The degradation of p-nitrophenol (PNP) by Moraxella and Pseudomonas spp. involves an initial monooxygenase-catalyzed removal of the nitro group. The resultant hydroquinone is subject to ring fission catalyzed by a dioxygenase enzyme. We have isolated a strain of an Arthrobacter sp., JS443, capable of degrading PNP with stoichiometric release of nitrite. During induction of the enzymes required for growth on PNP, 1,2,4-benzenetriol was identified as an intermediate by gas chromatography-mass spectroscopy (GC-MS) and radiotracer studies. 1,2,4-Benzenetriol was converted to maleylacetic acid, which was further degraded by the beta-ketoadipate pathway. Conversion of PNP to 1,2,4-benzenetriol is catalyzed by a monooxygenase system in strain JS443 through the formation of 4-nitrocatechol, 4-nitroresorcinol, or both. Our results clearly indicate the existence of an alternative pathway for the biodegradation of PNP.
Bacteria that metabolize p-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol. Here we describe the complete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil in India. Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol. Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol. 4-Nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite. The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography. Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol. One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol and nitroblue tetrazolium. Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole. Our results indicate that the two monooxygenations of PNP to THB are catalyzed by a single two-component enzyme system comprising a flavoprotein reductase and an oxygenase.
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.
Benzenetriol dioxygenase; p-Nitrophenol; Phylogenetic analysis
para-Nitrophenol (PNP), a priority environmental pollutant, is hazardous to humans and animals. However, the information relating to the PNP degradation pathways and their enzymes remain limited.
Pseudomonas sp.1-7 was isolated from methyl parathion (MP)-polluted activated sludge and was shown to degrade PNP. Two different intermediates, hydroquinone (HQ) and 4-nitrocatechol (4-NC) were detected in the catabolism of PNP. This indicated that Pseudomonas sp.1-7 degraded PNP by two different pathways, namely the HQ pathway, and the hydroxyquinol (BT) pathway (also referred to as the 4-NC pathway). A gene cluster (pdcEDGFCBA) was identified in a 10.6 kb DNA fragment of a fosmid library, which cluster encoded the following enzymes involved in PNP degradation: PNP 4-monooxygenase (PdcA), p-benzoquinone (BQ) reductase (PdcB), hydroxyquinol (BT) 1,2-dioxygenase (PdcC), maleylacetate (MA) reductase (PdcF), 4-hydroxymuconic semialdehyde (4-HS) dehydrogenase (PdcG), and hydroquinone (HQ) 1,2-dioxygenase (PdcDE). Four genes (pdcDEFG) were expressed in E. coli and the purified pdcDE, pdcG and pdcF gene products were shown to convert HQ to 4-HS, 4-HS to MA and MA to β-ketoadipate respectively by in vitro activity assays.
The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies identified 4-NC, HQ, 4-HS, and MA as intermediates in the degradation pathway of PNP by Pseudomonas sp.1-7. This is the first conclusive report for both 4-NC and HQ- mediated degradation of PNP by one microorganism.
para-Nitrophenol; Catabolism; Hydroquinone pathway; Hydroxyquinol pathway; Pseudomonas
We have isolated two soil bacteria (identified as Arthrobacter aurescens TW17 and Nocardia sp. strain TW2) capable of degrading p-nitrophenol (PNP) and numerous other phenolic compounds. A. aurescens TW17 contains a large plasmid which correlated with the PNP degradation phenotype. Degradation of PNP by A. aurescens TW17 was induced by preexposure to PNP, 4-nitrocatechol, 3-methyl-4-nitrophenol, or m-nitrophenol, whereas PNP degradation by Nocardia sp. strain TW2 was induced by PNP, 4-nitrocatechol, phenol, p-cresol, or m-nitrophenol. A. aurescens TW17 initially degraded PNP to hydroquinone and nitrite. Nocardia sp. strain TW2 initially converted PNP to hydroquinone or 4-nitrocatechol, depending upon the inducing compound.
Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported.
Maleylacetate reductase (EC 18.104.22.168) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P21212, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively.
maleylacetate reductase; Burkholderia sp. strain SJ98
The crystallization and preliminary crystallographic analysis of a para-nitrophenol 4-monooxygenase PnpA from Pseudomonas putida DLL-E4 are presented.
Para-nitrophenol 4-monooxygenase (PnpA) plays an important role in bacterial degradation of para-nitrophenol by oxidative release of the nitro group from the aromatic ring to form p-benzoquinone. In order to understand the structural basis of the function of this enzyme, PnpA was cloned, expressed in Escherichia coli and purified. PnpA was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The PnpA crystals belonged to space group P212121, with unit-cell parameters a = 54.47, b = 77.56, c = 209.17 Å, and diffracted to 2.24 Å resolution.
para-nitrophenol 4-monooxygenase; Pseudomonas putida DLL-E4
The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5′-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5′ end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5′ double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5′ end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5′ fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.
Microbiological analyses of activated sludge reactors after repeated exposure to 100 mg of p-nitrophenol (PNP) per liter resulted in the isolation of three Pseudomonas species able to utilize PNP as a sole source of carbon and energy. Cell suspensions of the three Pseudomonas sp., designated PNP1, PNP2, and PNP3, mineralized 70, 60, and 45% of a 70-mg/liter dose of PNP in 24, 48, and 96 h, respectively. Mass-balance analyses of PNP residues for all three cultures showed that undegraded PNP was less than 1% (less than 50 micrograms); volatile metabolites, less than 1%; cell residues, 8.4 to 14.9%; and water-soluble metabolites, 1.2 to 6.7%. A mixed culture of all three PNP-degrading Pseudomonas sp. was immobilized by adsorption onto diatomaceous earth biocarrier in a 1.75-liter Plexiglas column. The column was aerated and exposed to a synthetic waste stream containing 629 to 2,513 mg of PNP per liter at flow rates of 2 to 15 ml/min. Chemical loading studies showed that the threshold concentration for acute toxicity of PNP to the immobilized bacteria was 2,100 to 2,500 mg/liter. Further studies at PNP concentrations of 1,200 to 1,800 mg/liter showed that greater than 99 and 91 to 99% removal of PNP was achieved by immobilized bacteria at flow rates of 10 and 12 ml/min, respectively. These values represent hydraulic retention times of 48 to 58 min and PNP removal rates of 0.99 to 1.1 mg/h per g of biocarrier at 25 degrees C under optimal conditions. This study shows the successful use of immobilized bacteria technology to remove high concentrations of PNP from aqueous waste streams.
Polystyrene nanoparticles (PNP) cross rat alveolar epithelial cell monolayers via non-endocytic transcellular pathways. To evaluate epithelial cell type-specificity of PNP trafficking, we studied PNP flux across Madin Darby canine kidney cell II monolayers (MDCK-II). Effects of calcium chelation (EGTA), energy depletion (sodium azide (NaN3) or decreased temperature), and endocytosis inhibitors methyl-β-cyclodextrin (MBC), monodansylcadaverine and dynasore were determined. Amidine-modified PNP cross MDCK-II 500 times faster than carboxylate-modified PNP. PNP flux did not increase in the presence of EGTA. PNP flux at 4°C and after treatment with NaN3 decreased 75% and 80%, respectively. MBC exposure did not decrease PNP flux, whereas dansylcadaverine- or dynasore-treated MDCK-II exhibited ~80% decreases in PNP flux. Confocal laser scanning microscopy revealed intracellular colocalization of PNP with clathrin heavy chain. These data indicate that PNP translocation across MDCK-II (1) occurs via clathrin-mediated endocytosis and (2) is dependent upon PNP physicochemical properties. We conclude that uptake/trafficking of nanoparticles into/across epithelia is dependent both on properties of the nanoparticles and the specific epithelial cell type.
epithelial transport; endocytosis; clathrin; dynamin; surface charge
The kinetics of simultaneous mineralization of p-nitrophenol (PNP) and glucose by Pseudomonas sp. were evaluated by nonlinear regression analysis. Pseudomonas sp. did not mineralize PNP at a concentration of 10 ng/ml but metabolized it at concentrations of 50 ng/ml or higher. The Ks value for PNP mineralization by Pseudomonas sp. was 1.1 micrograms/ml, whereas the Ks values for phenol and glucose mineralization were 0.10 and 0.25 micrograms/ml, respectively. The addition of glucose to the media did not enable Pseudomonas sp. to mineralize 10 ng of PNP per ml but did enhance the degradation of higher concentrations of PNP. This enhanced degradation resulted from the simultaneous use of glucose and PNP and the increased rate of growth of Pseudomonas sp. on glucose. The Monod equation and a dual-substrate model fit these data equally well. The dual-substrate model was used to analyze the data because the theoretical assumptions of the Monod equation were not met. Phenol inhibited PNP mineralization and changed the kinetics of PNP mineralization so that the pattern appeared to reflect growth, when in fact growth was not occurring. Thus, the fitting of models to substrate depletion curves may lead to erroneous interpretations of data if the effects of second substrates on population dynamics are not considered.
Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.
Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded β-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an FeII center for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O2, resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O2 is supported by model chemistry.
In gram-negative bacteria, a pathway for aerobic degradation of phenylacetic acid (PAA) that proceeds via phenylacetyl-coenzyme A (CoA) and hydrolytic ring fission plays a central role in the degradation of a range of aromatic compounds. In contrast, the PAA pathway and its role are not well characterized in gram-positive bacteria. A cluster including 13 paa genes encoding enzymes orthologous to those of gram-negative bacteria was identified on the chromosome of Rhodococcus sp. strain RHA1. These genes were transcribed during growth on PAA, with 11 of the genes apparently in an operon yielding a single transcript. Quantitative proteomic analyses revealed that at least 146 proteins were more than twice as abundant in PAA-grown cells of RHA1 than in pyruvate-grown cells. Of these proteins, 29 were identified, including 8 encoded by the paa genes. Knockout mutagenesis indicated that paaN, encoding a putative ring-opening enzyme, was essential for growth on PAA. However, paaF, encoding phenylacetyl-CoA ligase, and paaR, encoding a putative regulator, were not essential. paaN was also essential for growth of RHA1 on phenylacetaldehyde, phenylpyruvate, 4-phenylbutyrate, 2-phenylethanol, 2-phenylethylamine, and l-phenylalanine. In contrast, growth on 3-hydroxyphenylacetate, ethylbenzene, and styrene was unaffected. These results suggest that the range of substrates degraded via the PAA pathway in RHA1 is somewhat limited relative to the range in previously characterized gram-negative bacteria.
A study was conducted to determine the role of concentration of the test chemical, of a second organic compound, and of mutation in the acclimation period before the mineralization of organic compounds in sewage. The acclimation period for the mineralization in sewage of 2 micrograms of 4-nitrophenol (PNP) per liter increased from 6 to 12 days in the presence of 10 mg of 2,4-dinitrophenol per liter. The extension of the acclimation period was equivalent to the time required for mineralization of 2,4-dinitrophenol. In contrast, the time for acclimation for the degradation of 2 micrograms of PNP per liter was reduced when 10 or 100 mg of phenol per liter was added. Lower phenol levels increased the acclimation period to 8 days. The length of the acclimation period for PNP mineralization decreased as the initial concentration of PNP increased from 2 micrograms to 100 mg/liter. The acclimation period for phenol mineralization was lengthened as the phenol concentration increased from 100 to 1,400 mg/liter. The length of the acclimation period for PNP and phenol biodegradation was reproducible, but it varied among replicates for the biodegradation of other nitro-substituted compounds added to sewage or lake water, suggesting that a mutation was responsible for acclimation to these other compounds. The acclimation period may thus reflect the time required for the destruction of toxins, and it also may be affected by the concentration of the test compound or the presence of other substrates.
Purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, has been expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique.
The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and α-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6 Å resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1 Å.
purine nucleoside phosphorylase; Streptococcus mutans; nucleotide-salvage pathway
Recombinant purine nucleoside phosphorylase from B. subtilis strain 168 has been crystallized at different pH values using the sitting-drop vapour-diffusion method.
Purine nucleoside phosphorylase (PNP; EC 22.214.171.124) is a key enzyme of the purine-salvage pathway. Its ability to transfer glycosyl residues to acceptor bases is of great biotechnological interest owing to its potential application in the synthesis of nucleoside analogues used in the treatment of antiviral infections and in anticancer chemotherapy. Although hexameric PNPs are prevalent in prokaryotes, some microorganisms, such as Bacillus subtilis, present both hexameric and trimeric PNPs. The hexameric PNP from B. subtilis strain 168, named BsPNP233, was cloned, expressed and crystallized. Crystals belonging to different space groups (P321, P212121, P6322 and H32) were grown in distinct conditions with pH values ranging from 4.2 to 10.5. The crystals diffracted to maximum resolutions ranging from 2.65 to 1.70 Å.
purine nucleoside phosphorylases; Bacillus subtilis strain 168
A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.
Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rifr and the gyrB gene leading to Nalr and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation.
A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5′-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3′-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3′ end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3′-to-5′ exonuclease. The finding of impediments to 3′-to-5′ degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.
Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability.
Campylobacter jejuni; polynucleotide phosphorylase; in vitro virulence tests; chick colonization; 2D-electrophoresis
p-Nitrophenol (4-NP) is recognized as an environmental contaminant; it is used primarily for manufacturing medicines and pesticides. To date, several 4-NP-degrading bacteria have been isolated; however, the genetic information remains very limited. In this study, a novel 4-NP degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was identified and characterized. The deduced amino acid sequences of npcB, npcA, and npcC showed identity with phenol 2-hydroxylase component B (reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32%), with 2,4,6-trichlorophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44%), and with hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp. strain BA-5-17 (76%), respectively. The npcB, npcA, and npcC genes were cloned into pET-17b to construct the respective expression vectors pETnpcB, pETnpcA, and pETnpcC. Conversion of 4-NP was observed when a mixture of crude cell extracts of Escherichia coli containing pETnpcB and pETnpcA was used in the experiment. The mixture converted 4-NP to hydroxyquinol and also converted 4-nitrocatechol (4-NCA) to hydroxyquinol. Furthermore, the crude cell extract of E. coli containing pETnpcC converted hydroxyquinol to maleylacetate. These results suggested that npcB and npcA encode the two-component 4-NP/4-NCA monooxygenase and that npcC encodes hydroxyquinol 1,2-dioxygenase. The npcA and npcC mutant strains, SDA1 and SDC1, completely lost the ability to grow on 4-NP as the sole carbon source. These results clearly indicated that the cloned npc genes play an essential role in 4-NP mineralization in R. opacus SAO101.
Phthalate isomers and their esters are used heavily in various industries. Excess use and leaching from the product pose them as major pollutants. These chemicals are toxic, teratogenic, mutagenic and carcinogenic in nature. Various aspects like toxicity, diversity in the aerobic bacterial degradation, enzymes and genetic organization of the metabolic pathways from various bacterial strains are reviewed here. Degradation of these esters proceeds by the action of esterases to form phthalate isomers, which are converted to dihydroxylated intermediates by specific and inducible phthalate isomer dioxygenases. Metabolic pathways of phthalate isomers converge at 3,4-dihydroxybenzoic acid, which undergoes either ortho- or meta- ring cleavage and subsequently metabolized to the central carbon pathway intermediates. The genes involved in the degradation are arranged in operons present either on plasmid or chromosome or both, and induced by specific phthalate isomer. Understanding metabolic pathways, diversity and their genetic regulation may help in constructing bacterial strains through genetic engineering approach for effective bioremediation and environmental clean up.
Phthalate esters; Toxicity; Biodegradation; Dioxygenases; Genetic regulation
Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into β-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.
Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.