Phenanthrene is degraded via either o-phthalic acid or 1, 2-dihydroxynaphthalene in bacteria. A s... more Phenanthrene is degraded via either o-phthalic acid or 1, 2-dihydroxynaphthalene in bacteria. A soil isolate Pseudomonas sp. strain PP2 degrades phenanthrene as the sole source of carbon, but failed to utilize naphthalene [Prabhu and Phale (2003) Appl Microbiol Biotechnol 61:342-351]. Analysis of the phenanthrene-grown culture spent media of this strain by gas chromatography-mass spectrometry (GC-MS) showed accumulation of o-phthalic acid. The cell-free extract prepared from this strain showed activity of 1-hydroxy-2-naphthoic acid dioxygenase (1-H-2-NADO). The extract showed conversion of 1-hydroxy-2-naphthoic acid and 2-carboxybenzaldehyde to o-phthalic acid, as analyzed by thin layer chromatography and GC-MS. However, it failed to grow or respire on o-phthalic acid. These results suggest that besides 1, 2-dihydroxynaphthalene pathway, the strain has a truncated o-phthalic acid pathway for phenanthrene metabolism and excretes o-phthalic acid as a dead-end product, indicating the c...
The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes t... more The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli BE.
Pseudomonas putida CSV86, a soil isolate, preferentially utilizes naphthalene over glucose as a s... more Pseudomonas putida CSV86, a soil isolate, preferentially utilizes naphthalene over glucose as a source of carbon and energy. We present the draft genome sequence, which is 6.4 Mb in size; analysis suggests the chromosomal localization of genes coding for naphthalene utilization. The operons coding for glucose and other aromatic compounds might also be annotated in another study.
Proceedings of the National Academy of Sciences, 1997
In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protr... more In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protruding into the -barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the highresolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 Å. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C͞A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (⌬116-120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.
Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene... more Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, phydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA Gly , integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation. Citation: Paliwal V, Raju SC, Modak A, Phale PS, Purohit HJ (2014) Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation. PLoS ONE 9(1): e84000.
Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and rec... more Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and recalcitrant. Microbes, however, have evolved the ability to utilize these highly reduced and recalcitrant compounds as a potential source of carbon and energy. Aerobic degradation of aromatics is initiated by oxidizing the aromatic ring, making them more susceptible to cleavage by ring-cleaving dioxygenases. A preponderance of aromatic degradation genes on plasmids, transposons, and integrative genetic elements (and their shuffling through horizontal gene transfer) have lead to the evolution of novel aromatic degradative pathways. This enables the microorganisms to utilize a multitude of aromatics via common routes of degradation leading to metabolic diversity. In this review, we emphasize the exquisiteness and relevance of bacterial degradation of aromatics, interlinked degradative pathways, genetic and metabolic regulation, carbon source preference, and biosurfactant production. We have also explored the avenue of metagenomics, which opens doors to a plethora of uncultured and uncharted microbial genetics and metabolism that can be used effectively for bioremediation.
Acinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeru... more Acinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or a-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADPdependent GDH (NADP-GDH), while cells grown on glucose, 2¾ yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon sourcedependent presence of two (GDH I and GDH II ), three (GDH A , GDH B and GDH C ) and one (GDH P ) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.
Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy vi... more Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy via the 'phthalic acid' route. The key enzyme, 1-hydroxy-2-naphthoic acid dioxygenase (1-HNDO, EC 1.13.11.38), was purified to homogeneity using a 3-hydroxy-2-naphthoic acid (3-H2NA)-affinity matrix. The enzyme was a homotetramer with a native molecular mass of 160 kDa and subunit molecular mass of~39 kDa. It required Fe(II) as the cofactor and was specific for 1hydroxy-2-naphthoic acid (1-H2NA), with K m 13.5 mM and V max 114 mmol min "1 mg "1 . 1-HNDO failed to show activity with gentisic acid, salicylic acid and other hydroxynaphthoic acids tested. Interestingly, the enzyme showed substrate inhibition with a K i of 116 mM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a K i of 24 mM. Based on the pH-dependent spectral changes, the enzyme reaction product was identified as 2-carboxybenzalpyruvic acid. Under anaerobic conditions, the enzyme failed to convert 1-H2NA to 2-carboxybenzalpyruvic acid. Stoichiometric studies showed the incorporation of 1 mol O 2 into the substrate to yield 1 mol product. These results suggest that 1-HNDO from Pseudomonas sp. strain PPD is an extradioltype ring-cleaving dioxygenase.
Pseudomonas putida CSV86 preferentially utilizes aromatics over glucose and cometabolizes them wi... more Pseudomonas putida CSV86 preferentially utilizes aromatics over glucose and cometabolizes them with organic acids. On aromatics plus glucose, CSV86 utilized aromatics first with concomitant appearance of transient metabolites such as salicylate, benzaldehyde and benzoate. Citrate was the main extracellular metabolite observed during glucose uptake. The strain showed simultaneous utilization of organic acids and aromatic compounds. Based on the metabolite analysis and growth profiles, we hypothesize that the repression of glucose utilization could be due to organic acid intermediates generated from aromatic compound metabolism. The online measurements indicate the instantaneous metabolic state of the culture. For example, the CO 2 evolution and agitation speed show peak values during the two growth phases in the diauxic growth while dissolved oxygen values show decrease at the corresponding durations. These measurements correlated well with the offline measurements but provided a better time resolution of the process.
Bacterial porins, which allow the passage of solutes across the outer bacterial membrane, are str... more Bacterial porins, which allow the passage of solutes across the outer bacterial membrane, are structurally well characterized. They therefore lend themselves to detailed studies of the determinants of ion flow through transmembraneous channels. In a comparative study, we have performed Brownian dynamics simulations to obtain statistically significant transfer efficiencies for cations and anions through matrix porin OmpF, osmoporin OmpK36, phosphoporin PhoE and two OmpF charge mutants. The simulations show that the electrostatic potential at the highly charged channel constriction serves to enhance ion permeability of either cations or anions, dependent on the type of porin. At the same time translocation of counterions is not severely impeded. At the constriction, cations and anions follow distinct trajectories, due to the segregation of basic and acidic protein residues. Simulated ion selectivity and relative conductance agree well with experimental values, and are dependent crucially on the charge constellation at the pore constriction. The experimentally observed decrease in ion selectivity and single channel conductance with increasing ionic strength is well reproduced and can be attributed to electrostatic shielding of the pore lining.
Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate a... more Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDH I was produced when cells were grown on isophthalate, while GDH II was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDH P , when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDH I and GDH II were found to be homotetramers, while GDH P was found to be a homohexamer. GDH II was more sensitive to inhibition by isophthalate (2.5-and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDH I . Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDH I and GDH II , in strain PP4. In strain ISP4, irrespective of the carbon source, the GDH P produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDH P from isophthalate-grown cells was 2.5-to 3-fold higher than that of GDH P from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDH P in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.
Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source ... more Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants K m and V max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 M and 9.5 and 5.1 mol min ؊1 mg ؊1 , respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The K i for 1-naphthol was determined to be 79.8 M. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes arom... more Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of Pseudomonas putida KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic-and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strain's ability to utilize aromatics and organic acids in preference to glucose.
Phthalate isomers and their esters are used heavily in various industries. Excess use and leachin... more 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.
Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dih... more Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dihydroxynaphthalene (1,2-DHN), salicylic acid and catechol. Enzyme activity versus growth profile and heat stability studies suggested the presence of two distinct hydroxylases, namely 1-hydroxy-2-naphthoic acid hydroxylase and salicylate hydroxylase. 1-Hydroxy-2-naphthoic acid hydroxylase was partially purified (yield 48%, fold 81) and found to be a homodimer with a subunit molecular weight of $34 kDa. The enzyme was yellow in color, showed UV-visible absorption maxima at 274, 375 and 445 nm, and fluorescence emission maxima at 527 nm suggested it to be a flavoprotein. The apoenzyme prepared by the acid-ammonium sulfate (2 M) dialysis method was colorless, inactive and lost the characteristic flavin absorption spectra but regained $90% activity when reconstituted with FAD. Extraction of the prosthetic group and its analysis by HPLC suggests that the holoenzyme contained FAD. The enzyme was specific for 1-H2NA and failed to show activity with any other hydroxynaphthoic acid analogs or salicylic acid. The K m for 1-H2NA in the presence of either NADPH or NADH remained unaltered (72 and 75 mM, respectively), suggesting dual specificity for the coenzyme. The K m for FAD was determined to be 4.7 mM. The enzyme catalyzed the conversion of 1-H2NA to 1,2-DHN only under aerobic conditions. These results suggested that 1-hydroxy-2-naphthoic acid hydroxylase is a flavoprotein monooxygenase specific for 1-H2NA and different from salicylate-1hydroxylase.
Combination of an origin repair mutagenesis system with a new mutS host strain increased the effi... more Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli B E host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and ben... more Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and benzoate as the sole source of carbon and energy. Compared with glucose, cells grew faster on aromatic compounds as well as on organic acids. The organism failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. Whole-cell oxygen uptake, enzyme activity and metabolic studies suggest that in strain CSV86 glucose utilization is exclusively by the intracellular phosphorylative pathway, while in Stenotrophomonas maltophilia CSV89 and P. putida KT2442 glucose is metabolized by both direct oxidative and indirect phosphorylative pathways. Cells grown on glucose showed five-to sixfold higher activity of glucose-6-phosphate dehydrogenase compared with cells grown on aromatic compounds or organic acids as the carbon source. Study of [ 14 C]glucose uptake by whole cells indicates that the glucose is taken up by active transport. Metabolic and transport studies clearly demonstrate that glucose metabolism is suppressed when strain CSV86 is grown on aromatic compounds or organic acids.
Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment cultur... more Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3-and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O 2 uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3-and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O 2 uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3-or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.
Phenanthrene is degraded via either o-phthalic acid or 1, 2-dihydroxynaphthalene in bacteria. A s... more Phenanthrene is degraded via either o-phthalic acid or 1, 2-dihydroxynaphthalene in bacteria. A soil isolate Pseudomonas sp. strain PP2 degrades phenanthrene as the sole source of carbon, but failed to utilize naphthalene [Prabhu and Phale (2003) Appl Microbiol Biotechnol 61:342-351]. Analysis of the phenanthrene-grown culture spent media of this strain by gas chromatography-mass spectrometry (GC-MS) showed accumulation of o-phthalic acid. The cell-free extract prepared from this strain showed activity of 1-hydroxy-2-naphthoic acid dioxygenase (1-H-2-NADO). The extract showed conversion of 1-hydroxy-2-naphthoic acid and 2-carboxybenzaldehyde to o-phthalic acid, as analyzed by thin layer chromatography and GC-MS. However, it failed to grow or respire on o-phthalic acid. These results suggest that besides 1, 2-dihydroxynaphthalene pathway, the strain has a truncated o-phthalic acid pathway for phenanthrene metabolism and excretes o-phthalic acid as a dead-end product, indicating the c...
The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes t... more The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli BE.
Pseudomonas putida CSV86, a soil isolate, preferentially utilizes naphthalene over glucose as a s... more Pseudomonas putida CSV86, a soil isolate, preferentially utilizes naphthalene over glucose as a source of carbon and energy. We present the draft genome sequence, which is 6.4 Mb in size; analysis suggests the chromosomal localization of genes coding for naphthalene utilization. The operons coding for glucose and other aromatic compounds might also be annotated in another study.
Proceedings of the National Academy of Sciences, 1997
In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protr... more In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protruding into the -barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the highresolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 Å. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C͞A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (⌬116-120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.
Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene... more Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, phydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA Gly , integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation. Citation: Paliwal V, Raju SC, Modak A, Phale PS, Purohit HJ (2014) Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation. PLoS ONE 9(1): e84000.
Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and rec... more Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and recalcitrant. Microbes, however, have evolved the ability to utilize these highly reduced and recalcitrant compounds as a potential source of carbon and energy. Aerobic degradation of aromatics is initiated by oxidizing the aromatic ring, making them more susceptible to cleavage by ring-cleaving dioxygenases. A preponderance of aromatic degradation genes on plasmids, transposons, and integrative genetic elements (and their shuffling through horizontal gene transfer) have lead to the evolution of novel aromatic degradative pathways. This enables the microorganisms to utilize a multitude of aromatics via common routes of degradation leading to metabolic diversity. In this review, we emphasize the exquisiteness and relevance of bacterial degradation of aromatics, interlinked degradative pathways, genetic and metabolic regulation, carbon source preference, and biosurfactant production. We have also explored the avenue of metagenomics, which opens doors to a plethora of uncultured and uncharted microbial genetics and metabolism that can be used effectively for bioremediation.
Acinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeru... more Acinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or a-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADPdependent GDH (NADP-GDH), while cells grown on glucose, 2¾ yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon sourcedependent presence of two (GDH I and GDH II ), three (GDH A , GDH B and GDH C ) and one (GDH P ) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.
Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy vi... more Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy via the 'phthalic acid' route. The key enzyme, 1-hydroxy-2-naphthoic acid dioxygenase (1-HNDO, EC 1.13.11.38), was purified to homogeneity using a 3-hydroxy-2-naphthoic acid (3-H2NA)-affinity matrix. The enzyme was a homotetramer with a native molecular mass of 160 kDa and subunit molecular mass of~39 kDa. It required Fe(II) as the cofactor and was specific for 1hydroxy-2-naphthoic acid (1-H2NA), with K m 13.5 mM and V max 114 mmol min "1 mg "1 . 1-HNDO failed to show activity with gentisic acid, salicylic acid and other hydroxynaphthoic acids tested. Interestingly, the enzyme showed substrate inhibition with a K i of 116 mM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a K i of 24 mM. Based on the pH-dependent spectral changes, the enzyme reaction product was identified as 2-carboxybenzalpyruvic acid. Under anaerobic conditions, the enzyme failed to convert 1-H2NA to 2-carboxybenzalpyruvic acid. Stoichiometric studies showed the incorporation of 1 mol O 2 into the substrate to yield 1 mol product. These results suggest that 1-HNDO from Pseudomonas sp. strain PPD is an extradioltype ring-cleaving dioxygenase.
Pseudomonas putida CSV86 preferentially utilizes aromatics over glucose and cometabolizes them wi... more Pseudomonas putida CSV86 preferentially utilizes aromatics over glucose and cometabolizes them with organic acids. On aromatics plus glucose, CSV86 utilized aromatics first with concomitant appearance of transient metabolites such as salicylate, benzaldehyde and benzoate. Citrate was the main extracellular metabolite observed during glucose uptake. The strain showed simultaneous utilization of organic acids and aromatic compounds. Based on the metabolite analysis and growth profiles, we hypothesize that the repression of glucose utilization could be due to organic acid intermediates generated from aromatic compound metabolism. The online measurements indicate the instantaneous metabolic state of the culture. For example, the CO 2 evolution and agitation speed show peak values during the two growth phases in the diauxic growth while dissolved oxygen values show decrease at the corresponding durations. These measurements correlated well with the offline measurements but provided a better time resolution of the process.
Bacterial porins, which allow the passage of solutes across the outer bacterial membrane, are str... more Bacterial porins, which allow the passage of solutes across the outer bacterial membrane, are structurally well characterized. They therefore lend themselves to detailed studies of the determinants of ion flow through transmembraneous channels. In a comparative study, we have performed Brownian dynamics simulations to obtain statistically significant transfer efficiencies for cations and anions through matrix porin OmpF, osmoporin OmpK36, phosphoporin PhoE and two OmpF charge mutants. The simulations show that the electrostatic potential at the highly charged channel constriction serves to enhance ion permeability of either cations or anions, dependent on the type of porin. At the same time translocation of counterions is not severely impeded. At the constriction, cations and anions follow distinct trajectories, due to the segregation of basic and acidic protein residues. Simulated ion selectivity and relative conductance agree well with experimental values, and are dependent crucially on the charge constellation at the pore constriction. The experimentally observed decrease in ion selectivity and single channel conductance with increasing ionic strength is well reproduced and can be attributed to electrostatic shielding of the pore lining.
Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate a... more Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDH I was produced when cells were grown on isophthalate, while GDH II was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDH P , when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDH I and GDH II were found to be homotetramers, while GDH P was found to be a homohexamer. GDH II was more sensitive to inhibition by isophthalate (2.5-and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDH I . Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDH I and GDH II , in strain PP4. In strain ISP4, irrespective of the carbon source, the GDH P produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDH P from isophthalate-grown cells was 2.5-to 3-fold higher than that of GDH P from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDH P in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.
Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source ... more Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants K m and V max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 M and 9.5 and 5.1 mol min ؊1 mg ؊1 , respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The K i for 1-naphthol was determined to be 79.8 M. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes arom... more Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of Pseudomonas putida KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic-and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strain's ability to utilize aromatics and organic acids in preference to glucose.
Phthalate isomers and their esters are used heavily in various industries. Excess use and leachin... more 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.
Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dih... more Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dihydroxynaphthalene (1,2-DHN), salicylic acid and catechol. Enzyme activity versus growth profile and heat stability studies suggested the presence of two distinct hydroxylases, namely 1-hydroxy-2-naphthoic acid hydroxylase and salicylate hydroxylase. 1-Hydroxy-2-naphthoic acid hydroxylase was partially purified (yield 48%, fold 81) and found to be a homodimer with a subunit molecular weight of $34 kDa. The enzyme was yellow in color, showed UV-visible absorption maxima at 274, 375 and 445 nm, and fluorescence emission maxima at 527 nm suggested it to be a flavoprotein. The apoenzyme prepared by the acid-ammonium sulfate (2 M) dialysis method was colorless, inactive and lost the characteristic flavin absorption spectra but regained $90% activity when reconstituted with FAD. Extraction of the prosthetic group and its analysis by HPLC suggests that the holoenzyme contained FAD. The enzyme was specific for 1-H2NA and failed to show activity with any other hydroxynaphthoic acid analogs or salicylic acid. The K m for 1-H2NA in the presence of either NADPH or NADH remained unaltered (72 and 75 mM, respectively), suggesting dual specificity for the coenzyme. The K m for FAD was determined to be 4.7 mM. The enzyme catalyzed the conversion of 1-H2NA to 1,2-DHN only under aerobic conditions. These results suggested that 1-hydroxy-2-naphthoic acid hydroxylase is a flavoprotein monooxygenase specific for 1-H2NA and different from salicylate-1hydroxylase.
Combination of an origin repair mutagenesis system with a new mutS host strain increased the effi... more Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli B E host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and ben... more Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and benzoate as the sole source of carbon and energy. Compared with glucose, cells grew faster on aromatic compounds as well as on organic acids. The organism failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. Whole-cell oxygen uptake, enzyme activity and metabolic studies suggest that in strain CSV86 glucose utilization is exclusively by the intracellular phosphorylative pathway, while in Stenotrophomonas maltophilia CSV89 and P. putida KT2442 glucose is metabolized by both direct oxidative and indirect phosphorylative pathways. Cells grown on glucose showed five-to sixfold higher activity of glucose-6-phosphate dehydrogenase compared with cells grown on aromatic compounds or organic acids as the carbon source. Study of [ 14 C]glucose uptake by whole cells indicates that the glucose is taken up by active transport. Metabolic and transport studies clearly demonstrate that glucose metabolism is suppressed when strain CSV86 is grown on aromatic compounds or organic acids.
Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment cultur... more Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3-and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O 2 uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3-and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O 2 uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3-or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.
Uploads
Papers by Prashant Phale