Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The epidemiology of Pseudomonas aeruginosa infections and colonizations was studied prospectively on a 12-bed medical intensive care unit. Patients were monitored for P. aeruginosa colonization by performing throat swabs or tracheal aspirates on admission and weekly thereafter over a period of 6 months. Cultures of possibly infected sites were taken as clinically indicated. Water samples from all patient care-related tap water outlets were collected in 2-weekly intervals and examined for the presence of P. aeruginosa. Strains isolated from patients and water samples were analysed by serotyping and random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing. During the 6-month period, 60 of 143 (42%) water samples contained P. aeruginosa at various levels ranging from 1 to >100 colony-forming units per 100ml sample. Genotypically, water samples contained 8 different clonotypes. Nine patients had infections due to P. aeruginosa and 7 patients were colonized. Isolates from patients showed a similar distribution of genotypes as did tap water isolates, and strains of identical genotype as patient strains had been isolated previously from tap water outlets in 8 out of 16 (50%) infection or colonization episodes. However, patients also harboured strains not previously isolated from tap water. Thus, in addition to tap water, other environmental or unknown reservoirs appeared to play a role for the epidemiology of P. aeruginosa infections on this ward. However, because tap water played a significant role for strain transmissions, we conclude that intensified water site care is justified.
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PMID:Common RAPD pattern of Pseudomonas aeruginosa from patients and tap water in a medical intensive care unit. 1674 Apr 15

Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.
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PMID:Role of Pseudomonas aeruginosa dinB-encoded DNA polymerase IV in mutagenesis. 1704 Oct 45

One hundred clinical isolates of Pseudomonas aeruginosa were serologically classified into 7 Jordanian serotypes (labeled JO1-JO7) Odeh, 2002, M.Sc. thesis, University of Jordan). Using the slide agglutination test, 4 of them (JO4, JO5, JO6, and JO7) were serologically matched with the International Antigenic Typing Scheme (IATS) strains (IATS 20, IATS 10, IATS 6, and IATS 11). One serotype (JO1) showed a weak cross-reaction with IATS 1. The remaining 2 local serotypes (JO2 and JO3) did not react with any of the 20 IATS strains. Serologic analysis data showed to a certain extent correlations with molecular data using genetic clustering and similarity indices generated by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Each of the 4 identified local serotypes formed a cluster with its serologically matched IATS strain with relatively high average similarity indices, whereas lower average similarity index was observed between IATS 1 and JO1, in consistence with the weak serologic reaction using the slide agglutination test. On the other hand, the 2 nontypeable serotypes (JO2 and JO3) formed 2 separate clusters that could not be matched to any of the IATS strain. Phenotypic and genotypic analyses suggest that JO2 and JO3, and possibly JO1, can be new serotypes of P. aeruginosa. RAPD-PCR was also used to study the relative relatedness among the 20 IATS strains. The IATS strains formed 2 main clusters. Half of the IATS strains formed one main cluster that included IATS 11-20. The remaining IATS strains (8 strains) formed the second main cluster, with the exception of IATS 4 and 9, where each formed a separate cluster.
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PMID:Serologic and molecular characterization of Pseudomonas aeruginosa Jordanian clinical isolates compared with the strains of International Antigenic Typing Scheme. 1750 95

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria.
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PMID:Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida. 1754 88

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida. The genome of this organism contains two uvrA genes, uvrA and uvrA2. Genetic studies on the effects of uvrA, uvrA2, uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida. However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB, uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2, uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida-besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.
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PMID:Dual role of NER in mutagenesis in Pseudomonas putida. 1772 Jun 31

The virulent Pseudomonas aeruginosa bacteriophage LUZ24 (45,625 bp) was isolated from hospital sewage. It belongs to the family of the Podoviridae, and carries a bidirectionally transcribed dsDNA genome delineated by two direct terminal repeats of 184 bp. In vitro transcriptional analysis identified seven sigma(70) promoters, revealing a bias towards stronger promoter strength in the late genomic region. Reverse transcription demonstrated in vivo splicing of a 668 bp Group I intron embedded inside the DNA polymerase gene. Using mass spectrometry, nine structural proteins were identified as part of the phage particle. The lytic characteristics of LUZ24 are evaluated against its genomic content, which displays an overall 71% sequence similarity to the temperate phage PaP3.
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PMID:The intron-containing genome of the lytic Pseudomonas phage LUZ24 resembles the temperate phage PaP3. 1851 45

A polymerase chain reaction-based approach was developed for species identification of Pseudomonas strains and for the direct detection of Pseudomonas populations in their natural environment. A highly selective set of primers (PsEG30F and PsEG790R), giving an amplicon of 760 nucleotides in length, was designed based on the internal conserved sequences of 33 selected rpoD gene sequences (the sigma 70 factor subunit of the DNA polymerase) of Pseudomonas type strains, representing the entire intrageneric phylogenetic clusters described in the genus. The utility of the primer set was verified on 96 Pseudomonas type strains and on another 112 recognised Pseudomonas strains. The specificity of the primer set was also tested against strains from species not belonging to the genus Pseudomonas. These primers were also shown to be useful for the direct detection of Pseudomonas species in environmental DNA after a cloning procedure. These results were compared in parallel with other cloning procedures described previously, based on the analysis of other genes (16S rDNA and ITS1) and also by using primers designed for rpoD on sequences from gamma-proteobacteria. All of the cultured Pseudomonas strains tested could be amplified with these novel primers, indicating that this method is also a useful tool for the specific analysis of Pseudomonas populations from environmental samples without the need for cultivation.
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PMID:An rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples. 1926 22

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF(ts1). The mutations that resulted in a temperature-sensitive phenotype in mSF(ts1) were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 degrees C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF(ts1) was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF(ts1) for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.
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PMID:Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon. 1961 13

Pseudomonas aeruginosa is a frequent cause of respiratory infections in cystic fibrosis (CF) patients. P. aeruginosa strains isolated from these patients have often a mucoid phenotype at advanced disease. This mucoid structure contains a dense amount of alginate type polysaccharide which facilitates bacterial attachment to lung epithelia and provides protection from the immune system due to biofilm formation. The aims of this study were to investigate the biofilm formation and the relation of this property with genotype and antibiotic susceptibilities of P. aeruginosa strains isolated from CF patients. The biofilm formation was determined by using the Congo Red agar and Christensen methods. RAPD-PCR (Random amplification of polymorphic DNA polymerase chain reaction) and disc diffusion methods were used for genotyping and antibiotic susceptibility testing, respectively. Biofilm production was found positive in 33.3% (20/60) of P. aeruginosa tested. While 9 of these 20 isolates were of mucoid colony morphotype, among the 40 biofilm negative isolates mucoid colony was detected in 16 of them. RAPD genotyping based on 70% similarity yielded 19 (A-S) clusters and subtypes related to five of these clusters (K1, K2, N1, N2, Q1, Q2, R1, R2, S1, S2) making up a total of 24 genotypes. Nine of these genotypes composed of biofilm positive isolates and 15 were biofilm negative ones. Most of the biofilm positive strains belonged to K1 (n = 5) and K2 (n = 6) genotypes while biofilm negative isolates were in the L (n = 8) and O (n = 7) genotypes. The comparison of antibiotic susceptibilities in both groups revealed no statistically significant difference (p > 0.0%). However, highest rate of resistance was detected for tobramycin and lowest rate for piperacillin/tazobactam. The data obtained from this study indicated that biofilm negative and positive P. aeruginosa isolates clustered in different groups. These results should be supported with larger scale multi-center studies which may provide information about P. aeruginosa dynamics in CF lungs.
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PMID:[Investigation of biofilm formation and relationship with genotype and antibiotic susceptibility of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis]. 2008 9

Pseudomonas aeruginosa biofilms exhibit increased antimicrobial resistance compared with planktonic isolates and are implicated in the pathogenesis of both acute and chronic lung infections. Whilst antibiotic choices for both infections are based on planktonic antibiotic susceptibility results, differences in biofilm-forming ability between the two diseases have not previously been explored. The aim of this study was to compare differences in biofilm formation and antibiotic resistance of P. aeruginosa isolated from intubated patients and from patients with chronic pulmonary disease associated with cystic fibrosis (CF). The temporal evolution of antibiotic resistance in clonal P. aeruginosa strains isolated from CF patients during periods of chronic infection and acute pulmonary exacerbation was also evaluated. Biofilm formation and biofilm antibiotic susceptibilities were determined using a modified microtitre plate assay and were compared with antibiotic susceptibility results obtained using traditional planktonic culture. Clonality was confirmed using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Pseudomonas aeruginosa isolates collected from intubated patients produced substantially more biofilms compared with CF isolates. There was considerable heterogeneity in biofilm-forming ability amongst the CF isolates and this was unrelated to pulmonary status. Biofilm antibiotic resistance developed rapidly amongst clonal CF isolates over time, whilst traditional antibiotic resistance determined using planktonic cultures remained stable. There was a significant positive correlation between imipenem/cilastatin and ceftazidime resistance and biofilm-forming ability. The variability in biofilm-forming ability in P. aeruginosa and the rapid evolution of biofilm resistance may require consideration when choosing antibiotic therapy for newly intubated patients and CF patients.
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PMID:Differences in biofilm formation and antimicrobial resistance of Pseudomonas aeruginosa isolated from airways of mechanically ventilated patients and cystic fibrosis patients. 2138 98


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