The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn5501 and Tn5502, are cryptic transposons of the Tn3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon.     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn 5501 and Tn 5502 , are cryptic transposons of the Tn 3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon. Received: 21 July 1997 / Accepted: 7 July 1998     

In vivo generation of R68.45-pPGH1 hybrid plasmids conferring a Phl+ (meta pathway) phenotype

Summary Plasmid pPGH1 originating from Pseudomonas putida strain H carries all the genes required for the degradation of phenol (or cresols) via the meta cleavage pathway. Besides mobilization of pPGH1 by a plasmid of the incompatibility group P-1, hybrid plasmids conferring the Phl⁺ phenotype could be selected, when R68.45 was the conjugative plasmid. The hybrids contain the complete R68.45 and part of pPGH1. Integration of Phl-DNA of pPGH1 into R68.45 occurred exclusively via the IS21 region of R68.45.Dedicated to Udo Taubeneck on the occasion of his 60th birthday     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Plasmid-coded degradation of salicylate using the catechol cleavage pathway of the host

Degradation rates of salicylate and phenol by Pseudomonas putida PpG1064 carrying the nahG gene on a multicopy plasmid were compared with those in NAH-carrying P. putida. Degradation rates of salicylate and phenol and the growth rate of the recombinant were higher than those in NAH-carrying P. putida in SP medium. The catechol 1,2 oxygenase activity of the recombinant in Sp medium was about twice that of the catechol 2,3 oxygenase and catechol 1,2 oxygenase activities of NAH-carrying P. putida. It was suggested that in simultaneous degradation of phenol and salicylate, the recombinant stimulated its ortho cleavage pathway and attained the higher degradation rates and growth rate.     

Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass

Summary A defined mixed culture of the yeast Cryptococcus elinovii H1 and the bacterium Pseudomonas putida P8 was immobilized by adsorption on activated carbon and sintered glass, respectively. Depending on its adsorption capacity for phenol the activated carbon system could completely degrade 17 g/l in batch culture, whereas the sintered glass system was able to degrade phenol up to 4 g/l. During semicontinuous degradation of phenol (1 g/l) both systems reached constant degradation times with the fourth batch that lasted 8 h when using the activated carbon system and 10 h in the sintered glass system. In the course of continuous degradation of phenol the activated carbon system reached a maximum degradation rate of 9.2 g l^–1 day^–1 compared to 6.4 g l^–1 day^–1degraded by the sintered glass system. 2-Hydroxymuconic acid semialdehyde could be identified and quantitatively determined as a metabolite of phenol degradation by P. putida P8. Increased membrane permeability under the influence of phenol was demonstrated by the examination of K⁺ efflux from P. putida P8. Offprint requests to: H.-J. Rehm     

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.     

Rationally engineered biotransformation of p‐nitrophenol

An operon encoding enzymes responsible for degradation of the EPA priority contaminant para‐nitrophenol (PNP) from Pseudomonas sp. ENV2030 contains more genes than would appear to be necessary to mineralize PNP. To determine some necessary genes for PNP degradation, the genes encoding the proposed enzymes in the degradation pathway (pnpADEC) were assembled into a broad‐host‐range, BioBricks‐compatible vector under the control of a constitutive promoter. These were introduced into Escherichia coli DH10b and two Pseudomonas putida strains, one with a knockout of the aromatic transport TtgB and the parent with the native transporter. The engineered strains were assayed for PNP removal. E. coli DH10b harboring several versions of the refactored pathway was able to remove PNP from the medium up to a concentration of 0.2 mM; above which PNP was toxic to E. coli. A strain of P. putida harboring the PNP pathway genes was capable of removing PNP from the medium up to 0.5 mM. When P. putida harboring the native PNP degradation cluster was exposed to PNP, pnpADEC were induced, and the resulting production of β‐ketoadipate from PNP induced expression of its chromosomal degradation pathway (pcaIJF). In contrast, pnpADEC were expressed constitutively from the refactored constructs because none of the regulatory genes found in the native PNP degradation cluster were included. Although P. putida harboring the refactored construct was incapable of growing exclusively on PNP as a carbon source, evidence that the engineered pathway was functional was demonstrated by the induced expression of chromosomal pcaIJF. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Kinetics of phenol degradation using Pseudomonas putida MTCC 1194

Pseudomonas putida (MTCC 1194) has been used to degrade phenol in water in the concentration range 100–1000?ppm. The inhibition effects of phenol as substrate have become predominant above the concentration of 500?ppm (5.31?mmoles/dm³). The optimum temperature and initial pH required for maximum phenol biodegradation were 30?°C and 7.00 respectively. From the degradation data the activation energy (E _a ) was found to be equal to 13.8?kcal/g mole substrate reacted. The most suitable inoculum age and volume for highest phenol degradation were 12?hrs and 7% v/v respectively. Surfactants had negligible effect on phenol biodegradation process for this microorganism. Monod model has been used to interpret the free cell data on phenol biodegradation. The kinetic parameters have been estimated upto initial concentration of 5.31?mmoles/dm³. μ _max and K _S gradually increased with higher concentration of phenol. However, beyond the phenol concentration of 5.31?mmoles/dm³, the inhibition became prominant. The μ _max has been to be a strong function of initial phenol concentration. The simulated and the experimental phenol degradation profiles have good correspondence with each other.     

Identification and characterisation of IS1383, a new insertion sequence isolated from Pseudomonas putida strain H

A new insertion sequence (IS1383) was identified on plasmids from Pseudomonas putida strain H and its nucleotide sequence was determined. IS1383 contains perfect terminal inverted repeats of 13-bp flanking a 1.4-kb internal sequence. A single significant open reading frame was identified that can encode a 342-amino acid polypeptide which was predicted to be highly basic and to have homology to polypeptides known from several other bacterial insertion sequences. At least six copies of IS1383 are present on the plasmids pPGH1 and pPGH2, whereas no copy could be detected on the chromosome of P. putida strain H. Target duplications did not flank the inverted repeats of any of the six IS1383 copies examined. Analysis of the integration sites of IS1383 revealed hints for a target specificity. Multiple sequence alignments of the transposases, the inverted repeats and the integration sites pointed to the assignment of IS1383 into a putative new family of insertion sequences defined as the IS1111 family.