... North Dakota Lignite by a Mixed Bacterial and a Mixed Bacterial/Fungal Culture A. MAKA,* VJ S... more ... North Dakota Lignite by a Mixed Bacterial and a Mixed Bacterial/Fungal Culture A. MAKA,* VJ SRIVASTAVA, JJ KILBANE 11, AND C. AKIN Institute of Gas Technology, Chicago, IL 60616 ... Applied Biochemistry and Biotechnology 71 5 Vol. 20/2 I, 1989 Page 2. 716 Maka et al. ...
This report was prepared as an account of work Sl_msored by an agency of the Urfitc, d States Gov... more This report was prepared as an account of work Sl_msored by an agency of the Urfitc, d States Government. Neither the United States Governnlent nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or rcslxmsibility for the accuracy, cx_mpleteness,or usefulness of any inforrnation, apparatus, product, or pr(Kzess disclosed, or represents that its use would not infringe priwately owned rights. Reference herein to any specific conamercial product, process, or service by trade name, trademark, manufacturer, or otherwise dcx:s not necessarily constitute or imply its endorsement, recorrmmndation, or favoring by the United States Governmerlt or any agency thereof. The views and opinions of authors ex. pressed herein do not necessarily state or reflect those of the United States Governnlertt or any agency thereof. This report has been reproduced directly from the best available copy.
The use of gas–liquid foams as a means of delivering chemicals to the subsurface is being conside... more The use of gas–liquid foams as a means of delivering chemicals to the subsurface is being considered as an aid to in-situ soil remediation schemes involving bioremediation, chemical oxidation and soil washing. Experiments were conducted to investigate the physics of foam flow in soils and to identify parameters that are important to allow foam injection at low pressures so as to avoid problems due to channelling and soil heaving. Gas–liquid foams of quality (gas-content) ranging from 87 to 99% were flowed through soils of permeability ranging from 0.09 to 900 darcy (0.09 to 900 μm2) in vertical columns 3 in. (7.6 cm) in diameter. Surfactant solutions used for foam generation included an aqueous anionic surfactant Standapol ES-2 and two ethanol-based surfactants developed for in-situ soil flushing. These foams behaved as highly viscous fluids in flowing through soils; the apparent viscosity increased with increasing soil permeability. Foams seem to break and regenerate. At steady state, there was a net accumulation of liquid in the pore space. Based on material balance calculations, liquid content in the soil ranged from 7 to 59%. This is much higher than the liquid content of the injected or produced foam. It was observed that pressure gradients for downflow were only a fraction of that for upflow. The results also suggest that low pressure gradients can be obtained by using foams of higher quality (gas content) and a foaming agent that provides good foamability but low foam stability.
The objective of this project was to develop a microbial culture capable of removing organic sulf... more The objective of this project was to develop a microbial culture capable of removing organic sulfur from coal. This objective was successfully achieved; thus (1) bacterial culture, capable of specifically metabolizing sulfur atoms present in organic molecules was developed and (2) using this culture, up to 91% removal of organic sulfur from coal was observed in a bioreactor. 25 refs., 30 figs., 13 tabs.
The work planned for this quarter included the isolation of pure bacterial cultures capable of de... more The work planned for this quarter included the isolation of pure bacterial cultures capable of desulfurizing organic substrates and the genetic study of those cultures through the isolation and analysis of mutations. All aspects of the project are proceeding well and are either on or ahead of schedule. Two pure cultures of bacteria, that are each capable of utilizing dibenzothiophene (DBT) as their sole source of sulfur, were isolated from the mixed culture IGTS7. These cultures have been identified as Rhodococcus rhodochrous and Bacillus sphaericus species and have been designated IGTS8 and IGTS9, respectively. The examination of all of these cultures confirmed the identities of IGTS8 and IGTS9 as Rhodococcus rhodochrous and Bacillus sphaericus, respectively, and revealed that IGTS8 and IGTS9 are apparently unique in their ability to utilize organically-bound sulfur. The metabolites of DBT produced by IGTS8 were analyzed by gas chromatography/mass spectroscopy. Genetic studies of IGTS8 have begun. Mutants resistant to high levels of three different antibiotics, and combinations of antibiotics have been obtained. Resistance to antibiotics is a selectable genetic trait that will help to unequivocally identify this particular strain of bacteria and will be used in future genetic experiments. Chemical mutagenesis and ultra violet light mutagenesismore » procedures are being optimized. 2 figs., 8 tabs.« less
ABSTRACT The overall program objective is to develop and evaluate environmentally benign agents o... more ABSTRACT The overall program objective is to develop and evaluate environmentally benign agents or products that are effective in the prevention, inhibition, and mitigation of microbially influenced corrosion (MIC) in the internal surfaces of metallic natural gas pipelines. The goal is to develop one or more environmentally benign (a.k.a. ''green'') products that can be applied to maintain the structure and dependability of the natural gas infrastructure. Previous testing indicated that the growth, and the metal corrosion caused by pure cultures of sulfate reducing bacteria were inhibited by hexane extracts of some pepper plants. This quarter tests were performed with mixed bacterial cultures obtained from natural gas pipelines. Treatment with the pepper extracts affected the growth and metabolic activity of the microbial consortia. Specifically, the growth and metabolism of sulfate reducing bacteria was inhibited. The demonstration that pepper extracts can inhibit the growth and metabolism of sulfate reducing bacteria in mixed cultures is a significant observation validating a key hypothesis of the project. Future tests to determine the effects of pepper extracts on mature/established biofilms will be performed next.
The Institute of Gas Technology (IGT) has been developing several techniques to improve biodegrad... more The Institute of Gas Technology (IGT) has been developing several techniques to improve biodegradation of town gas plant wastes such as multi-ring polynuclear aromatic hydrocarbons (PAH's). These techniques include the use of bioemulsifiers, chemical oxidants or biologically produced chemicals, PAH-degrading cultures developed by IGT through enrichment techniques, and appropriate co-metabolic and/or enzyme-inducing substrates. Depending upon the waste type and concentration,
The purpose of this project was to investigate the possibilities of developing an integrated phys... more The purpose of this project was to investigate the possibilities of developing an integrated physical/chemical/microbial process for the precombustion removal of sulfur from coal. Microorganisms are capable of specifically cleaving carbon-sulfur bonds and removing substantial amounts of organic sulfur from coal; however, the removal of organic sulfur from coal by microorganisms is hampered by the fact that, as a solid substrate, it is difficult to bring microorganisms in contact with the entirety of a coal sample. The suitability of physically/chemically treated coal samples for subsequent biodesulfurization was examined in this study.
The overall program objective is to develop and evaluate environmentally benign agents or product... more The overall program objective is to develop and evaluate environmentally benign agents or products that are effective in the prevention, inhibition, and mitigation of microbially influenced corrosion (MIC) in the internal surfaces of metallic natural gas pipelines. The goal is to develop one or more environmentally benign (a.k.a. "green") products that can be applied to maintain the structure and dependability of the natural gas infrastructure. Approach: Previous testing indicated that the growth, and the metal corrosion caused by pure cultures of sulfate reducing bacteria were inhibited by hexane extracts of some pepper plants. This quarter tests were performed with mixed bacterial cultures obtained from natural gas pipelines. Results: Treatment with the pepper extracts affected the growth and metabolic activity of the microbial consortia. Specifically, the growth and metabolism of sulfate reducing bacteria was inhibited.. Conclusions: The demonstration that pepper extracts can inhibit the growth and metabolism of sulfate reducing bacteria in mixed cultures is a significant observation validating a key hypothesis of the project. Future tests to determine the effects of pepper extracts on mature /established biofilms will be performed next.
DISTRt_t't.rTION OF THIS D()CL.;MEf,,i i" iS UNLIMITED DISCLAIMER This report was prepared as an ... more DISTRt_t't.rTION OF THIS D()CL.;MEf,,i i" iS UNLIMITED DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.
To develop molecular tools and examine inducible and constitutive gene expression in Thermus ther... more To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus. Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli. An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85 degrees C) from the dnaK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flavus was cloned and expressed in both E. coli and T. thermophilus HB27. This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85 degrees C) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible. The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species.
Biochemical and Biophysical Research Communications, Sep 1, 2002
A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to... more A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogencontaining heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2 kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37°C was determined to be 8:0 lmol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.
The purpose of this project was to investigate the possibilities of developing an integrated phys... more The purpose of this project was to investigate the possibilities of developing an integrated physical/chemical/microbial process for the precombustion removal of sulfur from coal. Microorganisms are capable of specifically cleaving carbon-sulfur bonds and removing substantial amounts of organic sulfur from coal; however, the removal of organic sulfur from coal by microorganisms is hampered by the fact that, as a solid substrate, it is difficult to bring microorganisms in contact with the entirety of a coal sample. The suitability of physically/chemically treated coal samples for subsequent biodesulfurization was examined in this study.
The Pseudomonas putida plasmid pAC25 specifies an inducible 3-chlorobenzoic acid degradation path... more The Pseudomonas putida plasmid pAC25 specifies an inducible 3-chlorobenzoic acid degradation pathway. 3-iodobenzoic acid is a poor substrate and fails to induce the enzymes of this pathway, even though 3-fluorobenzoic acid and 3-bromobenzoic acid can both serve as inducers and are good substrates. Through chemostatic selection we have isolated a derivative of pAC25 with altered regulation and substrate specificity such that 3-iodobenzoic acid can now serve as a sole carbon and energy source.
The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a... more The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a key role in the beta-ketoadipate pathway of benzoate degradation. A 10.2-kb EcoRI fragment isolated from a Pseudomonas putida genomic library complemented a mutant deficient in this enzyme. The MLEI coding gene, catB, was localized to a 1.6-kb fragment which was sequenced by the dideoxy chain termination method. MLEI was purified 25-fold from crude extracts of benzoate-grown P. putida PRS2015 harboring the cloned catB gene. Purified MLEI was greater than 95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit Mr was 40,000 which was in close agreement with the nucleotide sequence data. N-terminal sequence analysis of purified MLEI protein agreed with the N terminus predicted by the nucleotide sequence. Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene (K.L. Ngai, B.F., D.K. Chatterjee, L.N. Ornston, and A.M.C., unpublished), whose gene product catalyzes the analogous reaction in 3-chlorobenzoate degradation, showed significant homology. These results suggest that catB and clcB have diverged from a common ancestral gene.
... North Dakota Lignite by a Mixed Bacterial and a Mixed Bacterial/Fungal Culture A. MAKA,* VJ S... more ... North Dakota Lignite by a Mixed Bacterial and a Mixed Bacterial/Fungal Culture A. MAKA,* VJ SRIVASTAVA, JJ KILBANE 11, AND C. AKIN Institute of Gas Technology, Chicago, IL 60616 ... Applied Biochemistry and Biotechnology 71 5 Vol. 20/2 I, 1989 Page 2. 716 Maka et al. ...
This report was prepared as an account of work Sl_msored by an agency of the Urfitc, d States Gov... more This report was prepared as an account of work Sl_msored by an agency of the Urfitc, d States Government. Neither the United States Governnlent nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or rcslxmsibility for the accuracy, cx_mpleteness,or usefulness of any inforrnation, apparatus, product, or pr(Kzess disclosed, or represents that its use would not infringe priwately owned rights. Reference herein to any specific conamercial product, process, or service by trade name, trademark, manufacturer, or otherwise dcx:s not necessarily constitute or imply its endorsement, recorrmmndation, or favoring by the United States Governmerlt or any agency thereof. The views and opinions of authors ex. pressed herein do not necessarily state or reflect those of the United States Governnlertt or any agency thereof. This report has been reproduced directly from the best available copy.
The use of gas–liquid foams as a means of delivering chemicals to the subsurface is being conside... more The use of gas–liquid foams as a means of delivering chemicals to the subsurface is being considered as an aid to in-situ soil remediation schemes involving bioremediation, chemical oxidation and soil washing. Experiments were conducted to investigate the physics of foam flow in soils and to identify parameters that are important to allow foam injection at low pressures so as to avoid problems due to channelling and soil heaving. Gas–liquid foams of quality (gas-content) ranging from 87 to 99% were flowed through soils of permeability ranging from 0.09 to 900 darcy (0.09 to 900 μm2) in vertical columns 3 in. (7.6 cm) in diameter. Surfactant solutions used for foam generation included an aqueous anionic surfactant Standapol ES-2 and two ethanol-based surfactants developed for in-situ soil flushing. These foams behaved as highly viscous fluids in flowing through soils; the apparent viscosity increased with increasing soil permeability. Foams seem to break and regenerate. At steady state, there was a net accumulation of liquid in the pore space. Based on material balance calculations, liquid content in the soil ranged from 7 to 59%. This is much higher than the liquid content of the injected or produced foam. It was observed that pressure gradients for downflow were only a fraction of that for upflow. The results also suggest that low pressure gradients can be obtained by using foams of higher quality (gas content) and a foaming agent that provides good foamability but low foam stability.
The objective of this project was to develop a microbial culture capable of removing organic sulf... more The objective of this project was to develop a microbial culture capable of removing organic sulfur from coal. This objective was successfully achieved; thus (1) bacterial culture, capable of specifically metabolizing sulfur atoms present in organic molecules was developed and (2) using this culture, up to 91% removal of organic sulfur from coal was observed in a bioreactor. 25 refs., 30 figs., 13 tabs.
The work planned for this quarter included the isolation of pure bacterial cultures capable of de... more The work planned for this quarter included the isolation of pure bacterial cultures capable of desulfurizing organic substrates and the genetic study of those cultures through the isolation and analysis of mutations. All aspects of the project are proceeding well and are either on or ahead of schedule. Two pure cultures of bacteria, that are each capable of utilizing dibenzothiophene (DBT) as their sole source of sulfur, were isolated from the mixed culture IGTS7. These cultures have been identified as Rhodococcus rhodochrous and Bacillus sphaericus species and have been designated IGTS8 and IGTS9, respectively. The examination of all of these cultures confirmed the identities of IGTS8 and IGTS9 as Rhodococcus rhodochrous and Bacillus sphaericus, respectively, and revealed that IGTS8 and IGTS9 are apparently unique in their ability to utilize organically-bound sulfur. The metabolites of DBT produced by IGTS8 were analyzed by gas chromatography/mass spectroscopy. Genetic studies of IGTS8 have begun. Mutants resistant to high levels of three different antibiotics, and combinations of antibiotics have been obtained. Resistance to antibiotics is a selectable genetic trait that will help to unequivocally identify this particular strain of bacteria and will be used in future genetic experiments. Chemical mutagenesis and ultra violet light mutagenesismore » procedures are being optimized. 2 figs., 8 tabs.« less
ABSTRACT The overall program objective is to develop and evaluate environmentally benign agents o... more ABSTRACT The overall program objective is to develop and evaluate environmentally benign agents or products that are effective in the prevention, inhibition, and mitigation of microbially influenced corrosion (MIC) in the internal surfaces of metallic natural gas pipelines. The goal is to develop one or more environmentally benign (a.k.a. ''green'') products that can be applied to maintain the structure and dependability of the natural gas infrastructure. Previous testing indicated that the growth, and the metal corrosion caused by pure cultures of sulfate reducing bacteria were inhibited by hexane extracts of some pepper plants. This quarter tests were performed with mixed bacterial cultures obtained from natural gas pipelines. Treatment with the pepper extracts affected the growth and metabolic activity of the microbial consortia. Specifically, the growth and metabolism of sulfate reducing bacteria was inhibited. The demonstration that pepper extracts can inhibit the growth and metabolism of sulfate reducing bacteria in mixed cultures is a significant observation validating a key hypothesis of the project. Future tests to determine the effects of pepper extracts on mature/established biofilms will be performed next.
The Institute of Gas Technology (IGT) has been developing several techniques to improve biodegrad... more The Institute of Gas Technology (IGT) has been developing several techniques to improve biodegradation of town gas plant wastes such as multi-ring polynuclear aromatic hydrocarbons (PAH's). These techniques include the use of bioemulsifiers, chemical oxidants or biologically produced chemicals, PAH-degrading cultures developed by IGT through enrichment techniques, and appropriate co-metabolic and/or enzyme-inducing substrates. Depending upon the waste type and concentration,
The purpose of this project was to investigate the possibilities of developing an integrated phys... more The purpose of this project was to investigate the possibilities of developing an integrated physical/chemical/microbial process for the precombustion removal of sulfur from coal. Microorganisms are capable of specifically cleaving carbon-sulfur bonds and removing substantial amounts of organic sulfur from coal; however, the removal of organic sulfur from coal by microorganisms is hampered by the fact that, as a solid substrate, it is difficult to bring microorganisms in contact with the entirety of a coal sample. The suitability of physically/chemically treated coal samples for subsequent biodesulfurization was examined in this study.
The overall program objective is to develop and evaluate environmentally benign agents or product... more The overall program objective is to develop and evaluate environmentally benign agents or products that are effective in the prevention, inhibition, and mitigation of microbially influenced corrosion (MIC) in the internal surfaces of metallic natural gas pipelines. The goal is to develop one or more environmentally benign (a.k.a. "green") products that can be applied to maintain the structure and dependability of the natural gas infrastructure. Approach: Previous testing indicated that the growth, and the metal corrosion caused by pure cultures of sulfate reducing bacteria were inhibited by hexane extracts of some pepper plants. This quarter tests were performed with mixed bacterial cultures obtained from natural gas pipelines. Results: Treatment with the pepper extracts affected the growth and metabolic activity of the microbial consortia. Specifically, the growth and metabolism of sulfate reducing bacteria was inhibited.. Conclusions: The demonstration that pepper extracts can inhibit the growth and metabolism of sulfate reducing bacteria in mixed cultures is a significant observation validating a key hypothesis of the project. Future tests to determine the effects of pepper extracts on mature /established biofilms will be performed next.
DISTRt_t't.rTION OF THIS D()CL.;MEf,,i i" iS UNLIMITED DISCLAIMER This report was prepared as an ... more DISTRt_t't.rTION OF THIS D()CL.;MEf,,i i" iS UNLIMITED DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.
To develop molecular tools and examine inducible and constitutive gene expression in Thermus ther... more To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus. Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli. An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85 degrees C) from the dnaK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flavus was cloned and expressed in both E. coli and T. thermophilus HB27. This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85 degrees C) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible. The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species.
Biochemical and Biophysical Research Communications, Sep 1, 2002
A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to... more A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogencontaining heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2 kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37°C was determined to be 8:0 lmol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.
The purpose of this project was to investigate the possibilities of developing an integrated phys... more The purpose of this project was to investigate the possibilities of developing an integrated physical/chemical/microbial process for the precombustion removal of sulfur from coal. Microorganisms are capable of specifically cleaving carbon-sulfur bonds and removing substantial amounts of organic sulfur from coal; however, the removal of organic sulfur from coal by microorganisms is hampered by the fact that, as a solid substrate, it is difficult to bring microorganisms in contact with the entirety of a coal sample. The suitability of physically/chemically treated coal samples for subsequent biodesulfurization was examined in this study.
The Pseudomonas putida plasmid pAC25 specifies an inducible 3-chlorobenzoic acid degradation path... more The Pseudomonas putida plasmid pAC25 specifies an inducible 3-chlorobenzoic acid degradation pathway. 3-iodobenzoic acid is a poor substrate and fails to induce the enzymes of this pathway, even though 3-fluorobenzoic acid and 3-bromobenzoic acid can both serve as inducers and are good substrates. Through chemostatic selection we have isolated a derivative of pAC25 with altered regulation and substrate specificity such that 3-iodobenzoic acid can now serve as a sole carbon and energy source.
The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a... more The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a key role in the beta-ketoadipate pathway of benzoate degradation. A 10.2-kb EcoRI fragment isolated from a Pseudomonas putida genomic library complemented a mutant deficient in this enzyme. The MLEI coding gene, catB, was localized to a 1.6-kb fragment which was sequenced by the dideoxy chain termination method. MLEI was purified 25-fold from crude extracts of benzoate-grown P. putida PRS2015 harboring the cloned catB gene. Purified MLEI was greater than 95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit Mr was 40,000 which was in close agreement with the nucleotide sequence data. N-terminal sequence analysis of purified MLEI protein agreed with the N terminus predicted by the nucleotide sequence. Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene (K.L. Ngai, B.F., D.K. Chatterjee, L.N. Ornston, and A.M.C., unpublished), whose gene product catalyzes the analogous reaction in 3-chlorobenzoate degradation, showed significant homology. These results suggest that catB and clcB have diverged from a common ancestral gene.
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