Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid.

The Pseudomonas putida TOL plasmid pWW0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). Transconjugants are recipient cells that have received DNA from donor cells, whereas retrotransconjugants are donor bacteria that have received DNA from a recipient. The TOL plasmid pWW0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrated into the chromosome of other P. putida strains, a process that appears to involve a single conjugational event. The rate of retrotransfer (as well as of direct transfer) of the chromosomal marker is influenced by the location of the kanamycin marker on the chromosome and ranges from 10(-3) to less than 10(-8) retrotransconjugants per donor (transconjugants per recipient). The mobilized DNA is incorporated into the chromosome of the retrotransconjugants (transconjugants) in a process that seems to occur through recombination of highly homologous flanking regions. No interspecific mobilization of the chromosomal marker in matings involving P. putida and the closely related Pseudomonas fluorescens, which belongs to rRNA group I, was observed.     

The effect of lipophilic weak acids on the segregational stability of TOL plasmids in Pseudomonas putida

The effect of various lipophilic weak acids on the stability of certain TOL plasmids was investigated. Benzoate induced deletion of TOL plasmid DNA in Pseudomonas putida MT15, followed by loss of the plasmid; this effect was pH- and concentration-dependent, suggesting that undissociated benzoic acid was a more effective curing agent than the benzoate anion. Plasmid loss always approached a frequency of 100% after a lag and apparently depended on the prior occurrence of deletions, although deleted plasmid was stably maintained in the absence of the acid. m-Toluate, acetate and butyrate also induced deletions and plasmid loss at high frequencies, although these acids were less effective than benzoate. Benzoate inhibited the growth of plasmid-containing cells rather than permitting faster growth of cured cells on benzoate. Similar results were obtained with P. putida strains MT20 and MT84, which contain different TOL plasmids. We suggest that lipophilic weak acids induced deletions, possibly by excision of a transposon-like region, and disrupted the segregation of deleted plasmid.     

Enhanced tolerance to naphthalene and enhanced rhizoremediation performance for Pseudomonas putida KT2440 via the NAH7 catabolic plasmid

In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [(14)C]naphthalene, measured as the evolution of (14)CO(2), while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

A cleavage map of the TOL plasmid of Pseudomonas putida mt-2.

Summary A cleavage map of the TOL plasmid pWWO has been determined for the restriction endonucleases HindIII and XhoI. A number of techniques were employed including (i) digestion of purified cleavage products with a second enzyme; (ii) hybridisation of purified XhoI fragments to Southern blots of HindIII digest products and (iii) analysis of a number of deletion mutants.     

Chromosomal location of TOL plasmid DNA in Pseudomonas putida.

The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid. By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB. A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18. Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.     

Codon usage patterns suggest independent evolution of two catabolic operons on toluene-degradative plasmid TOL pWW0 of Pseudomonas putida

TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes responsible for the degradation of toluene. The structural genes for these catobolic enzymes are clustered into two operons—namely, the xylCMAB and xylXYZLTEGFJQKIH operons. We examined the codon usage patterns of these catabolic genes by measuring the codon-usage distances between pairs of these catabolic genes. The codon-usage distance, d, between gene 1 and gene 2 was defined as d = [(p _j – q _j )²]^1/2, where p _j > and q _j are the frequencies of the j-th codon in gene 1 and 2, respectively, j being any one of the 64 possible codons. We found that the genes in the same operon exhibit similar codon-usage patterns while genes in the different operons exhibit different codon bias. This observation suggests that genes in the same operon have coevolved, and that the ancestors of the xylCMAB and xylXYZLTEGFJQKIH operons evolved in different organisms. Correspondence to: S. Harayama     

Molecular relationships between pseudomonas INC P-9 degradative plasmids TOL, NAH, and SAL

We have examined the extent to which the degradative plasmids SAL, NAH, and TOL of the Inc P-9 incompatibility group share common DNA sequences. The homology we observe using 32P-labeled SAL and NAH DNA probes can be assigned to six regions of the TOL (pWWO) restriction endonuclease cleavage map. At least three of these regions are probably related to transfer and replication functions, whereas a fourth region is related to the common metacleavage pathway. Restriction endonuclease maps of the SAL and NAH plasmids are derived and the relationships between these plasmids discussed.     

Isolation and characterization of Pseudomonas putida R-prime plasmids

A number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient. For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved. Physical analysis of one R-prime showed that 3.5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.     

IncP-7 naphthalene-degradative plasmids from Pseudomonas putida

Abstract Seven naphthalene-degrading and two naphthalene and camphor-degrading Pseudomonas putida strains were isolated from marine sediments. Most of them carried two plasmids, of molecular size 60 and 200 kb. The naphthalene and salicylate metabolism determinants were transferred to a P. putida strain by conjugation, and the transconjugants acquired either both plasmids or only the 200-kb one. These plasmids appear to belong to the IncP-7 group and encode for catabolism of naphthalene and salicylate, but not camphor.     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.     

Regulation of the synthesis of the key enzymes for naphthalene catabolism in Pseudomonas putida and Pseudomonas fluorescens carrying the biodegradation plasmids NAH, pBS3, pBS2 and NPL-1

Regulation of the synthesis of key enzymes catalysing naphthalene catabolism was studied in Pseudomonas strains containing different plasmids of naphthalene biodegradation. The synthesis of naphthalene oxygenase, salicylate hydroxylase, catechol-1,2-oxygenase and cathechol-2,3-oxygenase was shown to be regulated in both the coordinated and non-coordinated manner.