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1.
NPL-1 and its derivative plasmid pBS106, which control the degradation of naphthalene and salicylate, were found to contain class II transposons of the Tn3 family. These transposons are involved in intraplasmid rearrangements, such as deletions and inversions, and can influence the expression of the catabolic and regulatory genes borne by biodegradation plasmids. The formation of a strong NahR-independent constitutive promoter by the inversion of a DNA fragment may be responsible for changing the character of naphthalene dioxygenase synthesis from inducible (in the case of plasmid NPL-1) to constitutive (in the case of plasmid NPL-41). The stability of plasmids NPL-1 and NPL-41 in the Pseudomonas putida strains grown on different substrates depends on the expression of the nah and tnp genes.  相似文献   

2.
The stability of biodegradation plasmids NPL-1 and NPL-41, which control the synthesis of enzymes for naphthalene oxidation to salicylate, was studied in Pseudomonas putida BSA under the conditions of its continuous cultivation with limitation in glucose or salicylate in the chemostat regime and without limitation in the pH-stat regime. Plasmid NPL-1, which controls the inducible synthesis of naphthalene oxygenase, is stable in the population of P. putida cells under the conditions of continuous cultivation on glucose, but is not stable in the course of cultivation on salicylate, an inductor of the naphthalene oxygenase synthesis. Plasmid NPL-41, which controls the constitutive synthesis of naphthalene oxygenase, is not stable in the population of P. putida cells under the conditions of continuous cultivation on glucose. The operation of genes, which control the oxidation of naphthalene to salicylate (nah), makes plasmids NPL-1 and NPL-41 unstable under the conditions of continuous cultivation in the absence of naphthalene from the medium, i.e. under the conditions when the expression of these genes is not necessary. In that case, cells containing plasmids with a deletion of nah-genes as well as cells without plasmids appear in the population of P. putida, which causes a decline in its futile energy and metabolic processes.  相似文献   

3.
T V Tso?  I A Kosheleva  A M Boronin 《Genetika》1986,22(11):2702-2712
The hybridization and restriction analysis of the plasmid pBS286 (73 Kb, the P-9 Inc group) as well as parental plasmids NPL-1, NPL-41 demonstrated that pBS286 plasmid (delta NPL-41::TnA) with the constitutive synthesis of naphthalene dioxygenase carried genes for naphthalene oxidation to salicylate and those participating in degradation of catechol. Restriction map of pBS286 using XhoI restriction endonuclease and that of the nah region using EcoRI, BamHI, SalI and XhoI were established. Structural peculiarities of nah genes from pBS286 are compared with previously described NAH7. Some nah genes were localized. An inverted DNA segment involved in nah gene regulation was shown to be closely linked to a proximal part of the nah1 operon or overlapped. Possible occurrence of a regulatory R locus in this region is suggested.  相似文献   

4.
Naphthalene oxidation by a parent and a mutant strain of Pseudomonas putida was studied. The parent strain contained a plasmid NPL-1 which controlled oxidation of naphthalene to salicylic acid and was capable of oxidizing salicylate. The mutant strain did not oxidize salicylate because of a mutation in salicylate hydroxylase; it contained also a mutant plasmid NPL-41 which determined constitutive synthesis of naphthalene oxygenase. Salicylic acid which accumulated as a product of naphthalene catabolism in the cultural broth of the wild strain was found to undergo further oxidation by the population of growing cells. The content of salicylic acid in the cultural broth of the mutant strain reached maximum and then remained constant. An anion-exchange resin was tested in order to prevent the inhibition of naphthalene oxygenase by salicylate and to increase the yield of salicylic acid. The transmissible character of the mutant plasmid NPL-41 makes it possible, with the aid of conjugation, to construct Pseudomonas strains which would oxidize naphthalene to salicylic acid without further degradation of this compound.  相似文献   

5.
Transconjugant strain of bacteria degraders of alkylnaphthalenesulphonate(ANS)--P Pseudomonas alcaligenes TR (NPL-41) was obtained under conditions of ANS selective pressure and flow cultivation of bacteria destructors of alkylbenzene sulphonate (ABS) P. alcaligenes TR (pABS) and naphthalene P. putida BS438 (NPL-41) and their immobilization on an inert carrier. Transconjugant P. alcaligenes TR(NPL-41) was prepared by means of conjugative transfer of plasmid NPL-41 from P. putida BS438 to P. alcaligenes TR. The check out of transconjugant under conditions of flow cultivation showed their ability to degradation of ANS. Investigation of the stability of plasmid NPL-41 in the transconjugant P. alcaligenes TR(NPL-41), obtained by molecular breeding under conditions of flow cultivation and by conjugative transfer, showed the transconjugant prepared by the former method to be more stable.  相似文献   

6.
Filonov  A. E.  Akhmetov  L. I.  Puntus  I. F.  Esikova  T. Z.  Gafarov  A. B.  Izmalkova  T. Yu.  Sokolov  S. L.  Kosheleva  I. A.  Boronin  A. M. 《Microbiology》2005,74(4):453-458
A genetically tagged, 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, inserted into naphthalene biodegradation plasmid pNF142 and containing the tetracycline resistance gene, 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 is found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically tagged strain is stable and competitive for 40 days. The transfer of labeled plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 526–532.Original Russian Text Copyright © 2005 by Filonov, Akhmetov, Puntus, Esikova, Gafarov, Izmalkova, Sokolov, Kosheleva, Boronin.  相似文献   

7.
The genetic systems that are responsible for naphthalene catabolism were analyzed in 18 naphthalene-degrading Pseudomonas fluorescens strains isolated from oil-contaminated soils in different regions of Russia. It was found that 13 strains contain plasmids, from 20 to 120 kb in size, at least 5 of which are conjugative and bear the catabolic genes responsible for the complete utilization of naphthalene and salicylate. Five plasmids belong to the P-7 incompatibility group, and two plasmids belong to the P-9 incompatibility group. The naphthalene biodegradation genes of P. fluorescens are highly homologous to each other. The study revealed a new group of the nahAc genes and two new variants of the nahG gene. The suggestion is made that the key genes of naphthalene biodegradation, nahAc and nahG, evolve independently and occur in P. fluorescens strains in different combinations.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 70–78.Original Russian Text Copyright © 2005 by Izmalkova, Sazonova, Sokolov, Kosheleva, Boronin.  相似文献   

8.
The object of the work was to study the functional expression of naphthalene and salicylic acid catabolism systems and the stability of naphthalene biodegradation plasmids NAH, pBS2, pBS3 and NPL-41 in Pseudomonas aeruginosa PAO. The catabolic systems of the plasmids were shown to be thermosensitive, with a slight variation between one another. The plasmids became unstable at a high temperature; the temperature of effective elimination was 41 degrees C for plasmids NPL-41 and pBS3, and 42 degrees C for plasmids NAH and pBS2. NAH and pBS2 produced a weak inhibiting effect while NPL-41 and pBS3 caused a strong inhibition of the PAO strain growth at 42 degrees C. As a result, many anomalous filamentous cells (partly in the state of lysis) appeared in the cultural broth. Only PAO cells that had lost their plasmid were capable of normal growth in a medium with MPA at an elevated temperature; this creates a convenient system for selection of clones that have lost the plasmids of naphthalene biodegradation. Some of these plasmids can inhibit growth of Pseudomonas strains at an elevated temperature; this fact should be taken into account when the capability of Pseudomonas to grow at a high temperature is used as a taxonomic feature.  相似文献   

9.
Genes for catechol 1,2- and 2,3-dioxygenases were cloned. These enzymes hold important positions in the ortho and meta pathways of the metabolism of aromatic carbons by microbial associations that consume the following volatile organic compounds in pilot minireactors: toluene, styrene, ethyl benzene, o-xylene, m-xylene, and naphthalene. Genes of both pathways were found in an association consuming m-xylene; only genes of the ortho pathway were found in associations consuming o-xylene, styrene, and ethyl benzene, and only genes of the meta pathway were found in associations consuming naphthalene and toluene. Genes of the ortho pathway (C12O) cloned from associations consuming o-xylene and ethyl benzene were similar to corresponding genes located on the pND6 plasmid of Pseudomonas putida. Genes of the ortho pathway from associations consuming o-xylene and m-xylene were similar to chromosomal genes of P. putida. Genes of the meta pathway (C23O) from associations consuming toluene and naphthalene were similar to corresponding genes formerly found in plasmids pWWO and pTOL.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 298–302.Original Russian Text Copyright © 2005 by Khomenkov, Shevelev, Zhukov, Kurlovich, Zagustina, Popov.  相似文献   

10.
Summary A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class 11 transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.Abbreviations Ap ampicillin - Cb carbenicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - Tc tetracycline - Tp trimethoprim  相似文献   

11.
Analysis of seven plasmids (77 to 135 kb in size) of the P-7 incompatibility group that are responsible for the biodegradation of naphthalene and salicylate has shown that the main natural host of IncP-7 plasmids is the species Pseudomonas fluorescens. The IncP-7 plasmids are structurally diverse and do not form groups, as is evident from their cluster analysis. The naphthalene catabolism genes of six of the IncP-7 plasmids are conservative and homologous to the catabolic genes of NAH7 and pDTG1 plasmids. The pAK5 plasmid contains the classical nahA gene, which codes for naphthalene dioxygenase, and the salicylate 5-hydroxylase gene (nagG) sequence, which makes the conversion of salicylate to gentisate possible.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 342–348.Original Russian Text Copyright © 2005 by Izmalkova, Sazonova, Sokolov, Kosheleva, Boronin.  相似文献   

12.
Most of the known naphthalene biodegradation plasmids determine the process of naphthalene degradation via salicylate and catechol using the meta pathway of catechol degradation. However, Pseudomonas putida strains with plasmids pBS2, pBS216, pBS217 and NPL-1 exert no activity of the enzymes involved in the meta pathway of catechol degradation. When 2-methylnaphthalene was added to the medium as a sole carbon source, mutants growing on this compound were isolated in the strains with the studied plasmids. Plasmid localization of the mutations was established using conjugation transfer as well as by obtaining spontaneous variants that had lost the ability to grow on 2-methylnaphthalene; the respective plasmid mutants were referred to as pBS101, pBS102, pBS103 and pBS105. The strains with the mutant plasmids were tested for the activity of the key enzymes involved in naphthalene catabolism and the activity of catechol-2,3-dioxygenase was found. The data allow one to arrive at the conclusion that plasmids pBS2, pBS216, pBS217 and NPL-1 contain silent genes for the meta pathway of catechol degradation, which are activated by the respective mutations.  相似文献   

13.
Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.  相似文献   

14.
The paper contains the data on the structure of NPL-1 plasmid controlling naphthalene biodegradation. The plasmid which pertains to the P-9 incompatibility group is transferrable conjugatively and is maintained stably within a wide range of gram-negative bacteria. The analysis of mutants and transposon derivatives of the plasmid made it possible to localize nah-genes in a DNA fragment, 23 kb in size. An inverted DNA segment of 4.2 kb was discovered which may participate in the regulation of nah-genes expression. The other peculiar features of NPL-1 were found distinguishing it from NAH and NAH7 plasmids described in literature.  相似文献   

15.
16.
Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-born genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.  相似文献   

17.
Sphingomonas yanoikuyae strain B1 is able to degrade a wider range of aromatic hydrocarbons than S. paucimobilis strain TNE12 can degrade. Various culture techniques were used to corroborate that B1 used m-xylene, biphenyl, toluene, naphthalene, and phenanthrene as sole carbon and energy sources. In contrast, TNE12 could not mineralize m-xylene, biphenyl, toluene, or naphthalene. However, fluoranthene served as carbon and energy source for TNE12 but not B1. Southern blots were performed using the cloned genomic region (approximately 23 kb) containing the degradative genes for the upstream pathways for biphenyl and m-xylene and a TOL plasmid-type meta operon from B1 as a probe against the Kpn I restriction-digested total DNA of TNE12. This 23 kb probe hybridized to three Kpn I-digested fragments of TNE12 DNA; thus significant homology existed between the aromatic hydrocarbon-degrading genes of B1 and TNE12. Further work with smaller probes revealed, however, that TNE12 DNA fragments did not hybridize with the probe containing the genes encoding for xylene monooxygenase and part of an aromatic dioxygenase. A recombinant plasmid, which contains only the genes for xylene monooxygenase, is able to complement TNE12 on m-xylene. These genes are, therefore, probably missing from TNE12. Hence, TNE12 cannot use monocylclic aromatics whereas B1 can. Pulsed field gel electrophoresis coupled with Southern blotting revealed that the aromatic degradative genes were on an approximately 240 kb plasmid of TNE12; the same genes in B1 are known to be chromosomal.  相似文献   

18.
Summary The genetic environment of plasmid-borne bla TEM mutant genes, encoding nine distinct TEM-type extended-spectrum -lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine bla TEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes bla TEM-1 or –2. A 6.6 kb DNA fragment of pCFF04 containing bla TEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that bla TEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was, in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with bla TEM-3.  相似文献   

19.
Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10–5–10–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.  相似文献   

20.
Summary To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from theStreptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in theStreptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterlessxylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes inS. griseofuscus giving operon fusions in some cases that drive expression of thexylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.  相似文献   

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