Plasmid-determined cadmium resistance in Pseudomonas putida GAM-1 isolated from soil.

Cadmium-resistant Pseudomonas putida GAM-1, which was able to grow in concentrations of CdCl2 as high as 7 mM, was isolated from soil in a rice paddy. This bacterium harbored a DNA plasmid of about 52 kilobases. The plasmid (pGU100) transformed Escherichia coli C600 to cadmium resistance. A cadmium-resistant transformant of E. coli C600 contained a plasmid corresponding to that seen in P. putida GAM-1. The transformant did not take up cadmium as well as P. putida GAM-1 did.     

Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida.

The plasmid pEST1412 contains the genes, pheA and pheB, encoding phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12]), respectively. Thse were originally cloned from the plasmid DNA of Pseudomonas sp. EST1001 [Kivisaar et al., Plasmid 24 (1990) 25-36]. Although pheA and pheB are cotranscribed using the promoter sequences derived from Tn4652 and the level of expression of C120 activities from pEST1412 was equal both in Escherichia coli and in Pseudomonas putida, the level of PMO activity measured in the cell-free extracts of E. coli was lower than that in P. putida. The nucleotide sequence of the 2.0-kb PstI-HindIII fragment of pEST1412 carrying pheA was determined. A 1821-bp ORF was found in this DNA. The structural gene (tfdB) encoding 2,4-dichlorophenol hydroxylase from pJP4 has been sequenced [Perkins et al., J. Bacteriol. 172 (1990) 2351-2359]. Comparison of the deduced amino acid sequences of tfdB and pheA revealed highly conserved regions in the protein products of these genes.     

Expression, localization, and functional analysis of polychlorinated biphenyl degradation genes cbpABCD of Pseudomonas putida.

Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression, localization, and functional analysis of polychlorinated biphenyl degradation genes cbpABCD of Pseudomonas putida.

Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and Export of Pseudomonas putida NTU-8 Creatinase by Escherichia coli Using the Chitinase Signal Sequence of Aeromonas hydrophila

The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.     

Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.     

Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593

Pseudomonas sp. strain 593, a soil bacterium, is able to use exogenous choline to synthesize phosphatidylcholine via phosphatidylcholine synthase (Pcs). A 2020 bp DNA fragment that hybridized to a Pcs probe was cloned. This fragment contained a large open reading frame (ORF) with two potential ATG start sites that would encode for 293 and 231 amino acid proteins. Fragments containing the two ORFs encoded Pcs when they were inserted into the expression vector pET23a and expressed under the control of the T7 promoter in Escherichia coli BL21(DE3) pLysS. However, when the two ORFs were inserted into the cloning vector pMD18-T and expressed without control of the plasmid promoter in E. coli DH5α, only the larger clone exhibited Pcs activity. This suggested that the larger fragment contained a native promoter driving expression of the smaller ORF. A promoter activity assay, in which DNA fragments were inserted into the promoter-probe plasmid pCB182 and β-galactosidase activity of E. coli transformants was tested, demonstrated that a promoter is indeed present in the DNA region. All results together indicate that the 696 bp ORF, not the larger 897 bp ORF, encodes the Pcs in Pseudomonas sp. strain 593 and carries a promoter in front of its 5' terminus.     

Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.     

An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression.

Pseudomonas aeruginosa strain 9169 has been reported to contain a plasmid that expresses resistance to carbenicillin (Cb), kanamycin (Km), and tetracycline (Tc) in Escherichia coli but resistance only to Cb in certain Pseudomonas recipients. The triply resistant plasmid in E. coli belonged to incompatibility (Inc) group P or P-1, whereas the singly resistant plasmid in P. aeruginosa was compatible with IncP-1 plasmids and other plasmids of established Inc specificity but incompatible with plasmid pSR1 that is here used to define a new Pseudomonas Inc group P-10. Additional physical and genetic studies showed that strain 9169 contained not one but two plasmids: IncP-1 plasmid R91a, determining the Cb Km Tc phenotype, and IncP-10 plasmid R91, determining Cb that differed in molecular weight and in EcoRI and BamHI restriction endonuclease recognition sites. Plasmid multiplicity rather than host effects on plasmid gene expression can account for differences in the phenotype of strain 9169 transconjugants to E. coli and P. aeruginosa.     

Functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of Pseudomonas origin

Abstract The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.     

Plasmid R68.45 and S-a transduction by the temperate bacteriophage 59 of Erwinia carotovora 268

Temperature bacteriophage 59 of Erwinia carotovera 268 had transduced extrachromosomal DNA: plasmids of R68.45 and S-a. Before plasmid transduction experiments the suitable donor strains of indicator culture Erwinia horticola 450 harbouring R68.45 and S-a were created. The frequency of plasmid R68.45 transfer from Pseudomonas putida to E. horticola 450-8 by conjugation was equal to 5 x 10(-8) per a donor cell and in the case of S-a--from E. coli C600 for the same recipient cells--was 2 x 10(-6). Bacteriophage 59 has transduced only separate markers of plasmid R68.45, since plasmid S-a is probably transduced by the phage as an intact unit.     

Transformation of bacteria with plasmid DNA by electroporation

The possibility of electric field-mediated transformation ("electroporation") of a gram-positive bacterium (Enterococcus faecalis) and two gram-negative bacteria (Escherichia coli and Pseudomonas putida) with plasmid DNA was investigated. E. faecalis protoplasts could be transformed by electroporation with a transformation frequency of 10(4) to 10(5) transformants/micrograms plasmid. Untreated--i.e., washed--cells of E. coli could be transformed with rates of 1 X 10(5) transformants/micrograms plasmid DNA. Transformation rates for P. putida cells were up to 3 X 10(4) if the method developed for E. coli was used. Detailed protocols for these systems, including the results of various optimization experiments, are given.     

Overexpression of salicylate hydroxylase and the crucial role of lys(163) as its NADH binding site

Expression systems for the sal gene encoding salicylate hydroxylase from Pseudomonas putida S-1 were examined and some constructs were expressed in these systems. By cultivation of Escherichia coli BL21 (DE3)/pSAH8 in LB medium at 37 degrees C with isopropyl-b-D-thiogalactopyranoside as the inducer, salicylate hydroxylase was overexpressed mainly in the form of inclusion bodies. Lower temperature cultivation at 20 degrees C after induction resulted in a large amount of the enzyme in the soluble form. The E. coli clone harboring the recombinant plasmid produced a 45 kDa protein that appeared to be electrophoretically and immunochemically identical to the P. putida enzyme and contained the same N-terminal amino acid sequence. This recombinant DNA product also exhibited properties characteristic of a flavoprotein and was fully functional as salicylate hydroxylase. Based on chemical modification of the salicylate hydroxylase from P. putida, Lys163 was previously proposed to be the NADH binding site. In this study, to obtain a better understanding of the predicted role of Lys163, this residue in the active center of salicylate hydroxylase was replaced with Arg, Gly, or Glu by conventional site-directed mutagenesis. Kinetic studies using these mutant enzymes and the recombinant enzyme revealed increases in apparent K(m) values for NADH in the order of wild-type enzyme > K163R > K163G > K163E, with some decreases in V(max). Examination of the recombinant enzyme and K163G indicated that the pH dependency of K(m) on NADH with pK(a) 10.5 is lost by mutation despite the lack of changes in V(max) values, suggesting a requirement for the lysine residue as the NADH binding site. Based on these results, Lys163 is proposed to play a role in the binding of NADH at the active site through an ionic bond rather than playing a role in catalysis.     

New alkane-responsive expression vectors for Escherichia coli and pseudomonas

We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB. The expression vectors were tested in several E. coli strains, P. putida GPo12 and P. fluorescens KOB2Delta1 with catechol-2,3-dioxygenase (XylE). Induction factors ranged between 100 and 2700 for pKKPalk in E. coli and pCom8 in Pseudomonas strains, but were clearly lower for pCom8, pCom9, and pCom10 in E. coli. XylE expression levels of more than 10% of total cell protein were obtained for E. coli as well as for Pseudomonas strains.     

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.     

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.