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.     

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.     

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.     

Control of catechol meta-cleavage pathway in Alcaligenes eutrophus

Alcaligenes eutrophus 335 (ATCC 17697) metabolizes phenol and p-cresol via a catechol meta-cleavage pathway. Studies with mutant strains, each defective in an enzyme of the pathway, showed that the six enzymes assayed are induced by the primary substrate. Studies with a putative polarity mutant defective in the expression of aldehyde dehydrogenase suggested that the structural genes encoding this and subsequent enzymes of the pathway exist in the same operon. From studies with mutant strains that constitutively synthesize catechol 2,3-oxygenase and subsequent enzymes and from the coordination of repression of these enzymes by p-toluate, benzoate, and acetate, it is proposed the catechol 2,3-oxygenase structural gene is situated in this operon (2,3-oxygenase operon). Studies with regulatory mutant strains suggest that the 2,3-oxygenase operon is under negative control.     

Characterization of the naphthalene-degrading bacterium, Rhodococcus opacus M213

Bacterial strain M213 was isolated from a fuel oil-contaminated soil in Idaho, USA, by growth on naphthalene as a sole source of carbon, and was identified as Rhodococcus opacus M213 by 16S rDNA sequence analysis and growth on substrates characteristic of this species. M213 was screened for growth on a variety of aromatic hydrocarbons, and growth was observed only on simple 1 and 2 ring compounds. No growth or poor growth was observed with chlorinated aromatic compounds such as 2,4-dichlorophenol and chlorobenzoates. No growth was observed by M213 on salicylate, and M213 resting cells grown on naphthalene did not attack salicylate. In addition, no salicylate hydroxylase activity was detected in cell free lysates, suggesting a pathway for naphthalene catabolism that does not pass through salicylate. Enzyme assays indicated induction of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase on different substrates. Total DNA from M213 was screened for hybridization with a variety of genes encoding catechol dioxygenases, but hybridization was observed only with catA (encoding catechol 1,2-dioxygenase) from R. opacus 1CP and edoD (encoding catechol 2,3-dioxygenase) from Rhodococcus sp. I1. Plasmid analysis indicated the presence of two plasmids (pNUO1 and pNUO2). edoD hybridized to pNUO1, a very large (approximately 750 kb) linear plasmid.     

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.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

Naphthalene plasmids in pseudomonads.

A rapid method beginning with the direct lysis of bacteria in alkaline sodium dodecyl sulfate was used to detect naphthalene plasmids in pseudomonads. The strains NCIB 9816, PG, ATCC 17483, and ATCC 17484, which can grow on naphthalene as the sole source of carbon and energy, were examined. All except ATCC 17483 contained more than one plasmid. ATCC 17483 did not contain any plasmids. The largest pair of plasmids found in each of NCIB 9816 and PG(NAH2 and NAH3, respectively) determined naphthalene metabolism and could be transferred by conjugation. This also transferred the unusually regulated meta pathway enzymes for catechol metabolism. NAH2 determines the constitutive production of low concentrations of catechol 2,3-dioxygenase and 2-hydroxymuconic acid semialdehyde dehydrogenase, and NAH3 determines the constitutive production of high concentration of these. NAH2 and NAH3 gave identical fragments on digestion with BamHI or HindIII, but these were quite different from those of NAH. Nonetheless, NAH2 and NAH3 hybridized with NAH.     

Growth of bacteria degrading naphthalene and salicylate at low temperatures

A total of 58 bacterial strains degrading naphthalene and salicylate were isolated from soil samples polluted with oil products, collected in different regions of Russia during winter and summer. The isolates were assessed for their ability to grow at low temperatures (4, 8, and 15 degrees C); bacteria growing at 4 degrees C in the presence of naphthalene or salicylate accounted for 65% and 53%, respectively, of the strains isolated. The strains differed in the temperature dependence of their growth rates. It was demonstrated that the type of expression of Nah+ phenotype at low temperatures depended on the combination of the host bacterium and the plasmid.     

Catechol oxygenases of Pseudomonas putida mutant strains.

Investigation of a mutant strain of Pseudomonas putida NCIB 10015, strain PsU-E1, showed that it had lost the ability to produce catechol 1,2-oxygenase after growth with catechol. Additional mutants of both wild-type and mutant strains PsU-E1 have been isolated that grow on catechol, but not on benzoate, yet still form a catechol 1,2-oxygenase when exposed to benzoate. These findings indicate that either there are separately induced catechol 1,2-oxygenase enzymes, or that there are two separate inducers for the one catechol 1,2-oxygenase enzyme. Comparisons of the physical properties of the catechol 1,2-oxygenases formed in response to the two different inducers show no significant differences, so it is more probable that the two proteins are the product of the same gene. Sufficient enzymes of the ortho-fission pathway are induced in the wild-type strain by the initial substrate benzoate (or an early intermediate) to commit that substrate to metabolism by ortho fission exclusively. A mechanism exists that permits metabolism of catechol by meta fission if the ortho-fission enzymes are unable to prevent its intracellular accumulation.     

Specificity of monohydric phenol oxidations by meta cleavage pathways in Pseudomonas aeruginosa T1

Summary The oxidation of several mono-hydric phenols by wild type and mutant strains of Pseudomonas aeruginosa T1 has been studied. The data suggest, that a non-specific enzyme sequence of the meta cleavage pathway is induced by all of these phenols, which can catalyze the oxidation of phenol and its analogues to pyruvate, a fatty acid and a carbonyl compound, according to the general scheme of Dagley et al. (1964). Mutants unable to grow on phenol (hydroxylase-negative), have been isolated, and they are also unable to grown on or oxidize the cresols and the xylenols. Revertants of these mutants regain the capacity to grow on all these phenols and are indistinguishable from the wild type. Induced-substrate relationships for the earlier enzymes of the pathway have been determined, e.g., phenol in addition to catechol and the methylcatechols is an inducer for catechol 2,3-oxygenase. Analysis of the enzymic content of cells grown in a variety of steadystate conditions shows (a) that the ratio of the specific activities of the phenol hydroxylase and catechol 2,3-oxygenase is constant for each of their analogous substrates; and (b) that induction and catabolite repression of catechol 2,3-oxygenase and the muconic semialdehyde hydrolyase are coordinate, but that control of the phenol hydroxylase is independent.Howard Hughes Medical Institute Investigator.     

Isolation and characterization of altered plasmids in mutant strains of Pseudomonas putida NCIB 9816

The ability of P. putida NCIB 9816 to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid, pDTG1. Derivatives of pDTG1 were obtained from cells after exposure to halogenated analogs of naphthalene or salicylate. The selection of mutants having a Nah-Sal- or a Nah-Sal+ phenotype could be enhanced by the addition of triphenyltetrazolium chloride to the indicator medium. Structurally modified plasmids were characterized by restriction endonuclease digestion and Southern hybridization experiments. The region of pDTG1 DNA that encodes the enzymes responsible for the conversion of naphthalene to salicylate was identified. The structural changes in mutant plasmids were correlated with the absence of essential enzymatic activities.     

Metabolism of naphthalene, 2-methylnaphthalene, salicylate, and benzoate by Pseudomonas PG: regulation of tangential pathways.

Naphthalene is metabolized by Pseudomonas PG through 1,2-dihydroxynaphthalene and salicylate to catechol, which is then degraded by the meta pathway. 2-Methylnaphthalene, but not 1-methylnaphthalene, also serves as a growth substrate and is metabolized by the same route, through 4-methylcatechol. The same nonspecific meta pathway enzymes appear to be induced by growth on either naphthalene or 2-methylnaphthalene. The level to which 2-hydroxymuconic semialdehyde hydrolase is induced is low and probably of no metabolic significance. Growth on salicylate or catechol, both intermediates of naphthalene degradation, or benzoate results in induction of the ortho pathway, the alternative route for catechol dissimilation. No induction of 1,2-dihydroxynaphthalene oxygenase was found in salicylate-grown cells. Anaerobic growth on a succinate-nitrate medium in the presence of various inducers indicates that cis, cis-muconate, or one of its metabolites is the inducer of the ortho pathway enzymes. The inducer or inducers of the early enzymes of naphthalene degradation and of the meta pathway enzymes must be an early intermediate of the naphthalene pathway above salicylate.     

Growth and plasmid-encoded naphthalene catabolism of Pseudomonas putida in batch culture

Abstract The growth characteristics of Pseudomonas putida plasmid-harbouring strains which catabolize naphthalene via various pathways in batch culture with naphthalene as the sole source of carbon and energy have been investigated. The strains under study were constructed using the host strain P. putida BS394 which contained various naphthalene degradation plasmids. The highest specific growth rate was ensured by the plasmids that control naphthalene catabolism through the meta-pathway of catechol oxidation. The strains metabolizing catechol via the ortho -pathway grew at a lower rate. The lowest growth rate was observed with strain BS291 harbouring plasmid pBS4 which controls naphthalene catabolism via the gentisic acid pathway. Various pathways of naphthalene catabolism appear to allow these strains to grow at various rates which should be taken into account when constructing efficient degraders of polycyclic aromatic compounds.     

Role of Catechol and the Methylcatechols as Inducers of Aromatic Metabolism in Pseudomonas putida

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.     

Isolation and characterisation of new Planococcus sp. strain able for aromatic hydrocarbons degradation

New Planococcus sp. strain S5 able to grow on salicylate or benzoate as sole carbon source was isolated from activated sludge adapted to sodium salicylate degradation. S5 was determined to be a strictly aerobic, gram-positive, catalase positive, oxidase negative, non-motile, non-spore forming coccus. The strain harboured a plasmid, named pLS5. The S5 strain when grown on salicylate expressed both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities and degraded this substrate by both the ortho and meta pathways while grown on benzoate expressed only catechol 1,2-dioxygenase activity. Curing of the plasmid from the strain showed that plasmid pLS5 was involved in salicylate degradation by the meta pathway.     

Propachlor degradation by a soil bacterial community.

Soil from a pesticide disposal site was used to enrich for microorganisms that degraded the acylanilide herbicide propachlor (2-chloro-N-isopropylacetanilide). After seven transfers of the enrichment, the culture contained about six strains. The highest yield of microbial biomass occurred if just two of these isolates, strains DAK3 and MAB2, were inoculated into a mineral salts medium containing propachlor. When only strain DAK3 was grown on propachlor, a metabolite (2-chloro-N-isopropylacetamide) was released into the medium. Strain MAB2 could grow on this metabolite. The results of morphological and physiological tests suggest that strains DAK3 and MAB2 most closely resemble species belonging to the genera Moraxella and Xanthobacter, respectively. Strain DAK3 can respire and grow on N-substituted acylanilides containing methyl, ethyl, or isopropyl substitutions, but is incapable of respiration or growth on acetanilide, aniline, or the acylanilide herbicides alachlor and metolachlor. Strain DAK3 appears to use the aromatic C atoms of propachlor for growth, as suggested by the growth yield on propachlor and the induction of catechol 2,3-oxygenase activity in acylanilide-grown cells.     

Propachlor degradation by a soil bacterial community.

Soil from a pesticide disposal site was used to enrich for microorganisms that degraded the acylanilide herbicide propachlor (2-chloro-N-isopropylacetanilide). After seven transfers of the enrichment, the culture contained about six strains. The highest yield of microbial biomass occurred if just two of these isolates, strains DAK3 and MAB2, were inoculated into a mineral salts medium containing propachlor. When only strain DAK3 was grown on propachlor, a metabolite (2-chloro-N-isopropylacetamide) was released into the medium. Strain MAB2 could grow on this metabolite. The results of morphological and physiological tests suggest that strains DAK3 and MAB2 most closely resemble species belonging to the genera Moraxella and Xanthobacter, respectively. Strain DAK3 can respire and grow on N-substituted acylanilides containing methyl, ethyl, or isopropyl substitutions, but is incapable of respiration or growth on acetanilide, aniline, or the acylanilide herbicides alachlor and metolachlor. Strain DAK3 appears to use the aromatic C atoms of propachlor for growth, as suggested by the growth yield on propachlor and the induction of catechol 2,3-oxygenase activity in acylanilide-grown cells.