Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.     

Plasmid-determined resistance to chromate in Pseudomonas aeruginosa

Abstract Resistance to chromate in five independent Pseudomonas aeruginosa clinical isolates was transferred by conjugation to P. aeruginosa strain PU21. All chromate-resistant transconjugants contained large plasmids that also conferred resistance to inorganic mercury. One of these plasmids, pUM505, increased the resistance to CrO₄²⁻ and decreased the accumulation of intracellular ⁵¹CrO₄²⁻ by the host cells as compared to the plasmidless strain PU21.     

Hybrid plasmid pBS251 containing genes for n-alkane degradation

The strain of Pseudomonas aeruginosa BS316 utilizing H-alkanes of the C6-C12 series (Alk+) harbours the 96 Md plasmid pBS250. The use of plasmid RP4 to mobilize Alk+ markers in conjugal transfer to Pseudomonas aeruginosa and Pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid RP4) and capable of growth on H-alkanes. A transconjugant from this series harbours plasmid pBS251, a derivative of plasmid RP4 containing the genes for octane and octanol catabolism. A fragment of DNA inserted into RP4 has a mol mass 3.8 Md, possesses two restriction sites for EcoRI, one site for PstI, is not restricted by SmaI and BamHI restriction endonucleases, and is localized in the region 4.5-5.7 Md on the physical map of plasmid RP4.     

Expression of Pseudomonas aeruginosa phage transposons in Pseudomonas putida PpG1 cells. II. Zygotic induction--a necessary condition for the formation of defective lysogens

The transfer of hybrid plasmid RP4::PT (where PT is the genome of a transposable phage specific for Pseudomonas aeruginosa) into recipient cells of P. putida strain PpG1 occurs with the same frequency as into P. aeruginosa, the homologous host for PT. Approximately 1/3 of all PpG1 exconjugants carrying RP4 markers lost the capability to produce viable PT phage. In contrast, in a cross with homologous recipient P. aeruginosa all exconjugant clones contained nondefective prophages in the hybrid plasmids. Zygotic induction is an obligatory condition for detection of PpG1 exconjugants with defective phages. The defective prophages in RP4::PT hybrid plasmids have deletions of different size; the other carry mutations indistinguishable from point mutations in an essential phage gene. Some of deletions also cover plasmid genes. At least some of the defective prophages, including deleted ones, have arisen in the recipient cells of P. putida after transfer of the hybrid plasmid.     

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.     

Conjugative transfer of plasmid RP1 to soil isolates of Pseudomonas fluorescens is facilitated by certain large RP1 deletions

Abstract The broad-host-range IncP plasmid RP1 could not be transferred by conjugation from Escherichia coli to Pseudomonas fluorescens strain CHA0. However, this conjugative transfer was possible with RP1 derivatives which had large deletions extending from the primase gene towards the Tra-2 region, thus lacking the kanamycin resistance gene and IS 21 . Such RP1 deletion derivatives permitted IncP cosmid mobilization to P. fluorescens CHA0 and could be used as vectors for transposon mutagenesis with a newly constructed Tn 5 derivative (carrying kanamycin and mercury resistance determinants) in strain CHA0 and another P. fluorescens soil isolate, strain S9.     

Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.

A host-vector system for Pseudomonas aeruginosa PAO was developed. Scattered regions of the strain PAO chromosome were cloned by direct selection for complementation of auxotrophs or from a DNA gene bank which contains over 1,000 independently isolated chromosome-vector recombinant plasmids. The use of partially digested chromosomal DNA facilitated the selection of a variety of strain PAO chromosomal markers. The progenitor of the vector was a small, multicopy plasmid, pRO1600, found in a PAO strain which had acquired RP1 in a mating experiment. The bacterial host range that could be determined by transformation of vectors produced from pRO1600 resembles that for plasmid RP1. Two derivative plasmids were formed: pRO1613, for cloning DNA cleaved with restriction endonuclease PstI, and pRO1614, which was formed by deleting part of pRO1613 and fusion with plasmid pBR322. Plasmid pRO1614 utilizes known cloning sites within the tetracycline resistance region of pBR322.     

Properties of RP4, an R Factor Which Originated in Pseudomonas aeruginosa S8

RP4, an R factor derived from Pseudomonas aeruginosa S8 and specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline, could be isolated as an extrachromosomal satellite of covalently closed circular DNA (CCC-DNA) of molecular weight about 62 million and of buoyant density 1.719 g/cm(3) (60% guanine plus cytosine). When RP4 was compared by hybridization with RP1, an R factor originating in another strain of P. aeruginosa, it was shown that RP4 contains most, if not all, of the base sequences of RP1, and also additional sequences not found in RP1.     

Properties of an R Factor Which Originated in Pseudomonas aeruginosa 1822

RP1, a group of genes specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline and originating in a strain of Pseudomonas aeruginosa, was freely transmissible between strains of P. aeruginosa, Escherichia coli, and Proteus mirabilis. Acquisition of the multiple drug resistance specified by RP1 by these strains was accompanied by acquisition of an extrachromosomal satellite of covalently closed circular deoxyribonucleic acid of molecular weight about 40 million daltons and of buoyant density 1.719 g/cm(3) (60% guanine plus cytosine).     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

R Plasmids R91 and R91a from Pseudomonas aeruginosa Share Only the Gene for Carbenicillin Resistance

Plasmid R91a of Pseudomonas aeruginosa strain 9169 is homologous with RP1. Plasmid R91 carried by the same strain is related in the Tn1 region but is otherwise unrelated to R91a.     

An assessment of the toxicity of metals to Pseudomonas aeruginosa PU21 (Rip64)

The toxicity of Co(II), Mn(II), Cd(II), and Zn(II) for Pseudomonas aeruginosa PU21, a Hg(II)-hyperresistant strain containing the mercury resistance mer operon, was determined. The metal tolerance of PU21 was strongly influenced by environmental conditions (e.g., existing metal, medium composition). Dose-response analysis on chronic and acute toxicity (e.g., EC(20), median effective dose EC(50), and slope factor B) of divalent cobalt, manganese, cadmium, and zinc cations in LB medium amended with citric acid phosphate buffered saline (CAPBS) suggested a toxicity series of Co > Mn approximately Zn > Cd for EC(50). In contrast, excluding the likely precipitate of Zn(II), the toxicity ranking in phosphate-buffered saline (PBS)-amended LB medium was Co > Cd > Mn. The metal toxicity in PBS, irrespective of metals, was greater than that in CAPBS. This might be attributed to the presence of citric acid in CAPBS as a chelating ligand donating electrons to hold free metals (e.g., Cd(2+), Zn(2+) tetrahedral ML(4) complex). The toxicity assessment established viable operation ranges (ca. 相似文献   

Structural instability of IncP-1 plasmids in Pseudomonas aeruginosa PAT involves interaction with plasmid pVS1

The structural instability exhibited by IncP-1 plasmids in Pseudomonas aeruginosa strain PAT was shown to be Rec+ dependent and involved interaction with the resident plasmid pVS1. Structural instability resulted from deletion of plasmid deoxyribonucleic acid at a frequency of ca. 10(-2)/cell per generation. Deletants could be stabilized by transduction into P. aeruginosa strain PAO, but in strain PAT deletants had only a transient existence, as continued deletion led eventually to the loss of the entire plasmid. The patterns of markers lost in PAT were used to demonstrate a marker order for R68 similar to that published elsewhere for RP4 (Barth and Grinter, J. Mol. Biol. 113:455-474, 1977), except that only one Tra region was found. R68 also exhibited Rec+-dependent structural instability in PAO(pVS1) derivatives but, unlike the case in PAT, instability was not accompanied by chromosome mobilization. We isolated deletants of pVS1 which were unable to promote structural instability.     

Comparison between chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin antioxidant properties and effects on rat liver CYP-catalysed drug metabolism

Ge-gen (Radix Puerariae; RP) is used in traditional oriental medicine for various medicinal purposes. The drug is the root of a wild leguminous creeper, Pueraria lobata (Willd) Ohwi. It possesses a high content of flavonoid derivatives, the most abundant of which is puerarin (PU). Here, using the enhanced chemiluminescence technique based on horseradish peroxidase and a luminol-oxidant-enhancer reagent, we evaluated in vitro the antioxidant activity of PU and RP crude extract. Both biological samples inhibited the steady-state chemiluminescent reaction in a dose-dependent fashion. However, different inhibition mechanism were postulated, since only RP behaved like conventional antioxidants. This activity was supposed to be due the presence of compounds other than PU in the crude extract. Using each of the specific substrates to different cytochrome P450 (CYP) isoforms or the regio- and stereo-selective hydroxylation of testosterone as polyfunctional probe we found that when intragastrically administered in male Wistar rats, PU (100 or 200 mg/kg b.w.) and RP (700 or 1,400 mg/kg b.w.) significantly altered hepatic CYP-linked monooxygenases. While both CYP content and NADPH-(CYP)-c-reductase activity were significantly increased in all situations, a complex pattern of CYP modulation was observed, including both induction (PU: CYP2A1, 1A1/2, 3A1, 2C11; RP: CYP1A2, 3A1, 2B1) and inactivation (PU and RP: CYP3A, 2E1, 2B1), the latter being due to either parental agents or metabolites, as demonstrated by in vitro studies. Overall, these findings indicate that RP contains compounds with potent antioxidant activity and that both PU and RP impairs CYP-catalysed drug metabolism.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.     

Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition.

Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.     

The dehalogenase gene dehI from Pseudomonas putida PP3 is carried on an unusual mobile genetic element designated DEH.

As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy. The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain PP3. DEH recombined with target plasmid DNAs at high frequencies (e.g. 3.8 x 10(-4) per RP4.5 plasmid transferred). The regulated expression of dehI was detected in P. putida, Pseudomonas aeruginosa, and Escherichia coli strains containing derivative plasmids of RP4.5 and pWW0 recombined with DEH. Movement of DEH from the unstable RP4 derivatives pNJ5000 and pMR5 resulted in the insertion of DEH into the chromosome of RecA+ strains of P. putida but not in RecA+ nor RecA- strains of E. coli. Rescue of DEH from the chromosome of P. putida KT2441 onto plasmid RP4 involved recombination at a frequency (2.7 x 10(-4) per RP4 plasmid transferred) comparable to that observed in strain PP3. The DEH element was not classified as a conventional transposon because it did not move as a discrete DNA fragment: dehI-containing inserts in plasmid DNA targets varied in size between 6 and 13 kb. In addition, DEH exhibited a marked preference for insertion into a specific site on the plasmid pWW0, but its transposition, independent of host recombinational systems, remains to be demonstrated. However, the transposonlike characteristics of DEH included the conservation of restriction endonuclease sites, high-frequency recombination with different target replicons (plasmid and chromosomal DNA), and promiscuous insertion into plasmid RP4-based replicons. Therefore, it is proposed that DEH is an unusual mobile genetic element.     

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.     

Genetic basis of non-transposition of Tn5 in Pseudomonas aeruginosa following mobilisation of RP4 Mob::Tn5 from Escherichia coli

Abstract A Tn 5 transposon mutagenesis system based on mobilization of the narrow-host-range plasmid pACYC184 from Escherichia coli by a chromosomally integrated promiscuous plasmid RP4 was found to be non-applicable to Pseudomonas aeruginosa recipients. Transposition following mobilization was based on cloning an RP4 DNA fragment (/ RP4 Mob) into pACYC184 and Tn 5 transposition into the fragment (/ RP4 Mob::Tn5). It was shown by DNA sub-cloning of RP4 Mob::Tn 5 on to a wide-host-range plasmid vector that mobilization was unaffected but that reduced survival of the vector or host following mobilization was responsible. However, mutagenesis was achieved by the provision of cloned RP4 Mob DNA in the P. aeruginosa recipients.