Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.     

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9.

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.     

Deoxyribonucleic Acid Homologies Among Some Pseudomonas Species

Phylogenetic relationships among a number of strains belonging to the genus Pseudomonas were explored by the use of in vitro deoxyribonucleic acid (DNA) hybridization. The fluorescent nomenspecies (P. fluorescens, P. putida, P. aeruginosa, P. cichorii, P. syringae, and related species), as well as the nonfluorescent species P. stutzeri, P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, were shown to belong to a single DNA homology complex which is isolated from other Pseudomonas species that have been studied [P. cepacia (= P. multivorans), P. caryophylli, P. marginata (= P. alliicola), P. pseudomallei, P. acidovorans, P. testosteroni, P. solanacearum, P. diminuta, P. facilis, P. delafieldii, P. saccharophila, P. palleronii]. A limited numerical analysis of the phenotypic properties of the examined strains supported, with some exceptions, their previous allocation to nomenspecies and biotypes. The internal structure and nomenclature of the "P. fluorescens homology complex" are discussed.     

Pseudomonas stutzeri and related species undergo natural transformation

Cells of Pseudomonas stutzeri are naturally transformed by homologous chromosomal DNA; they do not require chemical treatment to become competent. This capacity to undergo natural transformation was found to be shared by the closely related species P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, but was not detectable in strains of P. aeruginosa, P. perfectomarinus, P. putida, P. fluorescens, or P. syringae. P. stutzeri could be transformed either on plates or in liquid medium. Only double-stranded chromosomal DNA was effective; single-stranded DNA and plasmid DNA were not. DNA fragments larger than 10 kilobase pairs were more effective than smaller fragments. The transformation frequency was proportional to DNA concentration from 1 ng/ml to 1 microgram/ml; higher concentrations were saturating. The maximum frequency, about 10(-4) transformants per recipient cell, was obtained with cells from a culture in the early stationary growth phase. A variety of chromosomal mutations have been transformed, including mutations to auxotrophy and to antibiotic resistance. Other systems for genetic exchange in P. stutzeri have not yet been found; transformation offers a means for the genetic analysis of this metabolically versatile organism.     

Restriction analysis of Thiobacillus ferrooxidans DNA using pulsed field electrophoresis]

The DNA of Thiobacillus ferrooxidans digested by any of the five restriction endonucleases (DraI, EcoRI, Eco321, HindIII, XbaI) was studied by electrophoresis in the pulsating differently directed electric fields (PF). The influence of PF conditions on the sized row of the divided DNA fragments was studied. Only the XbaI restriction endonuclease was shown to cleave the Thiobacillus ferrooxidans DNA into a number of fragments permitting one to define the sizes of fragments and genome (no more than 2300 bp). The prospects of using the restriction analysis of Thiobacillus ferrooxidans wild type culture for improving its properties in obtaining heavy metals are discussed.     

Development of PCR assay to identify Pseudomonas fluorescens and its biotype

The paper provides a simple protocol that uses the polymerase chain reaction to amplify a specific portion of the 16S gene, allowing the recognition of Pseudomonas fluorescens from other group I Pseudomonas. The amplified DNA patterns of 16S rRNA and ITS1, from the restriction fragment length polymorphisms VspI, HaeIII and TaqI digestion, produced band patterns that distinguished the biotypes of Ps. fluorescens. In addition to distinguishing the biotypes C and 3 we used a phenotypical method for levan production.     

Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates).

The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.     

Temperate SM phage from Pseudomonas aeruginosa as a vector for cloning genetic information]

The recombinant bacteriophages with the genomes containing the DNA fragments of bacteria Erwinia chrysanthemi, including the pectatelyase gene, were constructed on the base of Pseudomonas aeruginosa temperate bacteriophage SM. The gene transferred into Pseudomonas aeruginosa PAO1 cells by transfection is expressed in the new bacterial host. The restriction maps of the recombinant bacteriophages are constructed and the position of an insert is defined. Bacteriophage SM was found to be capable of reproducing in Pseudomonas aeruginosa PAO1 cells when its DNA was shortened to 88% or increased to 111% of the normal genome length. Except for bacteriophage SM, the recombinant bacteriophage SM-2 with an unique restriction endonuclease site for XbaI can also be used as a vector for cloning. Bacteriophage SM capacity in cloning of heterological DNA at HindIII sites is not less than 8 Md, capacity of bacteriophage SM-2 is not less than 5 and 8 Md at XbaI and HindIII sites respectively.     

Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome

Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 Pac I, 53 Spe I, 71 Xba I) were placed on the physical map and complete maps of the circular chromosome were obtained for both Pac I and Spe I; only Xba I fragments linking Spe I fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by Southern hybridization and 31 genes were positioned including oriC , rDNA operons ( rnnA–E ), recA gacA and pyvD     

Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1

Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.     

Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1.

Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.     

萘质粒ND1.860HindⅢ片段的分子克隆和限制酶图谱

萘质粒ND1.860经限制性核酸内切酶HindⅢ完全消化和部分消化所产生的限制片段,分别在大肠杆菌质粒pBR322中克隆。通过对含有ND1.860HindⅢ片段的17个重组质粒进行限制酶分析,建立了ND1.860质粒的HindⅢ、EcoRⅠ和XbaⅠ种内切酶26个切点的酶切图谱。     

Electrophoretic analysis of the exoproducts of bacteria in the genus Pseudomonas

A high degree of similarity (exceeding 90%) between the electrophoregrams of the exoproducts of 31 Pseudomonas strains, including the standard species P. aeruginosa, P. maltophilia, P. putida, P. alcaligenes, P. testosteroni, P. diminuta, P. stutseri, P. fluorescens, has been shown by the method of electrophoresis in polyacrylamide gel.     

How quantitative is quantitative PCR with respect to cell counts?

Quantitative diagnostic PCR systems based upon rDNA targeted primer and probe combinations were developed for the detection of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, enterococci, Staphylococcus aureus, and Staphylococcus epidermidis. Primers and probes were designed in silico using the ARB software package (TU Munich) in combination with Primer Design software of PE Applied Biosystems. Purified genomic DNA or bacterial cells of target and reference organisms were used for the evaluation of the PCR assays applying the TaqMan technique on an ABI PRISM TM 7700 Sequence Detection System (PE Applied Biosystems). Sensitive, reliable and reproducible quantification of target rDNA could be achieved applying primer-probe combinations that mediate in vitro amplification of DNA fragments smaller than 100 base pairs. Large amounts of non target DNA (1 mg per sample) remarkably affected the quantification potential of the approach resulting in an underestimation of the amounts of target DNA. One of the principal goals was to use quantitative PCR to study the correlation of gene and cell numbers depending on the growth behavior of target organisms and to explore the potential to estimate cell numbers from target DNA quantification. A clear correlation of rDNA quantification and bacterial growth was observed, however, cell numbers cannot directly be estimated from quantitative PCR data, given that the cellular genome content varies with the growth phase of the organisms. In the case of Escherichia coli the cell numbers which could be assigned to a certain number of rDNA targets varied reasonably depending upon the growth phase of batch cultures.     

Characterization of Nonfermentative Nonfastidious Gram Negative Bacteria encountered in Medical Bacteriology

More than 90 morphological and physiological characters of 546 strains of nonfermentative, nonfastidious, Gram negative bacteria isolated from clinical specimens were examined to determine those features most useful for the identification of these bacteria. The species examined included Moraxella osloensis, Mor. lacunata, Acinetobacter anitratum, Ac. haemolyticus subsp. haemolyticus, Ac. haemolyticus subsp. alcaligenes, Ac. Iwoffi, Alcaligenes faecalis, Alc. odorans var. viridans, Pseudomonas fluorescens, Ps. putida, Ps. pseudomallei, Ps. maltophilia, Ps. stutzeri, Ps. acidovorans, Ps. alcaligenes , and atypical strains of Ps. aeruginosa.     

New approaches in genome analysis by pulsed-field gel electrophoresis: application to the analysis of Pseudomonas species

A general method for the evaluation of macrorestriction fragment patterns is presented and its applicability to the taxonomy of bacteria is demonstrated for 32 Pseudomonas species. Strains were differentiated at the species and subspecies level by genome size and macrorestriction fragment fingerprints of the chromosome that had been separated on pulsed-field gels. The relatedness of bacteria was ascertained from the similarity of AsnI, DraI, SpeI, SspI or XbaI fragment patterns. In general, the dendrograms calculated from the genome fingerprints corresponded with the phylogenetic classification obtained from phenotypic marker or nucleic acid hybridization analysis, but several exceptions were noted. The techniques and algorithms presented herein are generally applicable to the genome analysis of bacteria, lower eukaryotes, and DNA fragments cloned in yeast artificial chromosomes.     

DNA polymorphism in strains of the genus Brucella

Preparations of DNA from 23 Brucella strains including 19 reference strains were compared by restriction endonuclease analysis. Pulsed-field gel electrophoresis resulted in optimal resolution of fragments generated by digestion with low-cleavage-frequency restriction enzymes such as XbaI. By this technique, five electrophoretypes were distinguished in five reference strains of the different species, i.e., B. abortus, B. melitensis, B. suis, B. canis, and B. ovis. Minor profile differences allowed us to discriminate between most biovars within a species. However, the differences in the DNA patterns of different field strains of biovar 2 of B. melitensis were not sufficient to serve as markers for epidemiological studies. From the XbaI fragments, we were able to estimate the size of the genomes of B. abortus 544T and B. melitensis 16 MT. This method revealed a relationship between DNA fingerprints, species, and pathovars which could shed light on problems concerning the classification and evolution of members of the genus Brucella.     

Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA

Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.     

Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant.

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.