Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa

Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH₄)₂SO₄-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa.     

Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F

Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated. It was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine, beta-alanine or beta-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source. Only uridine, cytidine, beta-alanine, beta-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon. In solid medium, the pyrimidine analogue 5-fluorouracil or 5-fluorouridine could prevent P. fluorescens biotype F growth at a low concentration while a 20-fold higher concentration of 5-fluorocytosine, 5-fluorodeoxyuridine or 6-azauracil was necessary to block its growth. The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed. Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used. The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored. Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium. In contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol. A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NH4)2SO4 as the sole source of nitrogen.     

Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.     

Role of cytosine deaminase and β-alanine-pyruvate transaminase in pyrimidine base catabolism by Burkholderia cepacia

A determination of the possible role of the salvage enzyme cytosine deaminase or -alanine-pyruvate transaminase in the catabolism of the pyrimidine bases uracil and thymine by the opportunistic pathogen Burkholderia cepacia ATCC 25416 was undertaken. It was of interest to learn whether these enzymes were influenced by cell growth on pyrimidine bases and their respective catabolic products to the same degree as the pyrimidine reductive catabolic enzymes were. It was found that cytosine deaminase activity was influenced very little by cell growth on the pyrimidines tested. Using glucose as the carbon source, only B. cepacia growth on 5-methylcytosine as a nitrogen source increased deaminase activity by about three-fold relative to (NH₄)₂SO₄-grown cells. In contrast, the activity of –alanine-pyruvate transaminase was observed to be at least double in glucose-grown ATCC 25416 cells when pyrimidine bases and catabolic products served as nitrogen sources instead of (NH₄)₂SO₄. Transaminase activity in the B. cepacia glucose-grown cells was maximal after the strain was grown on either uracil or 5-methylcytosine as a nitrogen source compared to (NH₄)₂SO₄-grown cells. A possible role for -alanine-pyruvate transaminase in pyrimidine base catabolism by B. cepacia would seem to be suggested from the similarity in how its enzyme activity responded to cell growth on pyrimidine bases and catabolic products when compared to the response of the three reductive catabolic enzymes.     

Pyrimidine ribonucleoside catabolic enzyme activities ofPseudomonas pickettii

Pyrimidine ribonucleoside catabolic enzyme activities of the opportunistic pathogenPseudomonas pickettii were examined. Of the pyrimidine and related compounds tested, only dihydrouracil (nitrogen source) and ribose (carbon source) supported growth. Thin-layer chromatographic separation of the uridine and cytidine catabolities produced byP. pickettii extracts indicated that this pseudomonad contained nucleoside hydrolase activity. Its presence was confirmed by enzyme assay. Hydrolase activity was elevated in both glucose- and ribose-grown cells relative to succinate-grown cells. Nucleoside hydrolase activity was depressed when dihydrouracil served as a nitrogen source. Cytosine deaminase activity was present in extracts prepared from succinate-, glucose- or ribose-grown cells when (NH₄)₂SO₄ served as the nitrogen source although cells grown on glucose or ribose exhibited a higher enzyme activity. Cytosine deaminase activity was not detected in extracts prepared from cells grown on dihydrouracil as a nitrogen source. Both dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were measurable inP. pickettii. The dehydrogenase activity was higher with NADH than with NADPH as its nicotinamide cofactor when uracil served as its substrate. Carbon source did not affect dehydrogenase or dihydropyrimidinase activity greatly but both activities were diminished in cells grown on the nitrogen source dihydrouracil.     

Pyrimidine base and ribonucleoside catabolic enzyme activities of the Pseudomonas diminuta group

Pyrimidine base and ribonucleoside catabolic enzyme activities of the two type strains of the Pseudomonas diminuta group were investigated for taxonomic classification purposes. The presence of the pyrimidine salvage enzyme nucleoside hydrolase was indicated in both type strains following thin-layer chromatographic analysis. The presence of the hydrolase was also confirmed by enzyme assay. In addition, the activities of the pyrimidine salvage enzymes dihydropyrimidine dehydrogenase and dihydropyrimidinase were measurable in cell-free extracts of both P. diminuta and P. vesicularis. An absence of cytosine deaminase activity was found when assaying extracts of the two type strains. Nucleoside hydrolase and dihydropyrimidine dehydrogenase levels in P. vesicularis were influenced by carbon source while dihydropyrimidinase activity was observed to increase after P. diminuta growth on dihydrothymine as a nitrogen source.     

Pyrimidine ribonucleoside catabolism in Pseudomonas fluorescens biotype A

The pyrimidine ribonucleosides uridine or cytidine were shown to serve as a source of nitrogen or carbon for the growth of Pseudomonas fluorescens strain A126. After incubation of either pyrimidine ribonucleoside with extracts of this strain, the resultant catabolic products were detected by thin-layer chromatography. It was found that pyrimidine ribonucleoside catabolism in this pseudomonad involved the enzymes nucleoside hydrolase and cytosine deaminase. The specific activities of both these enzymes could be influenced by the nitrogen or carbon source present in the medium.     

Pathways of Pyrimidine Salvage in <Emphasis Type="Italic">Pseudomonas</Emphasis> and Former <Emphasis Type="Italic">Pseudomonas</Emphasis>: Detection of Recycling Enzymes Using High-Performance Liquid Chromatography

Pyrimidine salvage pathways are vital for all bacteria in that they share in the synthesis of RNA with the biosynthetic pathway in pyrimidine prototrophs, while supplying all pyrimidine requirements in pyrimidine auxotrophs. Salvage enzymes that constitute the pyrimidine salvage pathways were studied in 13 members of Pseudomonas and former pseudomonads. Because it has been established that all Pseudomonas lack the enzyme uridine/cytidine kinase (Udk) and all contain uracil phosphoribosyl transferase (Upp), these two enzymes were not included in this experimental work. The enzymes assayed were: cytosine deaminase [Cod: cytosine + H₂O → uracil + NH₃], cytidine deaminase [Cdd: cytidine + H₂O → uridine + NH₃], uridine phosphorylase [Udp: uridine + P_i ↔ uracil + ribose – 1 - P], nucleoside hydrolase [Nuh: purine/pyrimidine nucleoside + H₂O → purine/pyrimidine base + ribose], uridine hydrolase [Udh: uridine/cytidine + H₂O → uracil/cytosine + ribose]. The assay work generated five different Pyrimidine Salvage Groups (PSG) designated PSG1 – PSG5 based on the presence or absence of the five enzymes. These enzymes were assayed using reverse phase high-performance liquid chromatography techniques routinely carried out in our laboratory. Escherichia coli was included as a standard, which contains all seven of the above enzymes.     

Purification and some properties of cytosine deaminase from Salmonella typhimurium

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogenous. The molecular weight of cytosine deaminase was determined to be approx. 230 000 containing four identical subunits with each subunit having a molecular weight of 54 000. Cytosine deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50°C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The K_m and V values for cytosine were 0.74 mM and 47.16 μmole/min, respectively. As effectors of enzyme activity, PP_i stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171–1174).     

Reductive catabolism of uracil and thymine by Burkholderia cepacia

Catabolism of uracil and thymine in Burkholderia cepacia ATCC 25416 was shown to occur using a reductive pathway. The first pathway enzyme, dihydropyrimidine dehydrogenase, was shown to utilize NADPH as its nicotinamide cofactor. Growth of B. cepacia on pyrimidine bases as the nitrogen source instead of on ammonium sulfate increased dehydrogenase activity at least 32-fold. The second and third reductive pathway enzymes, dihydropyrimidinase and N-carbamoyl-β-alanine amidohydrolase, respectively, exhibited activities elevated more than 21-fold when pyrimidine or dihydropyrimidine bases served as the nitrogen source rather than ammonium sulfate. The pathway enzyme activities were induced after growth on 5-methylcytosine. Received: 17 January 1997 / Accepted: 5 May 1997     

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (^m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of ^m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung ⁺ host proficient in uracil excision repair.     

Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

Isolation of a Pseudomonas sp. Which Utilizes the Phosphonate Herbicide Glyphosate

A strain of bacteria has been isolated which rapidly and efficiently utilizes the herbicide glyphosate (N-phosphonomethylglycine) as its sole phosphorus source in a synthetic medium. The strain (PG2982) was isolated by subculturing Pseudomonas aeruginosa ATCC 9027 in a synthetic broth medium containing glyphosate as the sole phosphorus source. Strain PG2982 differs from the culture of P. aeruginosa in that it is nonflagellated, does not produce pyocyanin, and has an absolute requirement for thiamine. Strain PG2982 has been tentatively identified as a Pseudomonas sp. strain by its biochemical activities and moles percent guanine plus cytosine. Measurements of glyphosate with an amino acid analyzer show that glyphosate rapidly disappears from the medium during exponential growth of strain PG2982. In batch culture at 30°C, this isolate completely utilized 1.0 mM glyphosate in 96 h and yielded a cell density equal to that obtained with 1.0 mM phosphate as the phosphorus source. However, a longer lag phase and greater generation time were noted in the glyphosate-containing medium. Strain PG2982 can efficiently utilize glyphosate as an alternate phosphorus source.     

Degradation of Triphenyltin by a Fluorescent Pseudomonad

Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3′,4′,7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 μM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 μM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa ATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment.     

Pyrimidine,purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.This paper is dedicated to Professor John Ingraham, Department of Bacteriology, University of California, Davis, on the occasion of his retirement, in recognition of his many contributions in the field of bacterial growth and metabolism     

Ferritin and ferrihydrite nanoparticles as iron sources for Pseudomonas aeruginosa

Metabolism of iron derived from insoluble and/or scarce sources is essential for pathogenic and environmental microbes. The ability of Pseudomonas aeruginosa to acquire iron from exogenous ferritin was assessed; ferritin is an iron-concentrating and antioxidant protein complex composed of a catalytic protein and caged ferrihydrite nanomineral synthesized from Fe(II) and O₂ or H₂O₂. Ferritin and free ferrihydrite supported growth of P. aeruginosa with indistinguishable kinetics and final culture densities. The P. aeruginosa PAO1 mutant (ΔpvdDΔpchEF), which is incapable of siderophore production, grew as well as the wild type when ferritin was the iron source. Such data suggest that P. aeruginosa can acquire iron by siderophore-independent mechanisms, including secretion of small-molecule reductant(s). Protease inhibitors abolished the growth of the siderophore-free strain on ferritins, with only a small effect on growth of the wild type; predictably, protease inhibitors had no effect on growth with free ferrihydrite as the iron source. Proteolytic activity was higher with the siderophore-free strain, suggesting that the role of proteases in the degradation of ferritin is particularly important for iron acquisition in the absence of siderophores. The combined results demonstrate the importance of both free ferrihydrite, a natural environmental form of iron and a model for an insoluble form of partly denatured ferritin called hemosiderin, and caged ferritin iron minerals as bacterial iron sources. Ferritin is also revealed as a growth promoter of opportunistic, pathogenic bacteria such a P. aeruginosa in diseased tissues such as the cystic fibrotic lung, where ferritin concentrations are abnormally high.     

Synergistic effects of zinc oxide nanoparticles and various antibiotics combination against Pseudomonas aeruginosa clinically isolated bacterial strains

P. aeruginosa causes mostly both community-acquired and nosocomial infections, which leads to serious therapeutic challenges for treatment and requirement of appropriate therapeutic agent is needed which can combat antibiotic resistance. The research work was performed to investigate the effect of Zinc Oxide nanoparticles (ZnO NPs) in combination with Meropenem, Ciprofloxacin, and Colistin against clinical isolated strains of P. aeruginosa and ATCC 27853 strain.The minimum inhibitory concentration (MIC) of ZnO NPs and the antibiotics (Meropenem, Ciprofloxacin, and Colistin), was determined by the microdilution method and the results of MIC values were ranging between 1 and 16 µg/mL was found to be shown for antibiotics and ZnO NPs found to showed highest MIC values ranging from 2000 to 4000 µg/mL. The fractional inhibitory concentration index (FICI) was calculated using checkerboard method to test the combinations of ZnO NPs and the antibiotics (Meropenem, Ciprofloxacin, and Colistin), and among all the six P. aeruginosa clinical isolated strains P. aeruginosa (MRO-16-3 and MRO-16-4), showed FICI as 0.24 and 0.39 9, whereas P. aeruginosa ATCC 27853 strain showed FICI as 0.41 which indicates synergistic effect with Colistin.The time kill growth curve showed synergistic effect for the combination of Colistin and ZnO NPs against P. aeruginosa (MRO-16-3 and MRO-16-) strains. P. aeruginosa (MRO-16-3) was found to be highly sensitive to Colistin with an MIC of 2 µg/mL, which has shown to reduced bacterial growth to zero colonies after 24 h of incubation.In conclusion, combination of Colistin and ZnO NPs at appropriate dosage intervals might be beneficial as using therapeutic agent in treatment of P. aeruginosa ailments.     

Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol extracts from three Seseli L. taxa

The present study describes the total phenolic content, concentrations of flavonoids and in vitro antioxidant and antimicrobial activity of methanol extracts from Seseli pallasii Besser, S. libanotis (L.) Koch ssp. libanotis and S. libanotis (L.) Koch ssp. intermedium (Rupr.) P. W. Ball, growing wild in Serbia. The total phenolic content in the extracts was determined using Folin-Ciocalteu reagent and their amounts ranged between 84.04 to 87.52 mg GA (gallic acid)/g. The concentrations of flavonoids in the extracts varied from 4.75 to 19.37 mg Qu (quercetin)/g. Antioxidant activity was analyzed using DPPH reagent. Antioxidant activity ranged from 0.46 to 4.63 IC₅₀ (mg/ml) and from 1.98 to 2.19 mg VitC (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively, using BHA and VitC as controls. The antimicrobial activity of the extracts was investigated using a micro-well dilution assay for the most common human gastrointestinal pathogenic bacterial strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella enteritidis ATCC 13076, Bacillus cereus ATCC 10876, Listeria monocytogenes ATCC15313, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 10231. This finding suggests that Seseli species may be considered as a natural source of antioxidants and antimicrobial agents.     

AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.