612
ISOLATION PROCEDURES
[60]
in the metabolism of nicotine, 12 whereas NMb is unique among known rabbit P450 isozymes in that it hydroxylates testosterone mainly in the 15 position. Acknowledgments This work was supported by Grant DK-10339from the National Institutes of Health. 12D. E. Williams, X. Ding, and M. J. Coon, Biochem. Biophys. Res. Commun. 166, 945 (1990).
[60] B a c t e r i a l C y t o c h r o m e s P 4 5 0 : I s o l a t i o n and Identification By JULIAN A. PETERSON and J.-Y. L u Introduction The role of the members of the superfamily of proteins called cytochrome P450 in the oxidation of organic compounds has been well documented. Over 200 P450s have been identified, purified, and characterized, and they can be divided into many different gene families. These proteins have been found in essentially all aerobic organisms from prokaryotes to mammals.l All P450s which have been studied to date can be divided into two groups on the basis of the electron transfer partner which is involved in the provision of the required electrons for the functioning of P450. One group consists of those P450s which have a single flavoprotein reductase containing both FAD and FMN. Examples include P450s of the endoplasmic reticulum of higher organisms. There has been only one member of this group which has been carefully studied in a prokaryote, and that is the soluble, catalytically self-sufficient fatty acid to-hydroxylase of Bacillus megaterium, P450BM-3. A second group consists of P450s which have two electron transfer partners: (1) an FAD-containing reduced pyridine nucleotide dehydrogenase and (2) an iron-sulfur protein. The members of this group, which are found in higher organisms, are typically localized in mitochondria. Several examples have been found in microorganisms, and 1 D. W. Nebert, D. R. Nelson, M. J. Coon, R. W. Estabrook, R. Feyereisen, Y. FujiiKuriyama, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, J. C. Loper, R. Sato, M. R. Waterman, and D. J. Waxman, in "DNA and Cell Biology," Vol. 10, p. 1. Mary Ann Liebert, New York, 1991.
METHODSIN ENZYMOLOGY,VOL.206
Copyright© 1991by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[60]
BACTERIAL CYTOCHROMES P450
613
the best characterized is the enzyme involved in camphor metabolism, P450cam. The P450cam enzyme system has over the years proved to be an extremely powerful tool for structure-function studies. The data accumulated have been valuable for predicting how mammalian P450s might react during substrate binding, electron transfer, and oxygen activation. In addition, the three-dimensional structure of both classes of membrane-bound P450s has been predicted on the basis of sequence similarities to P450cam. 2 Although the extrapolated data seem sound, it is based on the structure of only P450cam. While much effort has been expended to obtain large amounts of mammalian P450s for comparable structure-function studies, to date none are available in the quantities or purity necessary. Several groups have begun recently to work to expand the number of examples of well-characterized soluble, bacterial P450s so that the general hypotheses regarding P450cam can be tested. The number of these prokaryotic P450s which have been identified is fairly large; however, less than I0 have been characterized to date and none of them as completely as P450cam. As the interest in the use of P450s as a tool for the oxidative modification of organic compounds increases, 3 the search for new forms of the enzyme with different substrate specificities will become important. The effort to identify candidate organisms can take at least two different directions: (1) examine culture collections for candidate organisms4-7; or (2) isolate new strains via culture enrichment techniques.S Culture enrichment techniques have proved to be useful in the isolation and identification of new sources of a wide variety of commercially valuable organisms 9 and should prove equally useful to investigators in biological oxidations. 3 In any effort to isolate new microbial strains which can metabolize compounds of interest, one must consider how widespread will be the 2 D. R. Nelson and H. W. Strobel, Biochemistry 28, 656 (1989). 3 H. G. Davies, R. H. Green, D. R. Kelly, and S. M. Roberts, in "Biotransformations in Preparative Organic Chemistry: The Use of Isolated Enzymes and Whole Cell Systems in Synthesis." Academic Press, New York, 1989. 4 J.-S. He, R. T. Ruettinger, H.-M. Liu, and A. J. Fulco, Biochim. Biophys. Acta 1009, 301 (1989). 5 R. H. Kanemoto, A. T. Powell, D. E. Akiyoshi, D. A. Regier, R. A. Kerstetter, E. W. Nester, M. C. Hawes, and M. P. Gordon, J. Bacteriol. 171, 2506 (1989). 6 M. Horii, T. Ishizaki, S.-Y. Paik, T. Manome, and Y. Murooka, J. Bacterial. 172, 3644
(1990). 7 C. A. Omer, R. Lenstra, P. J. Litle, C. Dean, J. M. Tepperman, K. J. Leto, J. A. Romesser, and D. P. O'Keefe, J. Bacteriol. 172, 3335 (1990). g J. H. Abul, R. I. Murray, K. K. Bhattacharyya, G. C. Wagner, and I. C. Gunsalus, J. Biol. Chem. 265, 1345 (1990). 9 n. Veldkamp, Methods Microbiol. 3A, 305 (1970).
614
ISOLATIONPROCEDURES
[60]
facility to oxidize these organic compounds and whether culture enrichment can be usefully applied. Clearly there are a variety of organic polymers which are persistent in the environment. The degradation of small organic compounds is much more favorable except in the cases where the compound is substituted with halogen atoms. In fact, mixtures of nitrogenor phosphate-based fertilizers were sprayed on some Alaskan beaches to promote the growth of preexisting microbes to oxidize the hydrocarbons present in the oil spilled from the Exxon Valdez in 1989. As described in this chapter, the probability of finding an organism which will oxidize the "target" hydrocarbon is quite high.
Materials and Methods
Spectrophotometric Assay for Cytochrome P450 in Whole Cells The typical spectrophotometric assay for P450 in turbid suspensions has been described, z° and it applies to the determination of P450 in whole bacterial cells. Special precautions may have to be taken, such as the inclusion of 7.5% (v/v) glycerol in the reaction mixture, to avoid settling of the cells during the course of the determination. This settling would result in false readings of absorbance. In the typical assay an appropriate dilution of the cells or the bacterial growth medium into 50 mM potassium phosphate buffer, pH 7.4 (with or without the glycerol) is treated with a few grains of sodium dithionite, which results in the reduction of the hemeproteins (as well as most other redox-active components of the cells). The cells are then divided between the sample and reference cuvettes and the baseline recorded from 500 to 400 nm. Carbon monoxide is then gently bubbled through the sample cuvette for approximately 30 sec. Care must be taken not to remove the cells from the cuvette in the bubbles which are introduced with the carbon monoxide. The difference spectrum of the carbon monoxide complex of the ferrous hemoproteins is then recorded. The spectrum should be recorded again until no further changes are observed. If the cells have been grown to the stationary phase on the organic compound of interest and the cell density is approximately 5 g wet weight per liter, the determination should be able to be done directly on the culture medium. This makes the assumption that the amount of P450 in the cells is greater than 0.2% of the soluble protein. In those cases where the P450 content is lower than 0.2%, the cells can be concentrated by l0 R. W. Estabrook, J. A. Peterson, J. Baron, and A. G. Hildebrandt, Methods Pharmacol. 2, 305 (1972).
[60]
BACTERIAL CYTOCHROMES P450
615
centrifugation at 5000 g for 5 min and then resuspended in 1/5 volume of the phosphate buffer.
Other Potential Assay or Screening Techniques The spectrophotometric assay procedure described above, which is typically utilized to determine the concentration of P450, is cumbersome to perform on the scale necessary to screen large numbers of candidate organisms. Thus, the utilization of other screening techniques was explored. Oligonucleotide Hybridization. In principle, an oligonucleotide probe prepared from a long, highly conserved region of interest (e.g., the hemebinding region or the I-helix region) ought to be able to be used to probe large numbers of colonies on agar plates for the presence of P450s. This would enable investigators to more rapidly screen culture isolates for the presence of colonies which might contain interesting P450s. In practice this procedure might not be very practical because of the great diversity at the nucleotide level even between P450s of the same class. In the case of prokaryotic P450s there is a consensus amino acid sequence H of AVEELLR between the I helix and the heme-binding region. If a series of oligonucleotides were synthesized which encodes all possible used codons for the amino acids used in this sequence, it would contain at least 512 different members for only 20 nucleotides. Obviously this mixed pool of oligonucleotides would not make a very good probe for bacterial P450s. Oligonucleotides might be synthesized to detect the presence of very closely related P450s, but even in this case, the known nucleotide sequences indicate that the probability of success is relatively low. Western Blots. Western blots using polyclonal antibodies prepared against specific bacterial P450s or even mammalian P450s probably cannot be used as a general screening tool for the presence of other P450s. It is now well recognized that the different subfamilies of mammalian P450s exhibit different antibody specificity, and hence there is no reason to expect that the various bacterial P450s will show any less of a diversity in their antigenic determinants. In fact, polyclonal antibodies prepared against P450cam will not cross-react with P4501in8 or P450terp. ~2 In summary, only the brute force screening procedure using the spectrophotometric measurement of the content of P450 in individual clones is a reliable measure of the presence of P450 in culture isolates. 11 Single-letter abbreviations for amino acids: A, alanine; E, glutamic acid; L, leucine; R, arginine; V, valine. 12j._y. Lu and J. A. Peterson, unpublished observations (1990).
616
ISOLATION PROCEDURES
[60]
Culture Enrichment Techniques The goal of culture enrichment is to select from the natural environment an organism which will fulfill specific needs. These "needs" are identified ahead of time and are used to select the desired bacterial strain: (1) the location which might harbor it; (2) the media which will provide a growth advantage; (3) the type of culture enrichment, closed or open; and (4) the growth conditions such as length of incubation, temperature, aeration, and agitation. In our laboratory, we wanted to isolate a new microbial strain which would oxidize water-insoluble natural product terpenes. It was presumed that the hydrocarbon would have to be oxidized by a P450-type of enzyme because of the waterinsoluble character of the compounds. The compounds were selected on the basis of their purity, availability, and cost. In our initial screen of soil samples, we used eight different monoterpenes. As described below, a-terpineol was only one of the compounds used, and the cells which grew on it were simply the first ones which were examined for P450 content.
Selection of Collection Site It should be obvious that the very best enrichment culture conditions will not select for a microorganism which does not already exist in the collection sample. In our laboratory, we wanted to select for an organism which would metabolize the natural product a-terpineol, which is used as an industrial perfuming agent. This compound is found in pine oil, and so samples were collected from several locations around Dallas County, TX, which would have been exposed to pine oils over the years. Since this natural product terpene was not accumulating, it was reasonable to assume that it was being degraded by some organism in addition to its slow evaporation from decaying vegetative matter. Two sites were initially chosen, both of which were relatively moist (swampy): (1) in a small pond which serves as a catch basin for a large stream draining a relatively large portion of North Dallas and (2) in a swamp in the floodplain of the Trinity River which flows through Dallas. After the sites have been selected, it is important to use sterile techniques when collecting samples. First, do not introduce laboratory strains of organisms into the collection sample. Second, some of the soil or water samples which are being collected may contain toxic or carcinogenic compounds which are being metabolized by the natural flora of the environment. We routinely use sterile surgical gloves, laboratory spatulas for digging, and plastic bags as containers for the samples. We have used successfully new Ziploc heavy-duty quart-size plastic bags. These are
[60]
BACTERIAL CYTOCHROMES P450
617
good because they are wide mouthed, and it is easy to insert soil and water mixtures into them. They can be easily sealed and stored in small boxes until return to the laboratory. They are essentially sterile and free of contaminating compounds as purchased. From each site chosen, approximately 100 g of a soil/water mixture is transferred to each plastic bag and labeled. Since the desired organisms are aerobic, the samples were not taken more than 1 inch deep in bottom sediments from the ponds or swamps. Growth Medium
Depending on the type of organism to be selected, a variety of different rich or minimal media can be utilized. Because we have been interested in the P450s of pseudomonads, we have used a minimal medium supplemented with a small amount of yeast extract and tryptone (Ref. 12 and Table I). So that growth is not limited by the availability of trace metals, a supplement is added to the medium after autoclaving. Rich media such as 2 × YT, LB, or TB 13 could be utilized in addition to the organic compound, but it must be remembered that organisms which grow on unusual organic compounds frequently have very long division times (>5 hr). If the medium is rich, other organisms might grow more rapidly than the desired strain; thus, the desired strain might be selected against simply by cell density pressures. However, working in the favor of selecting a desirable organism is the fact that most of the unusual organic compounds which are metabolized by P450s are inhibitors of the N A D H dehydrogenase of oxidative phosphorylation and hence inhibit bacterial growth. This gives a selective advantage to those organisms which can oxidize the compound and remove it from their immediate environment (detoxication). For each collection sample, the dirt and water are well mixed in the plastic bag and then approximately 10 ml of the mixture is transferred to each culture flask (250-ml Edenmeyer flask) containing 30 ml of the medium and an aliquot of the growth substrate (
ISOLATION PROCEDURES
[60]
in the metabolism of nicotine, 12 whereas NMb is unique among known rabbit P450 isozymes in that it hydroxylates testosterone mainly in the 15 position. Acknowledgments This work was supported by Grant DK-10339from the National Institutes of Health. 12D. E. Williams, X. Ding, and M. J. Coon, Biochem. Biophys. Res. Commun. 166, 945 (1990).
[60] B a c t e r i a l C y t o c h r o m e s P 4 5 0 : I s o l a t i o n and Identification By JULIAN A. PETERSON and J.-Y. L u Introduction The role of the members of the superfamily of proteins called cytochrome P450 in the oxidation of organic compounds has been well documented. Over 200 P450s have been identified, purified, and characterized, and they can be divided into many different gene families. These proteins have been found in essentially all aerobic organisms from prokaryotes to mammals.l All P450s which have been studied to date can be divided into two groups on the basis of the electron transfer partner which is involved in the provision of the required electrons for the functioning of P450. One group consists of those P450s which have a single flavoprotein reductase containing both FAD and FMN. Examples include P450s of the endoplasmic reticulum of higher organisms. There has been only one member of this group which has been carefully studied in a prokaryote, and that is the soluble, catalytically self-sufficient fatty acid to-hydroxylase of Bacillus megaterium, P450BM-3. A second group consists of P450s which have two electron transfer partners: (1) an FAD-containing reduced pyridine nucleotide dehydrogenase and (2) an iron-sulfur protein. The members of this group, which are found in higher organisms, are typically localized in mitochondria. Several examples have been found in microorganisms, and 1 D. W. Nebert, D. R. Nelson, M. J. Coon, R. W. Estabrook, R. Feyereisen, Y. FujiiKuriyama, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, J. C. Loper, R. Sato, M. R. Waterman, and D. J. Waxman, in "DNA and Cell Biology," Vol. 10, p. 1. Mary Ann Liebert, New York, 1991.
METHODSIN ENZYMOLOGY,VOL.206
Copyright© 1991by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[60]
BACTERIAL CYTOCHROMES P450
613
the best characterized is the enzyme involved in camphor metabolism, P450cam. The P450cam enzyme system has over the years proved to be an extremely powerful tool for structure-function studies. The data accumulated have been valuable for predicting how mammalian P450s might react during substrate binding, electron transfer, and oxygen activation. In addition, the three-dimensional structure of both classes of membrane-bound P450s has been predicted on the basis of sequence similarities to P450cam. 2 Although the extrapolated data seem sound, it is based on the structure of only P450cam. While much effort has been expended to obtain large amounts of mammalian P450s for comparable structure-function studies, to date none are available in the quantities or purity necessary. Several groups have begun recently to work to expand the number of examples of well-characterized soluble, bacterial P450s so that the general hypotheses regarding P450cam can be tested. The number of these prokaryotic P450s which have been identified is fairly large; however, less than I0 have been characterized to date and none of them as completely as P450cam. As the interest in the use of P450s as a tool for the oxidative modification of organic compounds increases, 3 the search for new forms of the enzyme with different substrate specificities will become important. The effort to identify candidate organisms can take at least two different directions: (1) examine culture collections for candidate organisms4-7; or (2) isolate new strains via culture enrichment techniques.S Culture enrichment techniques have proved to be useful in the isolation and identification of new sources of a wide variety of commercially valuable organisms 9 and should prove equally useful to investigators in biological oxidations. 3 In any effort to isolate new microbial strains which can metabolize compounds of interest, one must consider how widespread will be the 2 D. R. Nelson and H. W. Strobel, Biochemistry 28, 656 (1989). 3 H. G. Davies, R. H. Green, D. R. Kelly, and S. M. Roberts, in "Biotransformations in Preparative Organic Chemistry: The Use of Isolated Enzymes and Whole Cell Systems in Synthesis." Academic Press, New York, 1989. 4 J.-S. He, R. T. Ruettinger, H.-M. Liu, and A. J. Fulco, Biochim. Biophys. Acta 1009, 301 (1989). 5 R. H. Kanemoto, A. T. Powell, D. E. Akiyoshi, D. A. Regier, R. A. Kerstetter, E. W. Nester, M. C. Hawes, and M. P. Gordon, J. Bacteriol. 171, 2506 (1989). 6 M. Horii, T. Ishizaki, S.-Y. Paik, T. Manome, and Y. Murooka, J. Bacterial. 172, 3644
(1990). 7 C. A. Omer, R. Lenstra, P. J. Litle, C. Dean, J. M. Tepperman, K. J. Leto, J. A. Romesser, and D. P. O'Keefe, J. Bacteriol. 172, 3335 (1990). g J. H. Abul, R. I. Murray, K. K. Bhattacharyya, G. C. Wagner, and I. C. Gunsalus, J. Biol. Chem. 265, 1345 (1990). 9 n. Veldkamp, Methods Microbiol. 3A, 305 (1970).
614
ISOLATIONPROCEDURES
[60]
facility to oxidize these organic compounds and whether culture enrichment can be usefully applied. Clearly there are a variety of organic polymers which are persistent in the environment. The degradation of small organic compounds is much more favorable except in the cases where the compound is substituted with halogen atoms. In fact, mixtures of nitrogenor phosphate-based fertilizers were sprayed on some Alaskan beaches to promote the growth of preexisting microbes to oxidize the hydrocarbons present in the oil spilled from the Exxon Valdez in 1989. As described in this chapter, the probability of finding an organism which will oxidize the "target" hydrocarbon is quite high.
Materials and Methods
Spectrophotometric Assay for Cytochrome P450 in Whole Cells The typical spectrophotometric assay for P450 in turbid suspensions has been described, z° and it applies to the determination of P450 in whole bacterial cells. Special precautions may have to be taken, such as the inclusion of 7.5% (v/v) glycerol in the reaction mixture, to avoid settling of the cells during the course of the determination. This settling would result in false readings of absorbance. In the typical assay an appropriate dilution of the cells or the bacterial growth medium into 50 mM potassium phosphate buffer, pH 7.4 (with or without the glycerol) is treated with a few grains of sodium dithionite, which results in the reduction of the hemeproteins (as well as most other redox-active components of the cells). The cells are then divided between the sample and reference cuvettes and the baseline recorded from 500 to 400 nm. Carbon monoxide is then gently bubbled through the sample cuvette for approximately 30 sec. Care must be taken not to remove the cells from the cuvette in the bubbles which are introduced with the carbon monoxide. The difference spectrum of the carbon monoxide complex of the ferrous hemoproteins is then recorded. The spectrum should be recorded again until no further changes are observed. If the cells have been grown to the stationary phase on the organic compound of interest and the cell density is approximately 5 g wet weight per liter, the determination should be able to be done directly on the culture medium. This makes the assumption that the amount of P450 in the cells is greater than 0.2% of the soluble protein. In those cases where the P450 content is lower than 0.2%, the cells can be concentrated by l0 R. W. Estabrook, J. A. Peterson, J. Baron, and A. G. Hildebrandt, Methods Pharmacol. 2, 305 (1972).
[60]
BACTERIAL CYTOCHROMES P450
615
centrifugation at 5000 g for 5 min and then resuspended in 1/5 volume of the phosphate buffer.
Other Potential Assay or Screening Techniques The spectrophotometric assay procedure described above, which is typically utilized to determine the concentration of P450, is cumbersome to perform on the scale necessary to screen large numbers of candidate organisms. Thus, the utilization of other screening techniques was explored. Oligonucleotide Hybridization. In principle, an oligonucleotide probe prepared from a long, highly conserved region of interest (e.g., the hemebinding region or the I-helix region) ought to be able to be used to probe large numbers of colonies on agar plates for the presence of P450s. This would enable investigators to more rapidly screen culture isolates for the presence of colonies which might contain interesting P450s. In practice this procedure might not be very practical because of the great diversity at the nucleotide level even between P450s of the same class. In the case of prokaryotic P450s there is a consensus amino acid sequence H of AVEELLR between the I helix and the heme-binding region. If a series of oligonucleotides were synthesized which encodes all possible used codons for the amino acids used in this sequence, it would contain at least 512 different members for only 20 nucleotides. Obviously this mixed pool of oligonucleotides would not make a very good probe for bacterial P450s. Oligonucleotides might be synthesized to detect the presence of very closely related P450s, but even in this case, the known nucleotide sequences indicate that the probability of success is relatively low. Western Blots. Western blots using polyclonal antibodies prepared against specific bacterial P450s or even mammalian P450s probably cannot be used as a general screening tool for the presence of other P450s. It is now well recognized that the different subfamilies of mammalian P450s exhibit different antibody specificity, and hence there is no reason to expect that the various bacterial P450s will show any less of a diversity in their antigenic determinants. In fact, polyclonal antibodies prepared against P450cam will not cross-react with P4501in8 or P450terp. ~2 In summary, only the brute force screening procedure using the spectrophotometric measurement of the content of P450 in individual clones is a reliable measure of the presence of P450 in culture isolates. 11 Single-letter abbreviations for amino acids: A, alanine; E, glutamic acid; L, leucine; R, arginine; V, valine. 12j._y. Lu and J. A. Peterson, unpublished observations (1990).
616
ISOLATION PROCEDURES
[60]
Culture Enrichment Techniques The goal of culture enrichment is to select from the natural environment an organism which will fulfill specific needs. These "needs" are identified ahead of time and are used to select the desired bacterial strain: (1) the location which might harbor it; (2) the media which will provide a growth advantage; (3) the type of culture enrichment, closed or open; and (4) the growth conditions such as length of incubation, temperature, aeration, and agitation. In our laboratory, we wanted to isolate a new microbial strain which would oxidize water-insoluble natural product terpenes. It was presumed that the hydrocarbon would have to be oxidized by a P450-type of enzyme because of the waterinsoluble character of the compounds. The compounds were selected on the basis of their purity, availability, and cost. In our initial screen of soil samples, we used eight different monoterpenes. As described below, a-terpineol was only one of the compounds used, and the cells which grew on it were simply the first ones which were examined for P450 content.
Selection of Collection Site It should be obvious that the very best enrichment culture conditions will not select for a microorganism which does not already exist in the collection sample. In our laboratory, we wanted to select for an organism which would metabolize the natural product a-terpineol, which is used as an industrial perfuming agent. This compound is found in pine oil, and so samples were collected from several locations around Dallas County, TX, which would have been exposed to pine oils over the years. Since this natural product terpene was not accumulating, it was reasonable to assume that it was being degraded by some organism in addition to its slow evaporation from decaying vegetative matter. Two sites were initially chosen, both of which were relatively moist (swampy): (1) in a small pond which serves as a catch basin for a large stream draining a relatively large portion of North Dallas and (2) in a swamp in the floodplain of the Trinity River which flows through Dallas. After the sites have been selected, it is important to use sterile techniques when collecting samples. First, do not introduce laboratory strains of organisms into the collection sample. Second, some of the soil or water samples which are being collected may contain toxic or carcinogenic compounds which are being metabolized by the natural flora of the environment. We routinely use sterile surgical gloves, laboratory spatulas for digging, and plastic bags as containers for the samples. We have used successfully new Ziploc heavy-duty quart-size plastic bags. These are
[60]
BACTERIAL CYTOCHROMES P450
617
good because they are wide mouthed, and it is easy to insert soil and water mixtures into them. They can be easily sealed and stored in small boxes until return to the laboratory. They are essentially sterile and free of contaminating compounds as purchased. From each site chosen, approximately 100 g of a soil/water mixture is transferred to each plastic bag and labeled. Since the desired organisms are aerobic, the samples were not taken more than 1 inch deep in bottom sediments from the ponds or swamps. Growth Medium
Depending on the type of organism to be selected, a variety of different rich or minimal media can be utilized. Because we have been interested in the P450s of pseudomonads, we have used a minimal medium supplemented with a small amount of yeast extract and tryptone (Ref. 12 and Table I). So that growth is not limited by the availability of trace metals, a supplement is added to the medium after autoclaving. Rich media such as 2 × YT, LB, or TB 13 could be utilized in addition to the organic compound, but it must be remembered that organisms which grow on unusual organic compounds frequently have very long division times (>5 hr). If the medium is rich, other organisms might grow more rapidly than the desired strain; thus, the desired strain might be selected against simply by cell density pressures. However, working in the favor of selecting a desirable organism is the fact that most of the unusual organic compounds which are metabolized by P450s are inhibitors of the N A D H dehydrogenase of oxidative phosphorylation and hence inhibit bacterial growth. This gives a selective advantage to those organisms which can oxidize the compound and remove it from their immediate environment (detoxication). For each collection sample, the dirt and water are well mixed in the plastic bag and then approximately 10 ml of the mixture is transferred to each culture flask (250-ml Edenmeyer flask) containing 30 ml of the medium and an aliquot of the growth substrate (
