Biodegradation Plasmid Research Articles

Inheritance of IncP-9 Catabolic Plasmids in Pseudomonas Bacteria

This work investigated the capability of the conjugative transfer of epsilon-caprolactam biodegradation (CAP) plasmids, belonging to incompatibility (Inc) groups P-2, P-7, P-9, and IncP-9 naphthalene biodegradation (NAH) plasmids to various species of Pseudomonas bacteria, including strains of plant growth-promoting rhizobacteria (PGPR), and the character of plasmid inheritance in these strains. The frequency of the conjugative transfer of all CAP and NAH plasmids (irrespective of their Inc groups) into Pseudomonas PGPR was shown to be lower than that into P. putida soil strains by more than an order of magnitude. The low frequency of conjugative transfer was found not to be caused by secretion of antibiotically active metabolites by Pseudomonas PGPR strains. Stability of inheritance of catabolic plasmids under nonselective conditions was established to depend on their Inc group and bacterial host strain. Thus, all investigated plasmids were stably maintained in P. putida KT2442 and BS394 strains for at least 100 generations. However, only CAP plasmids of IncP-2 and IncP-7 groups were stably maintained in Pseudomonas PGPR cells. CAP and NAH plasmids of different IncP-9 subgroups were eliminated from cells of rhizosphere pseudomonads within several generations. Thus, the work first revealed inheritance features of IncP-9 catabolic plasmids in Pseudomonas PGPR cells.

Application of High-Performance Liquid Chromatography/High Resolution Mass Spectrometry to the Investigation of the Biodegradation and Transformation of Phenanthrene by a Plasmid Bearing Rhizosphere Bacteria Pseudomonas aureofaciens

Mass spectrometry was applied to identify metabolites and estimate the efficiency of phenanthrene biodegradation and transformation by rhizosphere bacteria Pseudomonas aureofaciens BS1393. Strains P. aureofaciens BS1393(pOV17) and P. aureofaciens BS1393(NPL-41) bearing various naphthalene biodegradation plasmids were used in the work. The strain BS1393(pOV17) contains the pOV17 wild type naphthalene biodegradation plasmid that determines the oxidation of naphthalene to Krebs cycle metabolites. The strain BS1393(NPL-41) contains the mutant plasmid NPL-41 governing the initial stages of naphthalene oxidation into salicylic acid. The limiting stages of phenanthrene biodegradation in bacteria with various plasmids have been identified according to the accumulation of intermediates. When bacteria were grown on phenanthrene, the main metabolites were as follows: (a) 2-hydroxy-2H-benzo[h]chromene-2-carboxylic acid/trans-4-(1-hydroxynaph-2-yl)-2-oxobut-3-enoic acid, (b) 1-hydroxy-2-naphthoic acid, and (c) salicylic acid. In the strain BS1393(pOV17), metabolite (а) was observed during 1–14 days of cultivation. Unlike it, in the strain BS1393(NPL-41), an insignificant amount of this metabolite was found after only 14 days. The availability of metabolite (b) in the growth of both strains was an evidence of the limited rate of its further decarboxylation. Metabolite (c) as a final product was found in the growth of the strain BS1393(NPL-41). Contrastingly, in the strain BS1393(pOV17), this metabolite was not found, which indicates the complete oxidation of phenanthrene.

Селекция мутантов по системе инициации репликации плазмиды pBS267 группы IncP-9

A minireplicon containing the rep gene and oriV site of the gamma subgroup of the IncP-9 caprolactam pBS267 biodegradation plasmid was cloned for the first time. It was established that a minimized variant of pBS267 plasmid cannot be sustained in E. coli and is inherited in an unstable way in bacteria Pseudomonas. Using in vitro mutagenesis, mutant variants of the minireplicon were produced, characterized by an increased number of copies in cells, the ability to replicate in E. coli, and relatively stable inheritance in P. putida cells. The obtained constructs are the basis for a study of the replication mechanisms of IncP-9 group plasmids, as well as use as vectors for molecular cloning in a wide range of gram-negative bacteria.

Transformation of low-molecular linear caprolactam oligomers by the caprolactam-degrading bacterium Pseudomonas putida BS394(pBS268)

A biosensor based on the most active caprolactam-degrading strain Pseudomonas putida BS394(pBS268) was used in the study of aerobic degradation of linear caprolactam oligomers by bacterial cells. The changes in the respiratory activity of the strain depend quantitatively on caprolactam dimer concentration, making it possible to develop biosensors for detection of caprolactam oligomers in aqueous media. Based on mass spectrometry data, the scheme of transformation of linear caprolactam oligomers by the degrader strain P. putida BS394(pBS268) was proposed for the first time. It was found that oxidative transamination to respective dicarbonic acids may be one of the mechanisms of transformation of linear caprolactam oligomers. According to the scheme proposed, the ability of the caprolactam-degrading strain to transform linear oligomers results from the broad substrate specificities of two enzymes of the caprolactam degradation pathway: 2-oxoglutarate-6-aminohexanoate transaminase and 6-oxohexanoate dehydrogenase. Transformation of linear oligomers is genetically controlled by the CAP biodegradation plasmid pBS268.

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.

Change of oil-degrading activity in microorganisms stored under laboratory conditions

Change of the oil-degrading activity was studied in psychrophilic microbial strains Rhodococcus spp. DS-07, DS-21 and Pseudomonas spp. DS-09, DS-22 maintained on various media: rich and synthetic with a selective agent. After 2.5 years of storage on rich medium, the oil-degrading activity decreased by 50-60%, whereas this decrease was insignificant in the medium with oil. Passages to selective medium with oil after the storage partly restored the activity. It was found that storage of oil-degrading microorganisms caused loss of biodegradation plasmids. Their recovery and long-term preservation demand the presence of the selective agent in the medium.

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.

Inheritance of biodegradation plasmids in the cells of homo- and heterologous hosts

A systematic analysis of the inheritance of D plasmids of the IncP-9 group (alpha-, beta-, gamma-, delta-, epsilon-, zeta-, eta-, theta-subgroups), IncP-7, as well as of those of undefined systematic affiliation in the cells of homologous (Pseudomonas putida) and heterologous (Escherichia coli) hosts was performed for the first time. For this purpose, mini-Tn5 transposons determining resistance to kanamycin (or streptomycin) were introduced into all the D plasmids under study. It has been established that all IncP-9 plasmids can be transmitted to the cells of a heterologous host E. coli (with the exception of plasmid pSVS15 from theta-subgroup). IncP-7 plasmids and those of undefined systematic affiliation do not possess this property and can be transmitted and stably inherited only in P. putida. The distinctive feature of most IncP-9 plasmids (alpha-, beta-, gamma-, delta-, epsilon-, and zeta-subgroups) is strict dependence of their inheritance on the temperature factor. At 37 degrees C, the plasmids of delta-, zeta-, and theta-subgroups are unstable in P. putida cells, while in E. coli nearly all plasmids of this systematic group are unstable. The exceptions are the plasmids of eta- and gamma-subgroups. Inheritance of these plasmids does not depend on temperature. At 28 degrees C and 37 degrees C, the eta plasmid is not maintained stably (inheritance stability is 2%), while the gamma plasmid has almost 100% stability under the same conditions.

The Construction and Monitoring of Genetically Tagged, 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.

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.

Effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens in the wheat rhizoplane and adjacent soil.

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 μg/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.

Degradation of 3-chlorobenzoate in soil by pseudomonads carrying biodegradative plasmids.

Degradation of continuously added 3-chlorobenzoate (3-CB) was studied in samples of chernozem soil. Soil columns were inoculated with Pseudomonas putida growing on 3-CB and carrying the biodegradation plasmid and with Pseudomonas aeruginosa incapable of growth on 3-CB and carrying the inserted biodegradation plasmid pBS 2 determining ortho-cleavage of the aromatic ring. While the 3-CB degradation was observed in both inoculated variants, the native microflora of the soil under study was incapable to degrade 3-CB. Among pseudomonads isolated from inoculated soil at different stages of cultivation and growth on 3-CB, some had the taxonomic features of P. putida as well as those differing in 1-5 characteristics. The study of the activities of the enzymes cleaving the aromatic ring revealed the presence of pyrocatechol 1,2-dioxygenase in the isolated strains only, as estimated by means of benzoate and 3-CB as substrates.