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Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.
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PMID:Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. 131 May 1

A new procedure is described to recombine plasmid-borne lacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flanking lacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncated lacZ and aadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carrying lacZ and aadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy in Pseudomonas putida the regulatory elements controlling expression of the XylS-activated Pm promoter of the TOL catabolic plasmid pWWO. We show here that the Pm promoter undergoes a XylS-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.
Mol Gen Genet 1992 May
PMID:A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. 131 99

A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.
Mol Microbiol 1992 Jul
PMID:Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. 132 24

Pseudomonas viridiflava is a soft-rotting pathogen of harvested vegetables that produces an extracellular pectate lyase (PL) responsible for maceration of plant tissue. A pel gene encoding PL was cloned from the genome of strain SJ074 and efficiently expressed in Escherichia coli. After a series of deletion subclonings and analysis by transposon mutagenesis, the pel gene was located in a 1.2-kb PstI-BglII genomic fragment. This fragment appears to contain a promoter at the PstI end required for pel gene expression. The PL produced by pectolytic E. coli clones is identical to those produced by strain SJ074 and by other strains of P. viridiflava in terms of molecular weight (42 kDa) and pI (9.7). A mutant of strain SJ074, designated MEI, which had Tn5 specifically inserted in the pel locus was constructed by site-directed mutagenesis. The MEI mutant produced 70- to 100-fold less PL than the wild type and failed to cause tissue maceration in plants. PL production and soft-rot pathogenicity in MEI and in a Pel- mutant previously isolated from strain SF312 were restored to the wild-type level by complementation in trans with the cloned pel gene. By using the 1.2-kb fragment as a probe, pel homologs were detected in four bacteria that are pathologically unrelated to P. viridiflava. These include three pathovars of P. syringae (pv. lachrymans, pv. phaseolicola, and pv. tabaci) and Xanthomonas campestris pv. malvacearum. No DNA fragments showing homology to pel of P. viridiflava were detected in genomic digests prepared from two strains of soft-rot erwinias.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Plant Microbe Interact
PMID:Cloning and characterization of a pectate lyase gene from the soft-rotting bacterium Pseudomonas viridiflava. 132 18

Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression. Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain. Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants. Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids. Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics. These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.
Mol Plant Microbe Interact
PMID:Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84. 132 19

Anaerobic growth of Pseudomonas aeruginosa on arginine depends on the arcDABC operon encoding the enzymes of the arginine deiminase pathway. The co-ordinate, anaerobic induction of these enzymes requires the FNR-like regulatory protein ANR, which activates the arc promoter lying upstream from arcD. By Northern hybridization experiments, three abundant arcA, arcAB and arcABC transcripts and three minor arcDA, arcDAB and arcDABC transcripts could be detected. The 5' ends of the arcA, arcAB and arcABC mRNAs were determined by S1 and primer extension mapping. These 5' ends appear to be generated by endonucleolytic cleavage (processing) in arcD mRNA rather than by a second promoter; this was concluded from the effects of insertion and deletion mutations in arcD. Intergenic inverted repeats between arcA and arcB as well as between arcB and arcC were shown to be involved in the formation of 3' ends of arc transcripts. Deletion of either intergenic region in the P. aeruginosa chromosome led to the loss of the arcA or arcAB transcript, respectively. Dot blot experiments revealed that arc mRNAs extracted from the wild-type strain had similar chemical half-lives in the arcA, arcB and arcC regions, ranging from 16 to 13 minutes. The half-life of arcD mRNA, by contrast, was significantly shorter, suggesting that this mRNA segment may be destabilized by the processing cuts within arcD. Deletion of the putative intergenic stem-loop structures did not result in a dramatic loss of arc mRNA stability. Thus, the intergenic hairpin structures do not contribute importantly to the overall mRNA stability; they might act primarily as partial transcription terminators and locally protect the 3' ends from exonuclease action. The expression levels of the four Arc proteins correlated approximately with the relative abundance of the corresponding mRNA segments. In conclusion, mRNA processing and, presumably, partial termination of transcription contribute to differential gene expression within the arc operon.
J Mol Biol 1992 Aug 20
PMID:RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa. 132 63

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri (ATCC 17588). Resonance assignments are proposed for all main-chain and most side-chain protons. Distance constraints were determined on the basis of nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scaler coupling constants. Twenty-four structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1033 interproton distance and 57 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for a loop region around residues 34-40, the first two residues at the N-terminus and the last two at the C-terminus, and some side chains located on the molecular surface. The average root mean squared deviation in position for equivalent atoms between the 24 individual structures and the mean structure obtained by averaging their coordinates is 0.54 +/- 0.08 A for the main-chain atoms and 0.97 +/- 0.09 A for all non-hydrogen atoms of residues 3-80 plus the heme group. These structures were compared to the X-ray crystallographic structure of an analogous protein, cytochrome c-551 from Pseudomonas aeruginosa [Matsuura, Takano, & Dickerson (1982) J. Mol. Biol. 156, 389-409). The main-chain folding patterns are very consistent, but there are some differences. The largest difference is in a surface loop segment from residues 34 to 40.
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PMID:Investigation of the solution conformation of cytochrome c-551 from Pseudomonas stutzeri. 132 5

Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrc genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed-field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.
Mol Microbiol 1992 Nov
PMID:Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes. 133 66

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.
Mol Ecol 1992 Aug
PMID:Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2,4-dichlorophenoxyacetate. 134 88

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.
Mol Ecol 1992 Oct
PMID:Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain. 134 90


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