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.     

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.     

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.     

Structural and functional variability of genetic systems for catabolizing polycyclic aromatic hydrocarbons in Pseudomonas putida strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1 operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

A comparative study of the NAH and TOL catabolic plasmids in Pseudomonas putida

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives. The NAH and TOL plasmids were found to be incompatible. Under the conditions used in these experiments the TOL plasmid transferred into some strains into which NAH was unable to transfer. The use of mutants to remove certain catabolic activities encoded by the bacterial host cell facilitated the allocation of growth genotypes to the NAH and TOL plasmids. TOL encoded the degradation of benzoate, m-toluate and p-toluate, whereas NAH encoded the degradation of naphthalene and salicylate. The other plasmid-associated growth phenotypes were partly plasmid-specified and partly specified by the host cell. The pH optimum of the catechol 2,3-dioxygenase specified by the TOL plasmid was approximately 6.7, whereas that of the NAH-encoded enzyme was approximately 8.3.     

Diversity of genetic systems responsible for biodegradation of naphthalene in Pseudomonas fluorescens strains

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 thirteen strains contain plasmids, from 20 to 120 kb in size, at least five 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.     

Molecular classification of IncP-9 naphthalene degradation plasmids

A large collection of naphthalene-degrading fluorescent Pseudomonas strains isolated from sites contaminated with coal tar and crude oil was screened for the presence of IncP-9 plasmids. Seventeen strains were found to carry naphthalene catabolic plasmids ranging in size from 83 to 120 kb and were selected for further study. Results of molecular genotyping revealed that 15 strains were closely related to P. putida, one to P. fluorescens, and one to P. aeruginosa. All catabolic plasmids found in these strains, with the exception of pBS216, pSN11, and p8909N-1, turned out to belong to IncP-9 beta-subgroup. Plasmids pBS216, pSN11, and p8909N-1 were identified as members of IncP-9 delta-subgroup. One plasmid, pBS2, contains fused replicons of IncP-9beta and IncP-7 groups. RFLP analyses of the naphthalene catabolic plasmids revealed that organisation of the replicon correlates well with the overall plasmid structure. Comparative PCR studies with conserved oligonucleotide primers indicated that genes for key enzymes of naphthalene catabolism are highly conserved among all studied plasmids. Three bacterial strains, P. putida BS202, P. putida BS3701, and P. putida BS3790, were found to have two different salicylate hydroxylase genes one of which has no similarity to the "classic" enzyme encoded by nahG gene. Discovery of a large group of plasmid with unique nahR suggested that the regulatory loop may also represent a variable part of the pathway for catabolism of naphthalene in fluorescent Pseudomonas spp.     

Maintenance and stability of introduced genotypes in groundwater aquifer material.

Three indigenous groundwater bacterial strains and Pseudomonas putida harboring plasmids TOL (pWWO) and RK2 were introduced into experimentally contaminated groundwater aquifer microcosms. Maintenance of the introduced genotypes was measured over time by colony hybridization with gene probes of various specificity. On the basis of the results of colony hybridization quantitation of the introduced organisms and genes, all introduced genotypes were stably maintained at approximately 10(5) positive hybrid colonies g-1 of aquifer microcosm material throughout an 8-week incubation period. Concomitant removal of the environmental contaminants, viz., toluene, chlorobenzene, and styrene, in both natural (uninoculated) and inoculated aquifer microcosms was also demonstrated. The results indicate that introduced catabolic plasmids, as well as indigenous organisms, can be stably maintained in groundwater aquifer material without specific selective pressure for the introduced genotypes. These results have positive implications for in situ treatment and biodegradation in contaminated aerobic groundwater aquifers.     

Maintenance and stability of introduced genotypes in groundwater aquifer material

Three indigenous groundwater bacterial strains and Pseudomonas putida harboring plasmids TOL (pWWO) and RK2 were introduced into experimentally contaminated groundwater aquifer microcosms. Maintenance of the introduced genotypes was measured over time by colony hybridization with gene probes of various specificity. On the basis of the results of colony hybridization quantitation of the introduced organisms and genes, all introduced genotypes were stably maintained at approximately 10(5) positive hybrid colonies g-1 of aquifer microcosm material throughout an 8-week incubation period. Concomitant removal of the environmental contaminants, viz., toluene, chlorobenzene, and styrene, in both natural (uninoculated) and inoculated aquifer microcosms was also demonstrated. The results indicate that introduced catabolic plasmids, as well as indigenous organisms, can be stably maintained in groundwater aquifer material without specific selective pressure for the introduced genotypes. These results have positive implications for in situ treatment and biodegradation in contaminated aerobic groundwater aquifers.     

Horizontal transfer of catabolic plasmids and naphthalene biodegradation in open soil

The horizontal transfer of naphthalene biodegradation plasmids and the parallel process of its microbial degradation were studied for the first time. The tagged naphthalene-degrading strains bearing labeled biodegradation plasmids were used for the monitoring of horizontal plasmid transfer in open soil. The population kinetics of microorganisms, the survival rate and competitiveness of introduced strains, and the transfer of biodegradation plasmids to indigenous strains were investigated. The transfer of the labeled plasmid pNF142::TnMod-OTc to the introduced plasmid-free recipient P. putida KT2442 and to indigenous soil microorganisms of the genus Pseudomonas was shown both under selection pressure (in the presence of naphthalene) and in its absence. The 16S rRNA gene sequencing showed that the soil strains that had acquired plasmids were close to the species P. lini, P. frederiksbergensis, P. jessenii, P. graminis, P. putida, and P. alcaligenes. Thus, direct evidence of dissemination of the naphthalene biodegradation plasmids in microbial populations in open soil under selective and nonselective conditions has been obtained.     

Structural and Functional Variation in Genetic Systems for Catabolism of Polycyclic Aromatic Hydrocarbons in Pseudomonas putida Strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

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.     

Incompatibility group P-7 plasmids responsible for biodegradation of naphthalene and salicylate in fluorescent pseudomonads

Analysis of seven plasmids (77 to 135 kbp 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.     

Influence of soil pollution on the composition of a microbial community

The abundance dynamics and composition of indigenous soil microbial communities were studied in soils polluted with naphthalene, dioctyl phthalate, diesel fuel, and crude oil. DGGE analysis of the 16S rRNA genes amplified from the total soil DNA revealed that the bacterial community of uncontaminated soil was more diverse and included no dominant species. In the soil samples polluted with the crude oil, diesel fuel, or dioctyl phthalate, Pseudomonas became the dominant bacteria since the third day of the experiment. In the soil polluted with naphthalene, two genera of bacteria (Pseudomonas and Paenibacillus) were dominant in population on the third day of the experiment, while on the 21th day of the experiment Arthrobacter became dominant. During the experiment, the average number of indigenous bacterial degraders increased approximately by two orders of magnitude. While the key genes of naphthalene catabolism, nahAc and nahH, were not detected in the pristine soil, they were found in a significant amount on the third day after naphthalene addition. Three degrader strains harboring the plasmids of naphthalene biodegradation (IncP-9 group) were isolated on the third day from the soil polluted with naphthalene. Two of these plasmids, although isolated from various degraders, were shown to be identical.     

Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.     

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.     

Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil.

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.     

Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil.

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.     

Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region

Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.     

Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients.

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.