Segregational and structural instability of recombinant plasmid carrying genes for naphthalene degrading pathway

The stability of recombinant plasmid carrying genes for naphthalene mineralization was determined. A strain of Pseudomonas putida capable of mineralizing naphthalene (Nap⁺) via salicylate (Sal⁺) was isolated, and all regulatory and structural genes for the whole pathway were found to be encoded on a 25 kb Eco RI fragment of an approximately 83 kb plasmid present in this strain. The 25 kb Eco RI fragment was cloned into a tetracycline-resistant (Tc^R) cloning vector pLAFR3 and the recombinant plasmid, pRKJ3 (Nap⁺, Sal⁺, Tc^R), thus obtained was transferred into the plasmid-free strain Pseudomonas putida KT2442 in order to test the stability of the plasmid. Plasmid pRKJ3 was found to be segregationally and/or structurally unstable, depending on the growth conditions. Two types of novel derivative strains having the phenotypes Nap⁻, Sal⁺, Tc^R and Nap⁻, Sal⁻, Tc^R with specific deletions of approximately 2 kb and 18 kb, respectively, were obtained.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Degradation of phenol and m-toluate in Pseudomonas sp. strain EST1001 and its Pseudomonas putida transconjugants is determined by a multiplasmid system.

The utilization of phenol, m-toluate, and salicylate (Phe+, mTol+, and Sal+ characters, respectively) in Pseudomonas sp. strain EST1001 is determined by the coordinated expression of genes placed in different plasmids, i.e., by a multiplasmid system. The natural multiplasmid strain EST1001 is phenotypically unstable. In its Phe-, mTol-, and Sal- segregants, the plasmid DNA underwent structural rearrangements without a marked loss of plasmid DNA, and the majority of segregants gave revertants. The genes specifying the degradation of phenol and m-toluate were transferable to P. putida PaW340, and in this strain a new multiplasmid system with definite structural changes was formed. The 17-kilobase transposable element, a part of the TOL plasmid pWWO present in the chromosome of PaW340, was inserted into the plasmid DNA in transconjugants. In addition, transconjugant EST1020 shared pWWO-like structures. Enzyme assays demonstrated that ortho-fission reactions were used by bacteria that grew on phenol, whereas m-toluate was catabolized by a meta-fission reaction. Salicylate was a functional inducer of the enzymes of both pathways. The expression of silent metabolic pathways of phenol or m-toluate degradation has been observed in EST1001 Phe- mTol+ and Phe+ mTol- transconjugants. The switchover of phenol degradation from the ortho- to the meta-pathway in EST1033 also showed the flexibility of genetic material in EST1001 transconjugants.     

The construction and monitoring of genetically marked, plasmid-containing, naphthalene-degrading strains in soil

A genetically marked, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142::TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain's fate in model soil systems to be monitored, whereas a minitransposon, built in naphthalene biodegradation plasmid pNF142, contains the tetracycline resistance gene and makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene was found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically marked strain is stable and competitive for 40 days. The transfer of marked plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.     

Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.     

Genetic engineering of Pseudomonas putida KT2442 for biotransformation of aromatic compounds to chiral cis-diols

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylations of aromatic compounds. Pseudomonas putida KT2442 (pSPM01) harboring TDO genes could effectively biotransform a wide-range of aromatic substrates into their cis-diols products. In shake-flask culture, approximately 2.7gl(-1) benzene cis-diols, 8.8gl(-1) toluene cis-diols and 6.0gl(-1) chlorobenzene cis-diols were obtained from the biotransformation process. Furthermore, vgb gene encoding Vitreoscilla hemoglobin protein (VHb) which enhances oxygen microbial utilization rate under low dissolved oxygen concentration was integrated into P. putida KT2442 genome. The oxidation ability of the mutant strain P. putida KTOY02 (pSPM01) harboring TDO gene was increased in the presence of VHb protein. As a result, approximately 3.8, 15.1 or 6.8gl(-1) different cis-diols production was achieved in P. putida KTOY02 (pSPM01) grown in shake-flasks when benzene, toluene or chlorobenzene was used as the substrate. The above results indicate that P. putida KT2442 could be used as a cell factory to biotransform aromatic compounds.     

NahY, a Catabolic Plasmid-Encoded Receptor Required for Chemotaxis of Pseudomonas putida to the Aromatic Hydrocarbon Naphthalene

Pseudomonas putida G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene, nahY, was found to be cotranscribed with meta cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The nahY gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A P. putida G7 nahY mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of nahY on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.     

Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil

To generate mutants with altered lipopolysaccharides (LPS) of the wild-type Pseudomonas putida KT2442, we used the mini-Tn 5luxAB -Km transposon. A mutant was found among luminescent colonies and selected as a negative clone in enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O-antigen of P. putida LPS. The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced. Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O-antigen polymerase (Wzy) of Salmonella enterica (muenchen). The wild-type gene was rescued by polymerase chain reaction (PCR), cloned into a broad-host-range plasmid and used to carry out complementation assays. The cloned gene was able to restore the wild-type phenotype of the P. putida wzy mutant. We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL :: xylE cassette. The wzy mutants of P. putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains. We analysed the ability of wzy , oprL and wzy oprL mutants of P. putida to colonize soil. In comparison with the wild-type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures.     

Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.

A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.     

苜蓿中华根瘤菌与耐盐有关DNA片段的亚克隆和测序分析

将苜蓿中华根瘤菌(Sinorhizobium meliloti)042B与耐盐有关的4kb ClaⅠ DNA片段克隆在pML122上,用HindⅢ酶切下其2.4kb DNA片段,回收后与pBBR1\|MCS2连接,然后转化大肠杆菌(Escherichia coli)DH5α,筛选到转化子GS2〖DK〗。将残留在pML122上16kb ClaⅠ\|HindⅢ DNA片段连同质粒一起回收,让其自连,转化大肠杆菌S17-1,得到转化子GS0。以GS0为供体,042B的盐敏感突变株GZ17为受体,进行二亲本杂交,没有得到接合子。以GS2为供体,GZ17为受体,在辅助质粒pRK2013的协助下,进行三亲本杂交,筛选到接合子GG2,获得2.4kb HindⅢ与耐盐有关的DNA片段。将此片段连接到测序载体pGEM\|7Zf(+)上进行测序。测序结果表明,该24kb HindⅢ DNA片段含有3个开放阅读框(ORF)。在此基础上再一次亚克隆,获得19kb与耐盐有关的DNA片段。     

Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51.

Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.     

Plasmid- and chromosome-mediated dissimilation of naphthalene and salicylate in Pseudomonas putida PMD-1.

Pseudomonas putida PMD-1 dissimilates naphthalene (Nah), salicylate (Sal), and benzoate (Ben) via catechol which is metabolized through the meta (or alpha-keto acid) pathway. The ability to utilize salicylate but not naphthalene was transferred from P. putida PMD-1 to several Pseudomonas species. Agarose gel electrophoresis of deoxyribonucleic acid (DNA) from PMD-1 and Sal+ exconjugants indicated that a plasmid (pMWD-1) of 110 megadaltons is correlated with the Sal+ phenotype; restriction enzyme analysis of DNA from Sal+ exconjugants indicated that plasmid pMWD-1 was transmitted intact. Enzyme analysis of Sal+ exconjugants demonstrated that the enzymes required to oxidize naphthalene to salicylate are absent, but salicylate hydroxylase and enzymes of the meta pathway are present. Thus, naphthalene conversion to salicylate requires chromosomal genes, whereas salicylate degradation is plasmid encoded. Comparison of restriction digests of plasmid pMWD-1 indicated that it differs considerably from the naphthalene and salicylate degradative plasmids previously described in P. putida.     

Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci.

Pseudomonas syringae pv. tabaci BR2 produces tabtoxin and causes wildfire disease on tobacco and bean plants. Approximately 2,700 Tn5 insertion mutants of a plasmid-free strain, PTBR 2.024, were generated by using suicide plasmid pGS9. Of these Tn5 mutants, 8 were no longer pathogenic on tobacco plants and 10 showed reduced symptoms. All of the eight nonpathogenic mutants caused typical wildfire disease symptoms on bean plants. Two of the nonpathogenic mutants failed to produce tabtoxin. The eight nonpathogenic mutants have Tn5 insertions into different EcoRI and SalI restriction fragments. The EcoRI fragments containing Tn5 from the eight nonpathogenic mutants were cloned into vector pTZ18R or pLAFR3. A genomic library of the parent strain was constructed in the broad-host-range cosmid pLAFR3. Three different cosmid clones that hybridized to the cloned Tn5-containing fragment from one of the nonpathogenic mutants, PTBR 4.000, were isolated from the genomic library. These clones contained six contiguous EcoRI fragments (a total of 57 kilobases [kb]). A 7.2-kb EcoRI fragment common to all three restored pathogenicity to mutant PTBR 4.000. None of the six EcoRI fragments hybridized to Tn5-containing fragments from the other seven mutants. The 7.2-kb fragment was conserved in P. syringae pv. tabaci and P. syringae pv. angulata, but not in other pathovars or strains. Because the mutants retained pathogenicity on bean plants and because of the conservation of the 7.2-kb EcoRI fragment only in pathovars of tobacco, we suggest that genes on the fragment might be related to host specificity.     

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.     

Transformation and expression of plasmid from Nocardioides sp. to Pseudomonas putida

Nocardioides sp. isolated from contaminated soil showed the presence of sulphur oxidizing (SO) genes in the plasmid pSB1 (34·2 kb). The presence of SO genes was confirmed by transformation to a plasmid-free Pseudomonas putida strain followed by hybridization studies.     

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.     

Cloning of genes degrading 3-chlorobenzoate from Pseudomonas putida strain 87]

The ability of Pseudomonas putida strain 87 to catabolize 3-chlorobenzoate was shown to be mediated by genes of pBS109 plasmid. The plasmid may be transferred by conjugation into P. aeruginosa PAO2175. It seems possible that the pBS109 plasmid codes for pyrocatechase II specific for halogenated catechol, but not catechol. The genes specifying utilization of 3-chlorobenzoate from pBS109 plasmid were cloned in the 5.5 kb BgIII fragment by using broad-host cloning system. The resulting pBS110 plasmid was transferred into P. putida, which results in utilization of 3-chlorobenzoate by transconjugants.     

Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDN1).

A 9,233-bp HindIII fragment of the aromatic amine catabolic plasmid pTDN1, isolated from a derivative of Pseudomonas putida mt-2 (UCC22), confers the ability to degrade aniline on P. putida KT2442. The fragment encodes six open reading frames which are arranged in the same direction. Their 5' upstream region is part of the direct-repeat sequence of pTDN1. Nucleotide sequence of 1.8 kb of the repeat sequence revealed only a single base pair change compared to the known sequence of IS1071 which is involved in the transposition of the chlorobenzoate genes (C. Nakatsu, J. Ng, R. Singh, N. Straus, and C. Wyndham, Proc. Natl. Acad. Sci. USA 88:8312-8316, 1991). Four open reading frames encode proteins with considerable homology to proteins found in other aromatic-compound degradation pathways. On the basis of sequence similarity, these genes are proposed to encode the large and small subunits of aniline oxygenase (tdnA1 and tdnA2, respectively), a reductase (tdnB), and a LysR-type regulatory gene (tdnR). The putative large subunit has a conserved [2Fe-2S]R Rieske-type ligand center. Two genes, tdnQ and tdnT, which may be involved in amino group transfer, are localized upstream of the putative oxygenase genes. The tdnQ gene product shares about 30% similarity with glutamine synthetases; however, a pUC-based plasmid carrying tdnQ did not support the growth of an Escherichia coli glnA strain in the absence of glutamine. TdnT possesses domains that are conserved among amidotransferases. The tdnQ, tdnA1, tdnA2, tdnB, and tdnR genes are essential for the conversion of aniline to catechol.     

Conjugal transfer of a TOL-like plasmid and extension of the catabolic potential of Pseudomonas putida F1

Strain mX was isolated from a petrol-contaminated soil, after enrichment on minimal medium with 0.5% (v/v) meta-xylene as a sole carbon source. The strain was tentatively characterized as Pseudomonas putida and harboured a large plasmid (pMX) containing xyl genes involved in toluene or meta-xylene degradation pathways via an alkyl monooxygenase and a catechol 2,3-dioxygenase. This new TOL-like plasmid was stable over two hundred generations and was self-transferable. After conjugal transfer to P. putida F1, which possesses the Tod chromosomal toluene biodegradative pathway, the transconjugant P. putida F1(pMX) was able to grow on benzene, toluene, meta-xylene, para-xylene, and ethylbenzene compounds as the sole carbon sources. Catechol 2,3-dioxygenases of the transconjugant cells presented a more relaxed substrate specificity than those of parental cells (strain mX and P. putida F1).     

Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442

Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the beta-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.