Microbial degradation of quinoline and methylquinolines

Several bacterial cultures were isolated that are able to degrade quinoline and to transform or to degrade methylquinolines. The degradation of quinoline by strains of Pseudomonas aeruginosa QP and P. putida QP produced hydroxyquinolines, a transient pink compound, and other undetermined products. The quinoline-degrading strains of P. aeruginosa QP and P. putida QP hydroxylated a limited number of methylquinolines but could not degrade them, nor could they transform 2-methylquinoline, isoquinoline, or pyridine. Another pseudomonad, Pseudomonas sp. strain MQP, was isolated that could degrade 2-methylquinoline. P. aeruginosa QP was able to degrade or to transform quinoline and a few methylquinolines in a complex heterocyclic nitrogen-containing fraction of a shale oil. All of the quinoline- and methylquinoline-degrading strains have multiple plasmids including a common 250-kilobase plasmid. The 225-, 250-, and 320-kilobase plasmids of the P. aeruginosa QP strain all contained genes involved in quinoline metabolism.     

Hydroxylation and biodegradation of 6-methylquinoline by pseudomonads in aqueous and nonaqueous immobilized-cell bioreactors.

Selective culturing of pseudomonads that could degrade quinoline led to enrichment cultures and pure cultures with expanded substrate utilization and transformation capabilities for substituted quinolines in immobilized and batch cultures. Immobilized cells of the pseudomonad cultures rapidly transformed quinolines to hydroxyquinolines in bioreactors and were able to tolerate higher substrate concentrations compared with batch cultures. After prolonged incubation on a mixture of quinoline and 6-methylquinoline, a quinoline-degrading culture of Pseudomonas putida developed the ability to biodegrade 6-methylquinoline, which initially was resistant to microbial attack, as a sole source of carbon and energy. 6-Methylquinoline was also degraded in a nonaqueous solution by this strain of P. putida when a solution of 6-methylquinoline in decane was flowed through an immobilized-cell fixed-bed bioreactor.     

Recruitment of a chromosomally encoded maleylacetate reductase for degradation of 2,4-dichlorophenoxyacetic acid by plasmid pJP4.

When Pseudomonas aeruginosa PAO1c or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes enzymes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not grow on TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6- and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1.     

Identification of small RNAs involved in nitrogen fixation in Anabaena sp. PCC 7120 based on RNA-seq under steady state conditions

The aim of the present study was to investigate the tolerance of five new Achromobacter and Pseudomonas strains to kerosene and to establish if the production of several secondary metabolites increases or not when these bacteria were grown in the presence of kerosene. The biodegradation of kerosene by isolated bacteria was also investigated in this study. Five Proteobacteria were isolated from different samples polluted with petroleum and petroleum products. Based on their morphological, biochemical, and molecular characteristics, isolated bacteria were identified as Achromobacter spanius IBBPo18 and IBBPo21, Pseudomonas putida IBBPo19, and Pseudomonas aeruginosa IBBPo20 and IBBPo22. All these bacteria were able to tolerate and degrade kerosene. Higher tolerance to kerosene and degradation rates were observed for P. aeruginosa IBBPo20 and IBBPo22, compared with that observed for A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19. All these bacteria were able to produce several secondary metabolites, such as surfactants and pigments. Glycolipid surfactants produced by P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 have a very good emulsification activity, and their activity increased when they were grown in the presence of kerosene. The production of rhamnolipid surfactants by P. aeruginosa IBBPo20 and IBBPo22 was confirmed by detection of rhlAB gene involved in their biosynthesis. Pyocyanin and pyoverdin pigments were produced only by P. aeruginosa IBBPo20 and IBBPo22, while carotenoid pigments were produced by all the isolated bacteria. Significant changes in pigments production were observed when P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 were grown in the presence of kerosene. Due to their ability to tolerate and degrade kerosene, and also to produce several secondary metabolites, the isolated bacteria could be used in the bioremediation of kerosene-polluted environments.     

Isolation and characterization of Pseudomonas aeruginosa R'' plasmids constructed by interspecific mating

Plasmid R68.45 was used to construct R' plasmids carrying a maximum of 4 to 5 map minutes of the Pseudomonas aeruginosa PAO chromosome by interspecific mating, using P. putida PPN as the recipient. These R' plasmids were used to determine the map location of the amiE locus and to identify tentatively a number of P. putida auxotrophic mutations. Some of these R' plasmids could not be maintained in recombination-deficient P. aeruginosa strains.     

Effect of plasmids from various incompatibility groups on the development of bacteriophages specific to Pseudomonas aeruginosa and Pseudomonas putida

The aim of this work was to study the effect of plasmids belonging to different incompatibility groups on the growth of bacteriophages in Pseudomonas aeruginosa and Pseudomonas putida strains. The growth of bacteriophages was shown to be limited most often due to the presence in cells of plasmids belonging to the P-2 incompatibility group. Plasmids of the Inc P-2 group differed from one another in the spectrum of bacteriophages whose growth they limited. Phages whose growth was suppressed in strains containing plasmids of the P-5, P-9 or P-10 incompatibility groups were found. Some plasmids showed no specific interaction with bacteriophages. The plasmids investigated differed in the studied trait in P. aeruginosa and P. putida cells. In contrast to P. aeruginosa PAO, P. putida PpGI plasmid containing cells did not maintain the growth of donor-specific bacteriophages and, to a lesser degree, limited the growth of phages specific for P. putida PpGI.     

TOL plasmid in Pseudomonas aeruginosa PAO: thermosensitivity of self-maintenance and inhibition of host cell growth.

The TOL plasmid originally isolated in Pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of Pseudomonas, i.e., P. putida, P. fluorescens, and P. aeruginosa, except for a strain of P. aeruginosa, strain PAO. The same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. In addition, the transmissibility of the TOL plasmid from strain PAO to P. putida was low when the plasmid was modified by the donor. By using P. aeruginosa PAO carrying the TOL plasmid, the stability and genetic expression of the plasmid as well as its effect on the host cell growth were examined. Thus the self-maintenance of the plasmid was found to be thermosensitive. Furthermore, the TOL plasmid inhibited the growth of strain PAO at high temperature, accompanied by the formation of some filamentous cells. These thermosensitive properties of the TOL plasmid were host dependent and not exhibited in another strain of P. aeruginosa.     

A comparative study of acquired amidase activity in Pseudomonas species

Pseudomonas putida PP3 carrying dehalogenases I and II and Pseudomonas aeruginosa PAU3 carrying dehalogenase I coded for by plasmid pUU2 were able to grow on 2-monochloropropionic acid (2MCPA). Neither strain utilized 2-chloropropionamide (2CPA) as a carbon or nitrogen source for growth. Mutations in both strains to 2Cpa+ phenotypes (designated P. putida PPW3 and P. aeruginosa PAU5, respectively) involved the expression of an acquired 2CPA-amidase activity. The amidase followed by dehalogenase reactions in these strains constituted a novel metabolic pathway for growth on 2CPA. P. putida PPW3 synthesized a constitutive amidase of molecular mass 59 kDa consisting of two identical subunits of 29 kDa. For those amides tested this acquired enzyme was most active against chlorinated aliphatic amides, although substrate affinities (Km) and maximum rates of activity (Vmax) were poor. P. aeruginosa PAU5 acquired a 2Cpa+ phenotype by overproducing the A-amidase normally used by this species to hydrolyse aliphatic amides. The A-amidase had only slight activity towards 2CPA. However, with constitutive synthesis the mutant grew on the chlorinated substrates. Chloroacetamide (CAA) was a toxic substrate analogue for these Pseudomonas strains. A strain resistant to CAA was isolated from P. aeruginosa PAU5 when exposed to 1-10 mM-CAA. This mutant, P. aeruginosa PAU6, synthesized an inducible A-amidase. CAA-resistance depended upon the simultaneous expression of CAA-inducible amidase and dehalogenase activities.     

Isolation of soil bacteria able to hydrolyze both organophosphate and carbamate pesticides

Two bacteria identified as Pseudomonas putida and Acinetobacter rhizosphaerae able to rapidly degrade the organophosphate (OP) fenamiphos (FEN) were isolated. Denaturating gradient gel electrophoresis analysis revealed that the two isolates were dominant members of the enrichment culture. Clone libraries further showed that bacteria belonging to α-, β-, γ-proteobacteria and Bacteroidetes were also present in the final enrichment but were not isolated. Both strains hydrolyzed FEN to fenamiphos phenol which was further transformed, only by P. putida. The two strains were using FEN as C and N source. Cross-feeding studies with other pesticides showed that P. putida degraded OPs with a P-O-C linkage and unexpectedly degraded the carbamates oxamyl and carbofuran being the first wild-type bacterial strain able to degrade both OPs and carbamates. The same isolate exhibited high bioremediation potential against spillage-level concentrations of aged residues of FEN and its oxidized derivatives.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light.

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

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.     

Isolation of plasmid deoxyribonucleic acid from Pseudomonas putida.

Conditions suitable for reproducible recovery of covalently closed circular deoxyribonucleic acid from strains of Pseudomonas putida containing degradative plasmids (CAM, SAL, OCT, etc.) have been defined. These degradative plasmids could not be isolated by the usual procedure, whereas RP1, an R factor of the P group, present in the isogenic strain of P. putida, was isolated equally well by either the usual procedure or the modified procedure. Characterization by electron microscopy of RP1 deoxyribonucleic acid confirmed the molecular weight (about 40 X 10(6)) previously determined by sucrose gradient centrifugation.     

Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight] h). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Recipient Range of IncP-7 Conjugative Plasmid pCAR2 from Pseudomonas putida HS01 is Broader than from Other Pseudomonas Strains

The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.     

IncP-7 naphthalene-degradative plasmids from Pseudomonas putida

Abstract Seven naphthalene-degrading and two naphthalene and camphor-degrading Pseudomonas putida strains were isolated from marine sediments. Most of them carried two plasmids, of molecular size 60 and 200 kb. The naphthalene and salicylate metabolism determinants were transferred to a P. putida strain by conjugation, and the transconjugants acquired either both plasmids or only the 200-kb one. These plasmids appear to belong to the IncP-7 group and encode for catabolism of naphthalene and salicylate, but not camphor.     

A strain of Pseudomonas aeruginosa growing on petroleum hydrocarbons

The strain Pseudomonas aeruginosa BS313 was used to isolate mutants that are capable to utilize octane as the sole carbon source. By means of conjugation plasmids of camphor (CAM) and naphthalene (pBS2) biodegradation were inserted into one of the mutant strains P. aeruginosa BS316. The resultant strain P. aeruginosa BS315 shows the capacity to degrade aliphatic, aromatic and cyclic oil hydrocarbons.     

Use of ureidopenicillins for selection of plasmid vector transformants in Pseudomonas aeruginosa and Pseudomonas putida.

Broad-host-range plasmids coding for beta-lactamase were successfully selected after transformation of Pseudomonas strains. Transformants of both Pseudomonas aeruginosa and Pseudomonas putida containing plasmid pRO1614 were isolated in media containing low concentrations of piperacillin. These strains were also susceptible to other ureidopenicillins. Similar selections of transformants with carbenicillin, ampicillin, or ticarcillin required high concentrations of antibiotics and yielded backgrounds of spontaneous resistant mutants.     

Ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria: evidence for the existence of new TOL plasmids.

Thirteen bacteria have been isolated from nine different soil samples by selective enrichment culture on m-toluate (m-methylbenzoate) minimal medium. Eight of these were classified as Pseudomonas putida, one as a fluorescent Pseudomonas sp., and four as nonfluorescent Pseudomonas sp. All 13 strains appeared to carry TOL plasmids superficially similar to that previously described in P. putida mt-2 in that: (i) all the wild-type strains could utilize toluene, m-xylene, and p-xylene as sole carbon and energy sources, (ii) these growth substrates were metabolized through the corresponding alcohols and aldehydes to benzoate, m-toluate, and p-toluate, respectively, and thence by the divergent meta (or alpha-ketoacid) pathway, and (iii) the isolates could simultaneously and spontaneously lose their ability to utilize the hydrocarbons, alcohols, aldehydes, and acids, particularly during growth on benzoate, giving rise to cured strains which could grow only on benzaldehyde and benzoate of the aromatic substrates by the alternative ortho (or beta-ketoadipate) pathway. Eight of the isolates were able to transfer their TOL plasmids into their own cured strains, but only five were able to transfer them in interstrain conjugation into the cured strains, but only five were able to transfer them in interstrain conjugation into the cured derivative of P. putida mt-2. However, P. putida mt-2 was able to transfer its TOL plasmid into 11 of the cured isolates, and eight of these were able to retransmit this foreign plasmid in intrastrain conjugation with their own cured derivatives. Three of the isolates, MT 14, MT 15, and MT 20, differed significantly from the others in that the wild-type strains dissimilated the p-methyl-substituted substrates poorly, and also, during growth on benzoate, in addition to the cured derivatives, they gave rise to derivatives with a phenotype intermediate between the cured and wild-type strains, the biochemical and genetic nature of which has not been elucidated.