisiae accumulate glycogen and readily mobilize it in a glucose-depleted medium
More LessMutant strains of Saccharomyces cerevisiae defective in respiration have been reported to be unable to store glycogen, as revealed by the iodine-staining method. In this report, it is shown that in contrast to this claim, mitochondrial respiratory mutants accumulated even more glycogen than wild-type cells during the fermentative growth on glucose. However, as soon as glucose was exhausted in the medium, these mutants readily and completely mobilized their glycogen content, contrary to wild-type cells which only transiently degraded this polymer. The mobilization of glycogen was a specific trait resulting from a defect in mitochondrial function that could not be suppressed by mutations in the cAMP- and Pho85 protein kinase-dependent nutrient-sensing pathways, and by other mutations which favour glycogen synthesis. To account for this mobilization, it was found that respiration-defective cells not only contained a less active glycogen synthase, but also a more active glycogen phosphorylase. Since glucose 6-phosphate (Glc6P) is a potent inhibitor of the phosphorylation and an activator of the dephosphorylation processes of glycogen synthase and glycogen phosphorylase, it is suggested that the drop in Glc6P observed at the onset of glucose depletion in respiration-deficient cells triggers this rapid and sustained glycogen mobilization. It is also proposed that this degradation provides the energy for the viability of respiratory mutants in glucose-starved medium.
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- Pseudomonas: Biology And Diversity
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An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures
Using the complete genome sequence of Pseudomonas aeruginosa PAO1, sequenced by the Pseudomonas Genome Project (ftp://ftp.pseudomonas.com/data/pacontigs.121599), a genome database (http://pseudomonas.bit.uq.edu.au/) has been developed containing information on more than 95% of all ORFs in Pseudomonas aeruginosa. The database is searchable by a variety of means, including gene name, position, keyword, sequence similarity and Pfam domain. Automated and manual annotation, nucleotide and peptide sequences, Pfam and SMART domains (where available), Medline and GenBank links and a scrollable, graphical representation of the surrounding genomic landscape are available for each ORF. Using the database has revealed, among other things, that P. aeruginosa contains four chemotaxis systems, two novel general secretion pathways, at least three loci encoding F17-like thin fimbriae, six novel filamentous haemagglutinin-like genes, a number of unusual composite genetic loci related to vgr/Rhs elements in Escherichia coli, a number of fix-like genes encoding a micro-oxic respiration system, novel biosynthetic pathways and 38 genes containing domains of unknown function (DUF1/DUF2). It is anticipated that this database will be a useful bioinformatic tool for the Pseudomonas community that will continue to evolve.
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Monitoring genome evolution ex vivo: reversible chromosomal integration of a 106 kb plasmid at two tRNALys gene loci in sequential Pseudomonas aeruginosa airway isolates
More LessThe GenBank accession numbers for the sequences reported in this paper are AF285416–AF285426.
The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics. Restriction mapping uncovered an 8·9 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus. These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P. aeruginosa clone C isolates. The breakpoints of the deletion in clone K and the attB–attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3’ end of the lysine tRNA structural genes (att site). pKLK106 sequentially recombined with either of the two tRNALys genes in clone K isolates. The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome; the att site of the phnAB–oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements. The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA. The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.
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Phylogeny of the replication regions of pPT23A-like plasmids from Pseudomonas syringae
More LessThe EMBL accession numbers for the sequences reported in this paper are AJ276998–AJ277021.
It was previously shown that most Pseudomonas syringae strains contain one or more plasmids with cross-hybridizing replication regions and other areas of homology, and these plasmids were designated the pPT23A-like family. The majority of these plasmids encode genes conferring epiphytic fitness or resistance to antibacterial compounds and those investigated in this study are essential for pathogenicity or increased virulence. The phylogeny of 14 pPT23A-like plasmids from five P. syringae pathovars was studied by comparing a fragment of the sequence of their repA genes (encoding a replicase essential for replication). In the phylogenetic tree obtained, four groups (≤88·8% identity between their members) could be identified. The first group contained the plasmids from three P. syringae pv. tomato strains, a P. syringae pv. apii strain and five out of the seven P. syringae pv. syringae strains, with identity ranging between 88·8 and 100%. The clustering of the pv. syringae strains did not reflect host specialization or previously reported phylogenetic relationships. The second group contained the plasmids from two strains of pv. glycinea and pv. tomato (95·5% identity), and it also included the previously sequenced replicon of a pathogenicity plasmid from P. syringae pv. phaseolicola. The plasmids from the remaining two pv. syringae strains were distantly related to the other plasmid sequences. Hybridization experiments using different genes or transposable elements previously described as plasmid-borne in P. syringae, showed that the gene content of highly related plasmids could be dissimilar, suggesting the occurrence of major plasmid reorganizations. Additionally, the phylogeny of the different native plasmids did not always correlate with the phylogeny of their harbouring strains, as determined by the analysis of extragenic repetitive consensus (ERIC) and arbitrarily primed PCR (AP-PCR) products. Collectively, these results suggest that pPT23A-like plasmids were, in most cases, acquired early during evolution.
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Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes
The GenBank accession numbers for the sequences determined in this work are: gyrB, D37926, D37297, D86005–D86019 and AB039381–AB039492; rpoD, D86020–D86036 and AB039493–AB039624.
Phylogenetic analysis of the genus Pseudomonas was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas (a total of 125 strains). Pseudomonas strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the ‘P. aeruginosa complex’, which included P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, P. oleovorans and P. pseudoalcaligenes, and the ‘P. stutzeri complex’, which included P. balearica and P. stutzeri. IGC II was further split into three subclusters that were designated the ‘P. putida complex’, the ‘P. syringae complex’ and the ‘P. fluorescens complex’. The ‘P. putida complex’ included P. putida and P. fulva. The ‘P. syringae complex’ was the cluster of phytopathogens including P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava and the pathovars of P. savastanoi and P. syringae. The ‘P. fluorescens complex’ was further divided into two subpopulations, the ‘P. fluorescens lineage’ and the ‘P. chlororaphis lineage’. The ‘P. fluorescens lineage’ contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the ‘P. chlororaphis lineage’ included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the ‘P. fluorescens complex’.
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Quantification of biofilm structures by the novel computer program comstat
The structural organization of four microbial communities was analysed by a novel computer program, COMSTAT, which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria, Pseudomonas putida, P. aureofaciens, P. fluorescens and P. aeruginosa, tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure – mean thickness, roughness, substratum coverage and surface to volume ratio – showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum, and formed flat, uniform biofilms. P. aureofaciens resembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0·03 mM, 0·1 mM or 0·5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover, biofilm roughness increased with lower citrate concentrations, whereas surface to volume ratio increased with higher citrate concentrations.
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Experimental reproducibility in flow-chamber biofilms
The structural organization of microbial communities is influenced by many factors, e.g. nutrient composition, shear stress and temperature. This paper presents a general method for quantitative comparison of biofilm structures and assessment of experimental reproducibility between independent biofilm experiments. By using a novel computer program, COMSTAT, biofilm structures of Pseudomonas aeruginosa and an isogenic rpoS mutant were quantified. The strains were tagged with the green fluorescent protein (GFP) and grown in flow chambers with a defined minimal medium as substrate. Three independent rounds of biofilm experiments were performed and in each round, each of the two variants was grown in two separate channels. Nine image stacks were acquired in each channel 146 h after inoculation. An analysis of variance model incorporating the factors experiment round, bacterial strain, channel number and image stack number was used to analyse the data calculated by COMSTAT. Experimental reproducibility was verified by estimating the magnitude of the variance of the effects round () and the interaction between bacterial strain and round (). Mean thickness of the wild-type and rpoS mutant biofilms was estimated at 6·31 μm (SE 0·81 μm) and 16·85 μm (SE 0·87 μm), respectively.
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Iron regulation of the hcnABC genes encoding hydrogen cyanide synthase depends on the anaerobic regulator ANR rather than on the global activator GacA in Pseudomonas fluorescens CHA0
More LessPseudomonas fluorescens CHA0 produces hydrogen cyanide (HCN), a secondary metabolite that substantially contributes to this strain’s biocontrol ability. Cyanogenesis is induced by oxygen-limiting conditions, but abolished by iron depletion. In P. fluorescens, the anaerobic regulator ANR and the global activator GacA are both required for the maximal expression of the HCN biosynthetic genes hcnABC. The molecular basis of this regulation by ANR and GacA was investigated under conditions of oxygen and iron limitation. A promoter deletion analysis using a translational hcnA′–′lacZ fusion revealed that a conserved FNR/ANR recognition sequence in the −40 promoter region was necessary and sufficient for the regulation by ANR in response to oxygen limitation. Stimulation of hcnA′–′lacZ expression by the addition of iron also depended on the presence of ANR and the FNR/ANR box, but not on GacA, suggesting that in addition to acting as an oxygen-sensitive protein, ANR also responds to iron availability. Expression of the translational hcnA′–′lacZ fusion remained GacA-dependent in hcn promoter mutants that were no longer responsive to ANR, in agreement with earlier evidence for a post-transcriptional regulatory mechanism under GacA control. These data support a model in which cyanogenesis is sequentially activated by ANR at the level of transcription and by components of the GacA network at the level of translation.
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Vanadium interferes with siderophore-mediated iron uptake in Pseudomonas aeruginosa
Vanadium is a metal that under physiological conditions can exist in two oxidation states, V(IV) (vanadyl ion) and V(V) (vanadate ion). Here, it was demonstrated that both ions can form complexes with siderophores. Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdine (PVD) and pyochelin (PCH). Vanadyl sulfate, at a concentration of 1–2 mM, strongly inhibited growth of P. aeruginosa PAO1, especially under conditions of severe iron limitation imposed by the presence of non-utilizable Fe(III) chelators. PVD-deficient mutants were more sensitive to vanadium than the wild-type, but addition of PVD did not stimulate their growth. Conversely, PCH-negative mutants were more resistant to vanadium than the wild-type strain. Both siderophores could bind and form complexes with vanadium after incubation with vanadyl sulfate (1:1, in the case of PVD; 2:1, in the case of PCH). Although only one complex with PVD, V(IV)–PVD, was found, both V(IV)– and V(V)–PCH were detected. V–PCH, but not V–PVD, caused strong growth reduction, resulting in a prolonged lag phase. Exposure of PAO1 cells to vanadium induced resistance to the superoxide-generating compound paraquat, and conversely, exposure to paraquat increased resistance to V(IV). Superoxide dismutase (SOD) activity of cells grown in the presence of V(IV) was augmented by a factor of two. Mutants deficient in the production of Fe-SOD (SodB) were particularly sensitive to vanadium, whilst sodA mutants deficient for Mn-SOD were only marginally affected. In conclusion, it is suggested that V–PCH catalyses a Fenton-type reaction whereby the toxic superoxide anion is generated, and that vanadium compromises PVD utilization.
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Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor
More LessFerric iron is an essential element for microbial growth but its water solubility in aerobic environments is considered to be low. Thus it is a limiting resource for which microbes must compete in natural habitats. Since competition for iron occurs at the level of individual cells, knowledge of the variability in iron bioavailability to such individuals is required to assess the nature of the competition in these habitats. Ferric iron availability to cells of Pseudomonas syringae was assessed by quantifying the fluorescence intensity of single cells harbouring a plasmid-borne transcriptional fusion of an iron-regulated promoter from a locus encoding a membrane receptor for a pyoverdine siderophore with a reporter gene encoding green fluorescent protein (GFP) following fluorescence microscopy. Cells of this iron biosensor exhibited iron-dependent GFP fluorescence that was inversely proportional to the amount of iron added to the media, and which differed by over 20-fold in iron-replete compared to iron-deplete culture media. Cells cultured in a medium of a given iron content exhibited a very narrow range of fluorescence intensities. In contrast, the fluorescence intensity of cells of the biosensor strain recovered from the rhizosphere or phylloplane of inoculated bean plants varied greatly. The distribution of fluorescence intensities was strongly right-hand skewed, with about 10% of the cells exhibiting substantially higher GFP fluorescence than that of the median cell. Cells of a positive control strain, harbouring a fusion of the constitutive nptII promoter with the gfp reporter gene, exhibited uniform GFP fluorescence both in culture media and on plants. These results indicate that there is substantial heterogeneity of iron biovailability to cells of P. syringae on plants, with only a small subset of cells experiencing low iron availability. Such heterogeneity places constraints on models of interactions of bacteria in natural habitats that are based on competition for limited iron.
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Regulatory interactions between the Hrp type III protein secretion system and coronatine biosynthesis in Pseudomonas syringae pv. tomato DC3000
More LessIn P. syringae, the co-ordinated regulation of different systems required for pathogenicity and virulence seems logical but has not been established. This question was addressed in the present study by analysing production of the phytotoxin coronatine (COR) in defined hrp/hrc mutants of P. syringae pv. tomato DC3000. COR was produced in vitro by mutants of DC3000 defective in hrcC, which encodes an outer-membrane protein required for type III-mediated secretion. When inoculated in plants, hrcC mutants produced chlorotic regions indicative of COR production, but lacked the necrotic lesions produced by the wild-type DC3000. Furthermore, a DC3000 mutant containing a polar mutation in hrcC, which inactivates hrcC, hrpT and hrpV, produced significantly higher amounts of COR than the wild-type strain in vitro. This mutant was able to produce COR earlier and at lower cell densities than the wild-type. The results indicate that the hrp/hrc secretion system is not required for COR production, but mutations in this system may have regulatory effects on the production of virulence factors such as COR.
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Temperature-responsive genetic loci in the plant pathogen Pseudomonas syringae pv. glycinea
More LessThe GenBank accession numbers for the nucleotide sequences of mutants 560, 561, 562, 563, 564, 568, 570, 574, 590, 591, 593, 596, 599, 601, 605, 608, 613, 617, 618, 626 and 632 determined in this work are AF274322–AF274342, respectively.
Plant-pathogenic bacteria may sense variations in environmental factors, such as temperature, to adapt to plant-associated habitats during pathogenesis or epiphytic growth. The bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, preferentially produces the phytotoxin coronatine at 18 °C and infects the host plant under conditions of low temperature and high humidity. A miniTn5-based promoterless glucuronidase (uidA) reporter gene was used to identify genetic loci of PG4180 preferentially expressed at 18 or 28 °C. Out of 7500 transposon mutants, 61 showed thermoregulated uidA expression as determined by a three-step screening procedure. Two-thirds of these mutants showed an increased reporter gene expression at 18 °C whilst the remainder exhibited higher uidA expression at 28 °C. MiniTn5-uidA insertion loci from these mutants were subcloned and their nucleotide sequences were determined. Several of the mutants induced at 18 °C contained the miniTn5-uidA insertion within the 32·8 kb coronatine biosynthetic gene cluster. Among the other mutants with increased uidA expression at 18 °C, insertions were found in genes encoding formaldehyde dehydrogenase, short-chain dehydrogenase and mannuronan C-5-epimerase, in a plasmid-borne replication protein, and in the hrpT locus, involved in pathogenicity of P. syringae. Among the mutants induced at 28 °C, insertions disrupted loci with similarities to a repressor of conjugal plasmid transfer, UV resistance determinants, an isoflavanoid-degrading enzyme, a HU-like DNA-binding protein, two additional regulatory proteins, a homologue of bacterial adhesins, transport proteins, LPS synthesis enzymes and two proteases. Genetic loci from 13 mutants did not show significant similarities to any database entries. Results of plant inoculations showed that three of the mutants tested were inhibited in symptom development and in planta multiplication rates. Temperature-shift experiments suggested that all of the identified loci showed a rather slow induction of expression upon change of temperature.
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The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase
The GenBank accession number for the sequence determined in this work is AF286536.
Several different species of Pseudomonas produce N-acylhomoserine lactones (AHLs), quorum-sensing signal molecules which are involved in the cell-density-dependent control of secondary metabolite and virulence gene expression. When Pseudomonas fluorescens F113 was cross-streaked against AHL biosensors capable of sensitively detecting either short (C4–C8) or long (C10–C14) acyl chain AHLs, no activity was detectable. However, by extracting cell-free stationary-phase culture supernatants with dichloromethane followed by reverse-phase HPLC, three distinct fractions were obtained capable of activating the AHL biosensors. Three AHLs were subsequently characterized using high-resolution MS and chemical synthesis. These were (i) N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3OH,C14:1-HSL), a molecule previously known as the Rhizobium leguminosarum small bacteriocin as a consequence of its growth inhibitory properties, (ii) N-decanoylhomoserine lactone (C10-HSL) and (iii) N-hexanoylhomoserine lactone (C6-HSL). A gene (hdtS) capable of directing synthesis of all three P. fluorescens AHLs in Escherichia coli was cloned and sequenced. In vitro transcription/translation of hdtS yielded a protein of approximately 33 kDa capable of directing the synthesis of 3OH,C14:1-HSL, C10-HSL and C6-HSL in E. coli. HdtS does not belong to either of the known AHL synthase families (LuxI or LuxM) and is related to the lysophosphatidic acid acyltransferase family. HdtS may therefore constitute a member of a third protein family capable of AHL biosynthesis.
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Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa
The pathogenesis of Pseudomonas aeruginosa is associated with expression of virulence factors, many of which are controlled by two N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Escherichia coli strains equipped with a luxR-based monitor system expressing green fluorescent protein (GFP) in the presence of exogenous AHL molecules were used to detect the production of AHLs from P. aeruginosa in vivo. Mice were challenged intratracheally with alginate beads containing P. aeruginosa and E. coli and killed on different days after the challenge. By means of confocal scanning laser microscopy, GFP-expressing E. coli bacteria could be detected in the lung tissues, indicating production and excretion of AHL molecules in vivo by the infecting P. aeruginosa. AHL signals were detected mainly in lung tissues exhibiting severe pathological changes. These findings support the view that expression of AHL molecules by P. aeruginosa during infection coincides with its pathogenesis.
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Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography–tandem mass spectrometry
More LessIsolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography–tandem mass spectrometry (μLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by μLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.
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migA, a quorum-responsive gene of Pseudomonas aeruginosa, is highly expressed in the cystic fibrosis lung environment and modifies low-molecular-mass lipopolysaccharide
Pseudomonas aeruginosa is an opportunistic human pathogen which poses a major threat to patients with cystic fibrosis (CF). Excessive amounts of mucus present in the lungs of CF patients promotes the colonization of P. aeruginosa. The migA gene, encoding a putative glycosyltransferase, has been shown to be highly inducible by respiratory mucus derived from CF patients. In this study, it is further demonstrated by population transcript analysis that the migA gene is highly expressed in the CF lung environment. Deletion analysis of the migA promoter identified a las-box-like sequence commonly found in promoters that are responsive to quorum sensing regulation. Further analysis of migA expression in quorum-sensing-defective strains, as well as its expression in response to autoinducer molecules, demonstrated that migA is regulated by the RhlI/RhlR quorum sensing regulatory system. Functionally, as the MigA sequence homology data suggested, the migA gene indeed affects the structure of LPS in P. aeruginosa. Increased expression of the migA gene results in a loss of core-plus-one LPS, while having no obvious effect on the long-chain O-antigen-bearing LPS. Although the exact biological role of the core-plus-one LPS is not clear, these experimental results suggest that migA up-regulation in the CF lung environment is part of the adaptive response which confers on P. aeruginosa a survival advantage.
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Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis
More LessA nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5′-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6–16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c 551, were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c 551, they underwent extensive cell death due to induction of apoptosis.
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Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of ExoS
More LessPseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Using a tissue culture system, invasive strains of P. aeruginosa were discovered to induce apoptosis at high frequency in HeLa and other epithelial and fibroblast cell lines. This apoptotic phenotype in the infected cells was determined by several criteria including (i) visual changes in cell morphology, (ii) induction of chromatin condensation and nuclear marginalization, (iii) the presence of a high percentage of cells with subG1 DNA content, and (iv) activation of caspase-3 activity. Induction of the type III secretion machinery, but not invasion of P. aeruginosa is required for induction of apoptosis. The apoptosis phenotype is independent of the cytoskeletal rearrangements that occur in the host cell early after infection. Mutants in P. aeruginosa exoS fail to induce apoptosis and complementation with wild-type exoS restored the apoptosis-inducing capacity, demonstrating that ExoS is the effector molecule. Analysis of exoS activity mutants shows that the ADP-ribosylating capacity of ExoS is essential for inducing the apoptotic pathway.
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Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides
More LessResistance to the polycationic antibiotic polymyxin B and expression of the outer-membrane protein OprH in the opportunistic pathogen Pseudomonas aeruginosa both involve the PhoP-PhoQ two-component regulatory system. The genes for this system form an operon with oprH, oprH-phoP-phoQ, that responds to Mg2+ starvation and PhoP levels. In this study, the Mg2+-regulated promoter for this operon was mapped upstream of oprH by primer-extension experiments. An oprH::xylE-GmR mutant H855 was constructed and measurement of the catechol 2,3-dioxygenase activity expressed from this transcriptional fusion provided evidence for a second, weak promoter for phoP-phoQ. Wild-type P. aeruginosa PAO1 strain H103 was found to exhibit Mg2+-regulated resistance to the α-helical antimicrobial cationic peptide CP28 in addition to its previously characterized resistance to polymyxin B. Resistance to this peptide was unchanged in the OprH-null mutant H855 and a PhoP-null mutant H851. In contrast, PhoQ-null mutant H854 demonstrated constitutive CP28 resistance. Northern blot analysis revealed constitutive expression of phoP in this strain, implicating PhoP-PhoQ in the resistance of P. aeruginosa to cationic peptides. Furthermore, all three null-mutant strains demonstrated increased resistance to the aminoglycoside antibiotics streptomycin, kanamycin and amikacin. Two additional mutant strains, H895 and H896, were constructed that carried unmarked deletions in oprH and were found to exhibit aminoglycoside susceptibility equivalent to that of the wild-type. This result provided definitive evidence that OprH is not involved in P. aeruginosa aminoglycoside resistance and that the changes in resistance in strain H855 and a previously reported oprH mutant were due to polar effects on phoP-phoQ rather than loss of OprH expression. A role for PhoP-PhoQ in resistance to aminoglycosides is envisaged that is distinct from that in resistance to cationic peptides and polymyxin B.
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Visualization of DNA–protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida
More LessThe ATP-dependent multimerization process undergone by the σ54-dependent activator XylR of the TOL plasmid pWW0 of Pseudomonas putida when bound to the upstream activating sequences (UAS) of the cognate Pu promoter was examined by transmission electron microscopy (TEM). To this end, supercoiled DNA templates were combined with increasing concentrations of the constitutive XylR variant XylRΔA, with or without ATP or its non-hydrolysable analogue ATPγS, and the resulting complexes were visualized by TEM. The different types of DNA–protein association were analysed and a statistical study of the frequency of the various forms was made. ATP appeared to establish an equilibrium between different molecular associations, as well as major changes in the physical shape of the DNA–protein complexes. The formation of higher nucleoprotein structures frequently bearing DNA bends became manifest. Such complexes often engaged otherwise separated UAS-containing plasmids, indicating that the ATP-driven multimer included XylR molecules recruited in trans. Whilst ATP caused the different types of XylR–DNA complex to occur at quite balanced frequencies, ATPγS appeared to displace the distribution predominantly towards the higher order forms. These data are compatible with the notion that each time ATP is hydrolysed the transcriptional activation complex is disassembled.
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Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide–enzyme complex
More LessThe propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes elastase, in Pseudomonas putida did not result in extracellular elastase activity, it has been suggested that the enzyme is not recognized by the Xcp secretion machinery of the heterologous host. Here, it is demonstrated that the proenzyme is normally processed in P. putida and that it is indeed not actively secreted by the Xcp machinery. Nevertheless, substantial amounts of the enzyme were detected in the extracellular medium. Co-immunoprecipitations revealed that the extracellular enzyme was associated with the propeptide, which explains the lack of enzymic activity. Since the propeptide–enzyme complex in P. putida apparently does not dissociate spontaneously, it is concluded that a host-specific factor is required to induce this event. Mutants were selected which showed extracellular elastase activity. Two mutations, located within the lasB gene, were further characterized. These mutations, resulting in the substitution of Ala and Thr at positions −15 and −153, respectively, of the propeptide (where position +1 is defined as the first residue of the mature enzyme) destabilized the propeptide–enzyme complex. It is concluded that Ala-15 and Thr-153 are required for the inhibitor function, but not for the chaperone function of the propeptide.
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- Systematics And Evolution
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Genetic relationships between clinical and environmental Vibrio cholerae isolates based on multilocus enzyme electrophoresis
More LessA total of 107 isolates of Vibrio cholerae, including 29 strains belonging to serogroup O139, were studied using multilocus enzyme electrophoresis (MLEE) to determine allelic variation in 15 housekeeping enzyme loci. All loci were polymorphic and 99 electrophoretic types (ETs) were identified from the total sample. No significant clustering of isolates was detected in the dendrogram generated from a matrix of coefficients of distances with respect to serogroup, biotype or country of isolation. The mean genetic diversity of this V. cholerae population (H=0·50) was higher than reported previously. Linkage disequilibrium analysis of the MLEE data showed a clonal structure for the entire population, but not in some of the population subgroups studied. This suggests an epidemic population structure. The results showed that the O139 strains were not clustered in a unique ET, in contrast to previous MLEE studies. This higher genetic variation of the O139 serogroup is concordant with ribotyping studies. The results also confirm that the O139 and O1 ElTor isolates are genetically more closely related to each other than to all the other subpopulations of V. cholerae studied.
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