Stability of the NPL-1 and NPL-41 plasmids of naphthalene biodegradation in Pseudomonas putida populations in continuous culture

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.     

Thermosensitivity of naphthalene biodegradation plasmids

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.     

Plasmid-borne Tn5 insertion mutation resulting in accumulation of gentisate from salicylate.

Plasmid-borne Tn5 insertion mutants of a Pseudomonas species which accumulated 2,5-dihydroxybenzoate (gentisate) following growth on 2-hydroxybenzoate (salicylate) were obtained from a pool of mutants that were unable to grow on naphthalene. One such mutant was characterized further. The ability of this mutant to oxidize gentisate was 100-fold less than the ability of a Nah+ Sal+ strain harboring the unmutagenized plasmid, although both strains oxidized and grew on salicylate. These bacteria were presumably able to metabolize salicylate via catechol, since they possessed an inducible, plasmid-encoded catechol 2,3-dioxygenase. Our results suggest that there is an alternate, plasmid-encoded route of salicylate degradation via gentisate and that some plasmid-associated relationship between this pathway and naphthalene oxidation exists.     

Plasmid-borne Tn5 insertion mutation resulting in accumulation of gentisate from salicylate

Plasmid-borne Tn5 insertion mutants of a Pseudomonas species which accumulated 2,5-dihydroxybenzoate (gentisate) following growth on 2-hydroxybenzoate (salicylate) were obtained from a pool of mutants that were unable to grow on naphthalene. One such mutant was characterized further. The ability of this mutant to oxidize gentisate was 100-fold less than the ability of a Nah+ Sal+ strain harboring the unmutagenized plasmid, although both strains oxidized and grew on salicylate. These bacteria were presumably able to metabolize salicylate via catechol, since they possessed an inducible, plasmid-encoded catechol 2,3-dioxygenase. Our results suggest that there is an alternate, plasmid-encoded route of salicylate degradation via gentisate and that some plasmid-associated relationship between this pathway and naphthalene oxidation exists.     

Effect of transposons on expression of genes for naphthalene biodegradation in Pseudomonas putida BS202(NPL-1) and derivative strains

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.     

Preparation of bacteria--degraders of alkylnaphthalenesulfonate by a molecular breeding method

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.     

The effect of transposons on the expression of the naphthalene biodegradation genes in Pseudomonas putida BS202(NPL-1) and derivative strains

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 Pseudomonas putida strains grown on different substrates depends on the expression of the nah and tnp genes.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 79–86.Original Russian Text Copyright © 2005 by Sokolov, Kosheleva, Filonov, Boronin.     

Nah-genes of Pseudomonas putida: molecular genetic analysis of the plasmid pBS286

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.     

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.     

Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba.

Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway. A strain of P. putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene. Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate). However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.     

Plasmid-mediated degradation of dibenzothiophene by Pseudomonas species.

The microbial transformation of dibenzothiophene (DBT) is of interest in the potential desulfurization of oil. We isolated three soil Pseudomonas species which oxidized DBT to characteristic water-soluble, sulfur-containing products. Two of our isolates harbored a 55-megadalton plasmid; growth in the presence of novobiocin resulted in both loss of the plasmid and loss of the ability to oxidize DBT. Reintroduction of the plasmid restored the ability to oxidize DBT to water-soluble products. The products resulting from the oxidation of DBT were characterized and included 3-hydroxy-2-formyl benzothiophene, 3-oxo-[3'-hydroxy-thionaphthenyl-(2)-methylene]-dihydrothionaph thene, and the hemiacetal and trans forms of 4-[2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoic acid. The products of DBT oxidation were inhibitory to cell growth and further DBT oxidation. DBT oxidation in our soil isolates was induced by naphthalene or salicylate and to a much lesser extent by DBT and was repressed by succinate.     

Plasmid-mediated degradation of dibenzothiophene by Pseudomonas species

The microbial transformation of dibenzothiophene (DBT) is of interest in the potential desulfurization of oil. We isolated three soil Pseudomonas species which oxidized DBT to characteristic water-soluble, sulfur-containing products. Two of our isolates harbored a 55-megadalton plasmid; growth in the presence of novobiocin resulted in both loss of the plasmid and loss of the ability to oxidize DBT. Reintroduction of the plasmid restored the ability to oxidize DBT to water-soluble products. The products resulting from the oxidation of DBT were characterized and included 3-hydroxy-2-formyl benzothiophene, 3-oxo-[3'-hydroxy-thionaphthenyl-(2)-methylene]-dihydrothionaph thene, and the hemiacetal and trans forms of 4-[2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoic acid. The products of DBT oxidation were inhibitory to cell growth and further DBT oxidation. DBT oxidation in our soil isolates was induced by naphthalene or salicylate and to a much lesser extent by DBT and was repressed by succinate.     

Expression of dibenzothiophene-degradative genes in two Pseudomonas species

The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers. This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp. HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype). Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b. Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b. Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains. DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes. Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.     

Mutants of the plasmid for biodegradation of naphthalene, determining catechol oxidation via the meta-pathway

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.     

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4.

Rhodococcus sp. strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy. Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene. In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp. strain B4. The key enzymes of naphthalene degradation in Rhodococcus sp. strain B4 have unusual cofactor requirements. The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.     

Effect of sodium salicylate on the population dynamics of a rhizospheric strain of Pseudomonas aureofaciens in soil and on wheat roots

The effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens BS1393 and its variant bearing the naphthalene biodegradation plasmid pBS216 was studied in the wheat rhizoplane and adjacent soil. Optimum salicylate concentration for the maintenance of the plasmid-bearing strain and for the normal growth of wheat was found to be 250 micrograms/g soil. When the soil was supplemented with salicylate, the population of P. aureofaciens BS1393(pBS216) in the wheat rhizoplane and adjacent soil was, respectively, 4- and 20-fold higher than that of the parent strain lacking the plasmid.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Physiological role of NahW, the additional salicylate hydroxylase found in Pseudomonas stutzeri AN10

The physiological role of NahW, the second salicylate hydroxylase of Pseudomonas stutzeri AN10, has been analysed by gene mutation and further complementation. When grown on naphthalene as a unique carbon and energy source, the nahW mutant showed a strong decrease in salicylate hydroxylase activity when compared with the wild-type strain, exhibited lower specific growth rates and accumulated salicylate in culture supernatants. Similarly, lower specific growth rates and salicylate accumulation were observed for the nahW mutant when growth on naphthalene supplemented with succinate or pyruvate. When P. stutzeri AN10 was grown in Luria–Bertani medium in the presence of salicylate, or was cultivated on minimal medium supplemented with salicylate as a unique carbon and energy source, an increase in the lag phase and a decrease in the specific growth rate were observed on increasing the salicylate concentrations, suggesting a plausible toxic effect. This toxic effect of salicylate was much more evident for the nahW mutant than for the wild-type strain. Complementation of the nahW mutant restored all growth parameters. These results indicate that NahW may have two functions in P. stutzeri AN10: (1) to improve its capacity to degrade naphthalene and (2) effectively convert the salicylate produced during naphthalene degradation to tricarboxylic acid cycle intermediates, preventing its toxic effect.     

Preliminary study on relationships among strains forming a bacterial community selected on naphthalene from a marine sediment

Two bacterial strains were isolated from a bacterial community formed of nine strains, selected from a marine sediment on a seawater medium with naphthalene as sole carbon source. The two strains studied in the present work were the only strains of this community able to grow in pure culture on naphthalene; therefore, they were called "primary" strains. The seven other strains were maintained in the community by using metabolic intermediates of the two primary strains; they were called "auxiliary" strains. Regulation of naphthalene metabolism was studied for the two primary strains. They oxidized naphthalene into catechol, which was degraded only by the meta pathway. For Pseudomonas Lav. 4, naphthalene oxygenase and salicylate hydroxylase were inducible; catechol 2,3-dioxygenase was constitutive. For Moraxella Lav. 7, naphthalene oxygenase was constitutive; salicylate hydroxylase and catechol 2,3-oxygenase were inducible. The Moraxella strain carries two cryptic plasmids, about 63- and 85-kb in molecular size. In the bacterial community culture medium, Moraxella Lav. 7 prevented accumulation of 2-hydroxymuconate semialdehyde formed by Pseudomonas Lav. 4. The auxiliary strains take up formic, acetic, pyruvic, propionic, and succinic acids released by the two primary strains.