JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1215-1222 0021-9193/91/031215-08$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 173, No. 3
Cloning and Analysis of s-Triazine Catabolic Genes from Pseudomonas sp. Strain NRRLB-12227 RICHARD W. EATONt* AND JEFFREY S. KARNS Pesticide Degradation Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
Beltsville, Maryland 20705 Received 20 September 1990/Accepted 25 November 1990
Pseudomonas sp. strain NRRLB-12227 degrades the s-triazine melamine by a six-step pathway which allows it to use melamine and pathway intermediates as nitrogen sources. With the plasmid pLG221, mutants defective in five of the six steps of the pathway were generated. TnS-containing-EcoRI fragments from these mutants were cloned and identified by selection for TnS-encoded kanamycin resistance in transformants. A restriction fragment from ammelide-negative mutant RE411 was used as a probe in colony hybridization experiments to identify cloned wild-type s-triazine catabolic genes encoding ammeline aminohydrolase, ammelide aminohydrolase, and cyanuric acid amidohydrolase. These genes were cloned from total cellular DNA on several similar, but not identical, HindHI fragments, as well as on a PstI fragment and a BgM fragment. Restriction mapp;ing and Southern hybridization analyses of these cloned DNA fragments suggested that these s-triazine catabolic genes may be located on a transposable element, the ends of which are identical 2.2-kb insertion sequences.
In 1982, 79 x 106 pounds (ca. 36 x 106 kg) of the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) were used in the United States, primarily with field corn and sorghum (14). This widespread use of atrazine and other s-triazines has caused some environmental concern in recent years because of the appearance of these compounds in groundwater. By 1988, atrazine had been detected in groundwater in 15 states both as a result of normal agricultural use and from point source contamination (29). The microbial metabolism of s-triazines has been studied by several groups, and this research has been reviewed recently (6). None of the microorganisms that have been studied is capable of rapidly and completely degrading atrazine, although Bekhi and Khan (1) recently reported the isolation of three Pseudomonas spp. which appeared to degrade atrazine very slowly. Genetic engineering may provide a means for constructing stable strains having higher degradation rates for s-triazines. For this teason, genetic studies of organisms capable of catalyzing some of the required steps were initiated. Pseudomonas sp. strain NRRLB-12227 was isolated by Cook and Hutter (8) with ammeline as the sole nitrogen source; it can transform various other s-triazines, including N-isopropylammeline, N-ethylammeline, melamine, ammelide, and cyanuric acid. A six-step pathway for the metabolismn of melamine to carbon dioxide and ammonia has been proposed (Fig. 1) (7, 21). All of the steps are hydrolytic, and all but the conversion of cyanuric acid to biuret release ammonia. No carbon or energy is derived from these compounds. In this study, we report the cloning and characterization of three closely linked genes encoding s-triazine catabolic enzymes in Pseudomonas sp. strain NRRLB12227. Restriction analysis of the cloned DNA fragments suggested that these three s-triazine catabolic genes mnay be located on a transposable element.
MATERIALS AND METHODS Strains and plasmids. Pseudomonas sp. strain NRRLB12227 (strain A; 8) (hereafter referred to as strain 12227) was obtained from the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., with the permission of the depositors. The Escherichia coli strains and plasmids used are listed in Table 1. The plasmids constructed in this study are described in Table 1 and Fig. 2 and 3. Chemicals and enzymes. Ammelide was a gift from Homer LeBaron, Ciba-Geigy Agricultural Division, Greensboro, N.C. Melamine was obtained from Aldrich Chemical Co., Milwaukee, Wis.; ammeline was obtained from K & K Laboratories, Inc., Plainview, N.Y.; cyanuric acid was obtained from Eastman Kodak Co., Rochester, N.Y.; and biuret was obtained from Fisher Scientific Co., Pittsburgh, Pa. Restriction endonucleases and DNA ligase were obtained from New England BioLabs, Beverly, Mass., and Bethesda Research Laboratories, Gaithersburg, Md., and used in accordance with manufacturer instructions. Media. The minimal medium was R medium (12) containing trace elements (17) and supplemented with succinate (0.1%) for cultivation of strain 12227 or glycerol (20 mM), methionine (0.3 mM), tryptophan (0.1 mM), and thiamine (0.05 mM) for cultivation of Escherichia coli LE392 (9). Media were solidified by adding 1.5% Bacto-Agar (Difco Laboratories, Detroit, Mich.). Media having s-triazines as sole nitrogen sources were prepared by eliminating the ammonium sulfate from R medium and adding filter-sterilized s-triazine solutions (8) to concentrations of 1.5 to 3 mM total nitrogen. These media were solidified with 1.5% Noble agar (Difco). LB medium was prepared from Lennox broth base (GIBCO, Grand Island, N.Y.) and solidified with 1.5% Bacto-Agar. Isolation of transposon mutants. Tn5 mutants of strain 12227 were isolated with the TnS donor E. coli LE392 (pLG221) as previously described (13). Mutant colonies that appeared on R medium plates supplemented with succinate and kanamycin (100 ,ug/ml) were picked onto a 10 by 10 grid on the same medium and, after overnight incubation at 30°C,
Corresponding author. Present address: Environmental Research Laboratory, U.S. Environmental Protection Agency, Sabine Island, Gulf Breeze, FL 32561-3999. *
1215
1216
J. BACTERIOL.
EATON AND KARNS NH2
N
N
H2Nl' i| i
( i 'UQi
II
I
E i!
X
rf 11I IPi
pRE481
1 kb* FIG. 4. Restriction enzyme cleavage maps of DNA fragments inserted into pBR322 to make pRE483 and pRE481. Plasmid pRE483 was obtained by inserting the 2.7-kb EcoRI-ClaI fragment from pRE453 (coordinates 22.3 to 32.6) into pBR322; pRE481 was obtained by inserting the 1.8-kb Sall fragment from pRE464 (coordinates 4.25 to 6.05) into pBR322. The arrow above the maps indicates the 2.2-kb repeated DNA segment.
filter, incubated with the 32P-labeled 1.5-kb ApaI-ClaI fragment from pRE483. All plasmids had a 0.6-kb ApaI-XmnI fragment (Fig. 5A), an 0.8-kb ApaI-PvuII fragment, and a 1.3-kb ApaI-AccI fragment that hybridized to the probe (Fig. 5B). Mutants unable to degrade both ammelide and cyanuric acid could have arisen either by TnS insertion or by a deletion resulting from recombination between the 2.2-kb repeated DNA segments or curing of a catabolic plasmid. To determine which of these possibilities occurred, we digested chromosomal DNAs from all of the ammelide-negative, cyanuric acid-negative mutants with EcoRI and, following electrophoresis through an agarose gel, hybridized them
ApaI/Xmnl Apal/Pvul abcdef g hi j bcdef g hi j
1221
B ja
ApaL/Acci AE2I/Xmnl Apal/Pvull bcdefghi jbcdef gh i j bcdef gh i
j
a6*~~~~~~~~4 0.
6* 6 1. 1.
0*4_
0.51
FIG. 5. Southern hybridization experiment done to determine the presence and extent of the repeated DNA segment in recombinant plasmids carrying s-triazine catabolic genes. Various plasmids were digested with ApaI-XmnI, ApaI-PvuII, or ApaI-AccI 32P-labeled 1.5-kb ApaI-ClaI fragment from pRE483. DNAs in each lane are as follows: a, 1-kb ladder (Bethesda Research Laboratories); b, pRE452; c, pRE460; d, pRE483; e, pRE451; f, pRE462; g, gel-purified 1.8-kb ApaI fragment (coordinates 25.65 to 27.14) from pRE468; h, pRE481; i, pRE467; and j, pRE470. (A) Ethidium bromide-stained agarose gel. (B) Autoradiogram of the nitrocellulose filter.
1222
EATON AND KARNS
possibly by homologous recombination between the 2.2-kb repeated DNA segments. The 4.6-kb PstI fragment that carries both genes failed to hybridize with total DNA from all of these mutants. Insertion sequence elements may be important in bringing about rapid evolutionary changes by causing genetic rearrangements that result in altered gene regulation, gene duplications, and the recombination of genes into novel units. In strain 12227, three closely linked s-triazine catabolic genes have been flanked by two insertion sequences. Data from the accompanying paper (11) suggest that the ammelide aminohydrolase gene and the cyanuric acid amidohydrolase gene have moved as a unit among three bacterial strains in the absence of the 2.2-kb repeated DNA segments and the ammeline aminohydrolase gene and that the latter have been inserted subsequently, with the resulting formation of a novel transposable element. ACKNOWLEDGMENT
J. BACTERIOL.
12.
13. 14. 15. 16.
17.
18.
This work was supported in part through a cooperative agreement with the Ciba-Geigy Corp., Greensboro, N.C. 19. 1.
2.
3.
4.
5.
6. 7. 8. 9. 10. 11.
REFERENCES Bekhi, R. M., and S. U. Khan. 1986. Degradation of atrazine by Pseudomonas: N-dealkylation and dehalogenation of atrazine and its metabolites. J. Agric. Food Chem. 34:746-749. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heynecker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. Boulnois, G. J., J. M. Varley, G. S. Sharpe, and F. C. H. Franklin. 1985. Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other Gram negative bacteria. Mol. Gen. Genet. 200:65-67. Braedt, G. 1988. Different reading frames are responsible for IS]-dependent deletions and recombination. Genetics 118:561570. Clewell, D. B., and D. R. Helinski. 1969. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. USA 62:1159-1166. Cook, A. M. 1987. Biodegradation of s-triazine xenobiotics. FEMS Microbiol. Rev. 46:93-116. Cook, A. M., P. Beilstein, H. Grossenbacher, and R. Hutter. 1985. Ring cleavage and degradative pathway of cyanuric acid in bacteria. Biochem. J. 231:25-30. Cook, A. M., and R. Hutter. 1981. s-Triazines as nitrogen sources for bacteria. J. Agric. Food Chem. 29:1135-1143. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Dretzen, G., M. Beliard, P. Sassone-Corsi, and P. Chambon. 1981. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem. 112:295-298. Eaton, R. W., and J. S. Karns. 1991. Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric
20. 21.
22. 23. 24. 25. 26. 27.
28.
29.
30.
acid amidohydrolase from three s-triazine-degrading bacterial strains. J. Bacteriol. 173:1363-1366. Eaton, R. W., and D. W. Ribbons. 1982. Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B. J. Bacteriol. 151:48-57. Eaton, R. W., and K. N. Timmis. 1986. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204. J. Bacteriol. 168:123-131. Gianessi, L. P. 1987. Lack of data stymies informed decisions on agricultural pesticides. Resources 89:1-4. Grindley, N. D. F., and R. R. Reed. 1985. Transpositional recombination in prokaryotes. Annu. Rev. Biochem. 54:863896. Grunstein, M., and D. Hogness. 1975. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72:3961-3965. Guirard, B. M., and E. E. Snell. 1981. Biochemical factors in growth, p. 79-111. In P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193199. lida, S., J. Meyer, and W. Arber. 1983. Prokaryotic IS elements, p. 159-221. In J. A. Shapiro (ed.), Mobile genetic elements. Academic Press, Inc., New York. Jorgensen, R. A., S. J. Roth, and W. S. Reznikoff. 1979. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 177:65-72. Jutzi, K., A. M. Cook, and R. Hutter. 1982. The degradative pathway of the s-triazine melamine. Biochem. J. 208:679-684. Kleckner, N. 1981. Transposable elements in prokaryotes. Annu. Rev. Genet. 15:341-404. Lederberg, E. M., and S. N. Cohen. 1974. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bacteriol. 119:1072-1074. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218. O'Connor, C. D., and G. 0. Humphreys. 1982. Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectors. Gene 20:219-229. Smith, G. E., and M. D. Summers. 1980. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl paper. Anal. Biochem. 109:123-129. Starlinger, P. 1980. IS elements and transposons. Plasmid 3:241-259. Triezenberg, S. J., C. Rushford, R. P. Hart, K. L. Berkner, and W. R. Folk. 1982. Structure of the Syrian hamster ribosomal DNA repeat and identification of homologous and nonhomologous regions shared by human and hamster ribosomal DNAs. J. Biol. Chem. 257:7826-7833. Williams, W. M., P. W. Holden, D. W. Parsons, and M. N. Lorber. 1988. Pesticides in ground water data base: 1988 interim report. U.S. Environmental Protection Agency, Washington, D.C. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.
Vol. 173, No. 3
Cloning and Analysis of s-Triazine Catabolic Genes from Pseudomonas sp. Strain NRRLB-12227 RICHARD W. EATONt* AND JEFFREY S. KARNS Pesticide Degradation Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
Beltsville, Maryland 20705 Received 20 September 1990/Accepted 25 November 1990
Pseudomonas sp. strain NRRLB-12227 degrades the s-triazine melamine by a six-step pathway which allows it to use melamine and pathway intermediates as nitrogen sources. With the plasmid pLG221, mutants defective in five of the six steps of the pathway were generated. TnS-containing-EcoRI fragments from these mutants were cloned and identified by selection for TnS-encoded kanamycin resistance in transformants. A restriction fragment from ammelide-negative mutant RE411 was used as a probe in colony hybridization experiments to identify cloned wild-type s-triazine catabolic genes encoding ammeline aminohydrolase, ammelide aminohydrolase, and cyanuric acid amidohydrolase. These genes were cloned from total cellular DNA on several similar, but not identical, HindHI fragments, as well as on a PstI fragment and a BgM fragment. Restriction mapp;ing and Southern hybridization analyses of these cloned DNA fragments suggested that these s-triazine catabolic genes may be located on a transposable element, the ends of which are identical 2.2-kb insertion sequences.
In 1982, 79 x 106 pounds (ca. 36 x 106 kg) of the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) were used in the United States, primarily with field corn and sorghum (14). This widespread use of atrazine and other s-triazines has caused some environmental concern in recent years because of the appearance of these compounds in groundwater. By 1988, atrazine had been detected in groundwater in 15 states both as a result of normal agricultural use and from point source contamination (29). The microbial metabolism of s-triazines has been studied by several groups, and this research has been reviewed recently (6). None of the microorganisms that have been studied is capable of rapidly and completely degrading atrazine, although Bekhi and Khan (1) recently reported the isolation of three Pseudomonas spp. which appeared to degrade atrazine very slowly. Genetic engineering may provide a means for constructing stable strains having higher degradation rates for s-triazines. For this teason, genetic studies of organisms capable of catalyzing some of the required steps were initiated. Pseudomonas sp. strain NRRLB-12227 was isolated by Cook and Hutter (8) with ammeline as the sole nitrogen source; it can transform various other s-triazines, including N-isopropylammeline, N-ethylammeline, melamine, ammelide, and cyanuric acid. A six-step pathway for the metabolismn of melamine to carbon dioxide and ammonia has been proposed (Fig. 1) (7, 21). All of the steps are hydrolytic, and all but the conversion of cyanuric acid to biuret release ammonia. No carbon or energy is derived from these compounds. In this study, we report the cloning and characterization of three closely linked genes encoding s-triazine catabolic enzymes in Pseudomonas sp. strain NRRLB12227. Restriction analysis of the cloned DNA fragments suggested that these three s-triazine catabolic genes mnay be located on a transposable element.
MATERIALS AND METHODS Strains and plasmids. Pseudomonas sp. strain NRRLB12227 (strain A; 8) (hereafter referred to as strain 12227) was obtained from the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., with the permission of the depositors. The Escherichia coli strains and plasmids used are listed in Table 1. The plasmids constructed in this study are described in Table 1 and Fig. 2 and 3. Chemicals and enzymes. Ammelide was a gift from Homer LeBaron, Ciba-Geigy Agricultural Division, Greensboro, N.C. Melamine was obtained from Aldrich Chemical Co., Milwaukee, Wis.; ammeline was obtained from K & K Laboratories, Inc., Plainview, N.Y.; cyanuric acid was obtained from Eastman Kodak Co., Rochester, N.Y.; and biuret was obtained from Fisher Scientific Co., Pittsburgh, Pa. Restriction endonucleases and DNA ligase were obtained from New England BioLabs, Beverly, Mass., and Bethesda Research Laboratories, Gaithersburg, Md., and used in accordance with manufacturer instructions. Media. The minimal medium was R medium (12) containing trace elements (17) and supplemented with succinate (0.1%) for cultivation of strain 12227 or glycerol (20 mM), methionine (0.3 mM), tryptophan (0.1 mM), and thiamine (0.05 mM) for cultivation of Escherichia coli LE392 (9). Media were solidified by adding 1.5% Bacto-Agar (Difco Laboratories, Detroit, Mich.). Media having s-triazines as sole nitrogen sources were prepared by eliminating the ammonium sulfate from R medium and adding filter-sterilized s-triazine solutions (8) to concentrations of 1.5 to 3 mM total nitrogen. These media were solidified with 1.5% Noble agar (Difco). LB medium was prepared from Lennox broth base (GIBCO, Grand Island, N.Y.) and solidified with 1.5% Bacto-Agar. Isolation of transposon mutants. Tn5 mutants of strain 12227 were isolated with the TnS donor E. coli LE392 (pLG221) as previously described (13). Mutant colonies that appeared on R medium plates supplemented with succinate and kanamycin (100 ,ug/ml) were picked onto a 10 by 10 grid on the same medium and, after overnight incubation at 30°C,
Corresponding author. Present address: Environmental Research Laboratory, U.S. Environmental Protection Agency, Sabine Island, Gulf Breeze, FL 32561-3999. *
1215
1216
J. BACTERIOL.
EATON AND KARNS NH2
N
N
H2Nl' i| i
( i 'UQi
II
I
E i!
X
rf 11I IPi
pRE481
1 kb* FIG. 4. Restriction enzyme cleavage maps of DNA fragments inserted into pBR322 to make pRE483 and pRE481. Plasmid pRE483 was obtained by inserting the 2.7-kb EcoRI-ClaI fragment from pRE453 (coordinates 22.3 to 32.6) into pBR322; pRE481 was obtained by inserting the 1.8-kb Sall fragment from pRE464 (coordinates 4.25 to 6.05) into pBR322. The arrow above the maps indicates the 2.2-kb repeated DNA segment.
filter, incubated with the 32P-labeled 1.5-kb ApaI-ClaI fragment from pRE483. All plasmids had a 0.6-kb ApaI-XmnI fragment (Fig. 5A), an 0.8-kb ApaI-PvuII fragment, and a 1.3-kb ApaI-AccI fragment that hybridized to the probe (Fig. 5B). Mutants unable to degrade both ammelide and cyanuric acid could have arisen either by TnS insertion or by a deletion resulting from recombination between the 2.2-kb repeated DNA segments or curing of a catabolic plasmid. To determine which of these possibilities occurred, we digested chromosomal DNAs from all of the ammelide-negative, cyanuric acid-negative mutants with EcoRI and, following electrophoresis through an agarose gel, hybridized them
ApaI/Xmnl Apal/Pvul abcdef g hi j bcdef g hi j
1221
B ja
ApaL/Acci AE2I/Xmnl Apal/Pvull bcdefghi jbcdef gh i j bcdef gh i
j
a6*~~~~~~~~4 0.
6* 6 1. 1.
0*4_
0.51
FIG. 5. Southern hybridization experiment done to determine the presence and extent of the repeated DNA segment in recombinant plasmids carrying s-triazine catabolic genes. Various plasmids were digested with ApaI-XmnI, ApaI-PvuII, or ApaI-AccI 32P-labeled 1.5-kb ApaI-ClaI fragment from pRE483. DNAs in each lane are as follows: a, 1-kb ladder (Bethesda Research Laboratories); b, pRE452; c, pRE460; d, pRE483; e, pRE451; f, pRE462; g, gel-purified 1.8-kb ApaI fragment (coordinates 25.65 to 27.14) from pRE468; h, pRE481; i, pRE467; and j, pRE470. (A) Ethidium bromide-stained agarose gel. (B) Autoradiogram of the nitrocellulose filter.
1222
EATON AND KARNS
possibly by homologous recombination between the 2.2-kb repeated DNA segments. The 4.6-kb PstI fragment that carries both genes failed to hybridize with total DNA from all of these mutants. Insertion sequence elements may be important in bringing about rapid evolutionary changes by causing genetic rearrangements that result in altered gene regulation, gene duplications, and the recombination of genes into novel units. In strain 12227, three closely linked s-triazine catabolic genes have been flanked by two insertion sequences. Data from the accompanying paper (11) suggest that the ammelide aminohydrolase gene and the cyanuric acid amidohydrolase gene have moved as a unit among three bacterial strains in the absence of the 2.2-kb repeated DNA segments and the ammeline aminohydrolase gene and that the latter have been inserted subsequently, with the resulting formation of a novel transposable element. ACKNOWLEDGMENT
J. BACTERIOL.
12.
13. 14. 15. 16.
17.
18.
This work was supported in part through a cooperative agreement with the Ciba-Geigy Corp., Greensboro, N.C. 19. 1.
2.
3.
4.
5.
6. 7. 8. 9. 10. 11.
REFERENCES Bekhi, R. M., and S. U. Khan. 1986. Degradation of atrazine by Pseudomonas: N-dealkylation and dehalogenation of atrazine and its metabolites. J. Agric. Food Chem. 34:746-749. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heynecker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. Boulnois, G. J., J. M. Varley, G. S. Sharpe, and F. C. H. Franklin. 1985. Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other Gram negative bacteria. Mol. Gen. Genet. 200:65-67. Braedt, G. 1988. Different reading frames are responsible for IS]-dependent deletions and recombination. Genetics 118:561570. Clewell, D. B., and D. R. Helinski. 1969. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. USA 62:1159-1166. Cook, A. M. 1987. Biodegradation of s-triazine xenobiotics. FEMS Microbiol. Rev. 46:93-116. Cook, A. M., P. Beilstein, H. Grossenbacher, and R. Hutter. 1985. Ring cleavage and degradative pathway of cyanuric acid in bacteria. Biochem. J. 231:25-30. Cook, A. M., and R. Hutter. 1981. s-Triazines as nitrogen sources for bacteria. J. Agric. Food Chem. 29:1135-1143. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Dretzen, G., M. Beliard, P. Sassone-Corsi, and P. Chambon. 1981. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem. 112:295-298. Eaton, R. W., and J. S. Karns. 1991. Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric
20. 21.
22. 23. 24. 25. 26. 27.
28.
29.
30.
acid amidohydrolase from three s-triazine-degrading bacterial strains. J. Bacteriol. 173:1363-1366. Eaton, R. W., and D. W. Ribbons. 1982. Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B. J. Bacteriol. 151:48-57. Eaton, R. W., and K. N. Timmis. 1986. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204. J. Bacteriol. 168:123-131. Gianessi, L. P. 1987. Lack of data stymies informed decisions on agricultural pesticides. Resources 89:1-4. Grindley, N. D. F., and R. R. Reed. 1985. Transpositional recombination in prokaryotes. Annu. Rev. Biochem. 54:863896. Grunstein, M., and D. Hogness. 1975. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72:3961-3965. Guirard, B. M., and E. E. Snell. 1981. Biochemical factors in growth, p. 79-111. In P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193199. lida, S., J. Meyer, and W. Arber. 1983. Prokaryotic IS elements, p. 159-221. In J. A. Shapiro (ed.), Mobile genetic elements. Academic Press, Inc., New York. Jorgensen, R. A., S. J. Roth, and W. S. Reznikoff. 1979. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 177:65-72. Jutzi, K., A. M. Cook, and R. Hutter. 1982. The degradative pathway of the s-triazine melamine. Biochem. J. 208:679-684. Kleckner, N. 1981. Transposable elements in prokaryotes. Annu. Rev. Genet. 15:341-404. Lederberg, E. M., and S. N. Cohen. 1974. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bacteriol. 119:1072-1074. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218. O'Connor, C. D., and G. 0. Humphreys. 1982. Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectors. Gene 20:219-229. Smith, G. E., and M. D. Summers. 1980. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl paper. Anal. Biochem. 109:123-129. Starlinger, P. 1980. IS elements and transposons. Plasmid 3:241-259. Triezenberg, S. J., C. Rushford, R. P. Hart, K. L. Berkner, and W. R. Folk. 1982. Structure of the Syrian hamster ribosomal DNA repeat and identification of homologous and nonhomologous regions shared by human and hamster ribosomal DNAs. J. Biol. Chem. 257:7826-7833. Williams, W. M., P. W. Holden, D. W. Parsons, and M. N. Lorber. 1988. Pesticides in ground water data base: 1988 interim report. U.S. Environmental Protection Agency, Washington, D.C. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.
