APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1992, p. 2531-2535
0099-2240/92/082531-05$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 58, No. 8
Molecular Cloning and Mapping of Phenol Degradation Genes from Bacillus stearothermophilus FDTP-3 and Their Expression in Escherichia coli FU-MIN DONG, LI-LI WANG, CHANG-MEI WANG, JIE-PING CHENG, ZHI-QING HE, ZU-JIA SHENG, AND REN-QUAN SHEN*
Institute of Genetics, Fudan University, Shanghai 200433, People's Republic of China Received 18 December 1991/Accepted 13 May 1992
Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacilus stearothermophilus and were mapped by subcloning and by use of a TnS insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida. Phenolic compounds are hazardous pollutants. Utilization and degradation of phenolic compounds by mesophiles were reported early in the 1950s (7). The degradation pathways and regulatory mechanisms in metabolism of phenol, cresols, toluene, and xylenes in mesophiles have been extensively studied during the past 30 years, especially in pseudomonads (2-4, 13, 14, 18). The benzene ring of toluene or related compounds may be cleaved by either the meta or the ortho pathway through the intermediate compound catechol or substituted catechols (17). However, not as much information is available on thermophiles. Buswell and Twomey isolated a strain of Bacillus stearothermophilus capable of utilizing phenol and cresols (6). Adams and Ribbons isolated a strain of B. stearothermophilus which degraded both phenol and benzoic acid (1). Gurujeyalakshmi and Oriel reported the isolation of a phenol-degrading B. stearothermophilus strain and the partial characterization of its phenol hydroxylase (10). In this paper, we report the catabolic pathway of phenol degradation in a strain, FDTP-3, of B. stearothermophilus isolated from the effluent of the Nanjing Oil Refinery (19a) and the cloning and mapping of two genes relevant to the phenol degradation pathway.
Preparation of total bacterial DNA was performed as described by Saito and Miura (19). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs or Sino-American Biotechnology Company. Reaction conditions were as described by Maniatis et al. (16). Isolation of DNA fragments from the gel was done as described by Guo and Wu (9). Southern transfer and hybridization were performed as described by Maniatis et al. (16). The biotin labelling kit and the nick translation kit were purchased from Bethesda Research Laboratories, Inc. Labelling of probes with biotinyl-11-dUTP and detection of homology were done as recommended by the manufacturer (4a). Total DNA of donor strain FDTP-3, plasmid DNA of pFDA11, pPL392 carrying genexylE from Pseudomonas putida (11), and other controls digested with BamHI were used for Southern transfer. Transformation. Preparation of competent E. coli cells and plasmid transformation were performed as described by Maniatis et al. (16). Preparation of crude enzyme extract. Bacterial strains were grown overnight in liquid rich medium, supplemented with antibiotics when necessary, and pelleted. The pellet was suspended in 0.1 M phosphate buffer (pH 7.6) and pelleted once again. Fifteen grams (wet weight) was resuspended in
MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. Culture media and culture temperature. The rich medium (pH 7.2) contained 10 g of polypeptone, 5 g of yeast extract, and 5 g of NaCl per liter. The minimal medium (pH 7.2) contained 2 g of glucose, 0.5 g of K2HPO4, 1 g of MgSO4 7H20, 1 g of NH4Cl, 10 ,ug of CuSO4. 5H20, 50 ,g of CaCl2. 6H20, 70 ,ug of ZnSO4 7H20, 0.4 mg of nicotinamide, 0.4 mg of thiamine, and 2 ,ug of biotin per liter. Chloramphenicol (5 ,ug/ml), ampicillin (AMP) (50 ,ug/ml), and kanamycin (KAN) (3 ,ug/ml) (which was used instead of neomycin) were added when necessary. All Escherichia coli strains were cultured at 37°C, and strain FDTP-3 was cultured at 60°C. DNA manipulation. Plasmid DNA extraction was performed as described by Birnboim and Doly (5). *
6.5 ml of the same buffer and was sonicated with a VirSonic 300 sonicator for 5 min. After being centrifuged for 15 min at 23,000 x g, the supernatant was used as the crude enzyme extract.
Detection of catechol 2,3-dioxygenase. FDTP-3 was spread rich medium and incubated overnight. A 0.5 M catechol solution was sprayed onto the plate. After the yellow color of the colonies (as well as the surrounding medium) appeared, after incubation at 60°C for 5 min, the plate was eluted with a small amount of phosphate buffer (0.1 M, pH 7.6) and centrifuged. The supernatant was scanned with a Shimadzu UV-240 spectrophotometer. The yellowish compound 2-hydroxymuconic semialdehyde (HMS) gives a characteristic absorption peak at 375 nm. Identification of catechol as an immediate degradation product of phenol. The crude extract prepared from E. coli K514(pTG402), which possesses catechol 2,3-dioxygenase activity like that of the mesophile P. putida (20), was used to on
Corresponding author. 2531
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APPL. ENVIRON. MICROBIOL.
DONG ET AL. TABLE 1. Bacterial strains and plasmids
Strain or
Source, reference,
Characteristics
plasmid
Donated by Q. Y. Zhou
FDTP-3 E. coli TG1
or derivation
supE hsd-5 thi (lac-proAB) F' [traD36 proAB' lacIq lacZ AM15] supE44 hsdS20 (r- mB) recA13 ara-14 proA2 lacYl galK2 rpsL20 xyl-5 mtl-l F- his trp tsx spc nalA AlacX74 ara A(lac-proAB) rpsL thi (480 lacZ AM15) A(srl-recA)306::Tn1O(Tcr) F' [traD36 proAB+ lacIq lacZ AM15] A(lac proB)Valr Tn5 F' lac+ pro' hsrK hsmK derivative of C600
Laboratory stock
pPL392
Apr
pTG402 pPGV5 pBR322 pFDC11 pFDC111 pFDA10
xylE Apr Cm' Apr Nmr Cms
pFDA11 pFDA13
Same as pFDA10 Apr pheA pheB
pFDA16
Apr pheB
pFDA17 pFDA21
Apr pheB Apr pheA
pBR322 cloned with 16.4-kb fragment from Tol plasmid, donated by S. Harayama (11) Donated by P. Dhaese; 20 Donated by P. Dhaese Laboratory stock 12 This work pFDC111 cloned with 6.4-kb fragment of DNA from FDTP-3 (this work) This work pUC18 cloned with 6.4-kb fragment from pFDA11 (unpublished data) Subcloned from pFDA11 with pUC119 as vector (unpublished data) Deletion derivative of pFDA16 (unpublished data) Subcloned from pFDA13 with pUC18 as vector (unpublished data)
HB101 ED2196 MV1184
GmlapX K514
Apr Tcr Cmr Nmr Apr Cmr Nmr Apr Cmr Km pheA pheB
identify catechol, the enzymatic reaction product of phenol, by the characteristic absorption peak of HMS. Assay of enzyme thermostability. The crude extract with proper dilution in phosphate buffer (0.1 M, pH 7.6) was inactivated at different temperatures for 15 min. A 0.5 M catechol solution was added to a final concentration of 2.5 mM. The optical density measurements at 37 and 55°C without inactivation were taken as 100% for mesophilic and thermophilic enzymes, respectively. Therefore, the optical density at 375 nm read from the scanned curve was taken as a measure of the enzyme activity remaining. Transposon mutagenesis. Transposon Tn5 with a genetic marker for neomycin resistance was used. Since the four PstI sites on Tn5 were known (15) and there is one PstI site on the multiple cloning site of pFDA13, the insertion site of TnS in pFDA13 was analyzed according to the size of the fragments obtained by digesting the plasmid with PstI and PstI-BamHI. RESULTS
Identification of the bacterial strain FDTP-3. FDTP-3 is a gram-positive, spore-forming, starch-hydrolyzing, catalasepositive thermophile. Negative tests included the VogesProskauer reaction, growth on rich medium with 5% NaCl or 0.02% sodium azide, and growth on rich medium of pH 5.7. The optimum growth temperature was within the range 60 to 65°C. FDTP-3 was thus identified as B. stearothermophilus by the methods of Gordon et al. (8). Carbon source. FDTP-3 can use glucose as the sole carbon source on minimal medium supplemented with vitamins and
Laboratory stock 11 Laboratory stock
Laboratory stock Donated by P. Dhaese
microelements. Among phenol, toluene, benzene, o-cresol, p-cresol, and benzoic acid, only phenol (0.05%) was shown to be used by FDTP-3 as the sole carbon source on the same medium. Catechol 2,3-dioxygenase activity of FDTP-3. The presence of catechol 2,3-dioxygenase activity in FDTP-3 was detected by scanning the eluent from the plate as described above. The curve reveals the characteristic absorption maximum of HMS at 375 nm. When distilled water was used instead of catechol solution, the eluent did not show the characteristic absorption maximum. The same characteristic absorption maximum was observed for the eluent of E. coli ED2196 harboring the plasmid pPL392, which carries gene xylE of P. putida encoding catechol 2,3-dioxygenase (11), while the eluent of E. coli HB101 harboring pBR322 gave no absorption maximum at 375 nm. It is known that the enzyme catechol 2,3-dioxygenase converts catechol to HMS. Therefore, it was concluded that FDTP-3 possesses catechol 2,3-dioxygenase activity. It is known that the enzymes taking part in the degradation of hydrocarbons in P. putida are all inducible. Catechol 2,3-dioxygenase of FDTP-3 was found to be strongly induced by phenol or benzoic acid but not by benzene, toluene, p-cresol, or o-cresol (data not shown). It is interesting that benzoic acid cannot be used as the sole carbon source by FDTP-3. It is a gratuitous inducer. Cloning of phenol degradation genes from FDTP-3. Phenol degradation genes from FDTP-3 were cloned on a shuttle plasmid, pFDC111 (Fig. 1), by ligating the BamHI digest of pFDC111 and that of the total DNA from FDTP-3. pFDC111 was constructed from pFDC11 (12). E. coli TG1 transformed
VOL. 58, 1992
PHENOL DEGRADATION GENES FROM B. STEAROTHERMOPHILUS
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a° a
.X.t >
I
a
w Eco RI
DNA ligase, T4
I
0
40
60
Temp
FIG. 2. Comparison of the thermostability of catechol 2,3-dioxfrom B. stearothernophilus FDTP-3 with that of catechol 2,3-dioxygenase from P. putida. Enzyme activities of extracts of both E. coli K514(pTG402) (-) and E. coli MV1184(pFDA17) (a) were determined after inactivation at different temperatures for 15 min. ygenase
FIG. 1. Construction of the shuttle plasmid pFDC111 used for cloning the phenol degradation genes. pPGV5 (7.7 kb) was completely digested with EcoRI, and pFDC11 (5.5 kb) was partially digested with EcoRI. The ligation mixture was transformed into E. coli HB101. Apr Cmr transformants with fragments inserted at different EcoRI sites were selected. pFDC111 (8.2 kb) was identified by digesting the plasmid with XbaI. Ba, BamHI; Bg, BgIII; E, EcoRI; H, HindIII; P, PstI; SI, Sall; Su, Sau3A; X, XbaI; cat, chloramphenicol acetyltransferase.
with the ligation mixture was spread on a rich medium plate supplemented with AMP. After the rich medium plate was sprayed with catechol solution, five yellow colonies were obtained from among 1,558 transformants. Two of the yellow transformants harboring recombinant plasmids pFDA10 and pFDA11 were further identified on a plate containing AMP and KAN. Agarose gel electrophoresis of pFDA10 and pFDA11 digested with HindIII or BamHI gave exactly the same pattern (data not shown). A 6.4-kb insert was found in both plasmids. With the biotinylated pFDA11 DNA as the probe, Southern hybridization was carried out against pPL392 or the total DNA of the donor strain. A hybridization signal between the donor DNA and the probe pFDA11 was evident, but only a very weak signal between pFDAll and pPL392 was observed (data not shown) despite the fact that they carry a gene encoding an enzyme with the same catalytic function. The thermostability of catechol 2,3-dioxygenase encoded by pFDA17 (a derivative of pFDA13 [see Fig. 4]) was compared with that of the enzyme encoded by pTG402 (20) (Fig. 2). It was found that the former enzyme was much more thermostable than the latter; thus, the origin of the cloned gene was further confirmed. Restriction endonuclease analysis of plasmid pFDA13 showed that the 6.4-kb insert has four HindIII sites, three ClaI sites, two EcoRI sites, one EcoRV site, and one PvuII site. No BglII, PstI, or SalI site was found (see Fig. 4).
0le
3~~~~~~~
o~~~~~
1 9() w.iv
300 .
o( ig
(raw)
FIG. 3. Absorption spectrum curves showing the conversion of phenol into catechol by cloned genes from FDTP-3. All organisms were cultured on rich medium supplemented with AMP. (Line 1) Catechol solution (final concentration, 3 mM) was added to the extract of E. coli(pFDA13), and the mixture was incubated at 60°C for 15 min. (Line 2) Phenol (final concentration, 3 mM) was added to the extract of E. coli(pFDA21), and the mixture was incubated at 60°C for 45 min; next, the extract of E. coli(pTG402) was added, and the mixture was incubated at 37°C for 30 min. (Line 3) Phenol (final concentration, 3 mM) was added to the extract of E. coli(pFDA21), and the mixture was incubated at 60°C for 45 min.
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DONG ET AL. E
H
C
i.9I..
e
i*
H
I
H
C
I I
I
phA
ph& aU
H
i .1
I
P/EV C
1t t
E
e
1.
-|-4
pFDA13 E
E
pFDA21
e1
H
pFDA 16
pFDA 17
FIG. 4. Physical maps of the cloned fragments of recombinant plasmids containing phenol degradation genes and genetic map showing the location of the inserted TnS. pFDA13 was constructed by digesting the 6.4-kb insert from pFDA1l with BamHI and subcloning the fragment into the BamHI sites of pUC18. pFDA21 was obtained by digesting pFDA13 with EcoRI and subcloning the 3.7-kb fragment into the EcoRI site of pUC18. pFDA16 is a recombinant plasmid carrying the 2.75-kb fragment (from BamHI to the second HindIII site) from the 6.4-kb insert subcloned in pUC119 in a stepwise manner. pFDA17 (1.3 kb) was derived from pFDA16 by shortening the left terminal with the ExoIII-mung bean nuclease system. The sites of Tn5 insertion are indicated as follows: A, insertion within the genespheA andpheB; A, insertion outside both genes. Restriction cleavage sites: B, BamHI; E, EcoRI; C, ClaI; H, HindIII; P, PstI; EV, EcoRV; P/EV, PvuII and EcoRV very close to one another.
Catechol is the immediate degradation product of phenol in FDTP-3. Colonies of FDTP-3 grown on rich medium containing phenol were yellow. That phenol is degraded to HMS through catechol was proved by studying the cloned genes. pFDA21 is a recombinant plasmid subcloned from pFDA13 carrying a 3.7-kb fragment bordered by the two EcoRI sites (see Fig. 4). The crude extract (or resting cells) of E. coli MV1184(pFDA21) incubated with phenol at 60°C for 45 min gave no absorption maximum at 375 nm (Fig. 3, line 3). However, on addition of the crude extract (or resting cells) of E. coli K514(pTG402), which carries the subcloned xylE gene from P. putida, an absorption maximum at 375 nm did appear after incubation at 37°C for 30 min (Fig. 3, line 2). This suggests that pFDA21 does not carry the catechol 2,3-dioxygenase gene but carries a gene encoding phenol hydroxylase that converts phenol to catechol, which is then converted to HMS by the enzyme catechol 2,3-dioxygenase, which exists in the extract of E. coli K514(pTG402). Therefore, it was suggested that FDTP-3 degrades phenol via the meta pathway as in P. putida, although it is not necessarily the only pathway. Mapping of the genes on the 6.4-kb cloned fragment by subcloning. The colonies of E. coli MV1184 harboring the multicopy plasmid pFDA13 grown on rich medium supplemented with AMP and phenol were yellow. This suggests that the cloned 6.4-kb fragment carries at least two genes, pheA and pheB, coding for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. pFDA16 and pFDA21 are subclones of the 6.4-kb fragment (Fig. 4). E. coli MV1184 (pFDA16) grown on rich medium supplemented with AMP and phenol was not yellow. However, a yellow color appeared when MV1184(pFDA16) was grown on rich medium supplemented with AMP but without phenol and then sprayed with catechol solution. It was inferred that gene pheB, coding for catechol 2,3-dioxygenase, resides on pFDA16. Colonies (as well as the surrounding medium) of E. oH
phenol hydiroxylase pheA
OH
coli MV1184(pFDA21) were brown when streaked on rich medium supplemented with AMP and phenol. Furthermore, when E. coli MV1184(pFDA21) was inoculated on rich medium supplemented with AMP and phenol adjacent to an E. coli strain with only catechol 2,3-dioxygenase activity, such as E. coli MV1184(pFDA16), a yellow color appeared in the region of the latter. It is inferred that the brown is due to the accumulation of catechol (from its precursor, phenol), which is autooxidized to quinone substances in the presence of air, and that the yellow color is due to the conversion of catechol to HMS by the nearby strain with catechol 2,3dioxygenase activity. It is therefore inferred that genepheA coding for phenol hydroxylase resides on pFDA21. Transposon mutagenesis of the recombinant plasmid pFDA13. pFDA13 was used to transform the TnS-containing E. coli TnS Gmlo3A. Yellow transformants were selected from rich medium supplemented with AMP, KAN, and phenol and were spread on the same medium and incubated overnight. Bacterial mass was collected, and the plasmid DNA was extracted and used to transform E. coli TG1. White, yellow, and brown colonies were noticed on rich medium supplemented with AMP, KAN, and phenol. The white colonies were believed to be those with TnS inserted within gene pheA, the brown colonies were believed to be those with TnS inserted within gene pheB, and the yellow colonies were believed to be those with TnS inserted anywhere except in pheA or pheB. The nature of the insertion mutants was further confirmed by the following observations. That the brown colonies are those with TnS inserted within the pheB gene was confirmed by the fact that they remained white on rich medium supplemented with AMP and KAN but without phenol even after being sprayed with catechol solution, while those with TnS inserted within gene pheA turned yellow under the same conditions. Furthermore, when mutants with Tn5 inserted within genes pheA and pheB were streaked side by side, a yellow color ap-
catechol 2
tOH
l3-dioxygcnltse
tiohexB
FIG. 5. Metabolic pathway of phenol in strain FDTP-3.
OOH OH
VOL. 58, 1992
PHENOL DEGRADATION GENES FROM B. STEAROTHERMOPHILUS
peared in the region of the former. These observations were compatible with those obtained by subcloning. To determine the location of each insertion in pFDA13::Tn5 mutant plasmids, plasmid DNA from each derivative was isolated and analyzed by cleavage with the restriction enzymes PstI and PstI-BamHI. The insertion map is shown in Fig. 4, and the locations of the two genes were demonstrated. DISCUSSION The results reported here suggest that the metabolic pathway of phenol in the strain FDTP-3 is as shown in Fig. 5. Attempts to detect the enzyme catalyzing the reaction following catechol 2,3-dioxygenase activity were unsuccessful. Catechol 2,3-dioxygenase is functionally the same as the enzyme encoded by xylE in P. putida, although its thermostability is quite different and the homology between the two genes is rather limited. Three different classes of insertion mutation can be visualized directly by the color developed on the plate. The site of Tn5 insertion seems to be unevenly distributed, particularly in the pheA gene. No polar mutant has been found; thus, we are not in a position to suggest whether these genes are organized into an operon or to suggest the direction of transcription.
2535
Acids Res. 7:1513-1523. 6. Buswell, J. A., and D. G. Twomey. 1975. Utilization of phenol and cresols by Bacillus stearothermophilus, strain PH24. J. Gen. Microbiol. 87:377-379. 7. Dagley, S., and M. D. Patel. 1957. Oxidation of p-cresol and related compounds by a Pseudomonas. Biochem. J. 66:227-233. 8. Gordon, R. E., W. C. Hayness, and C. H.-N. Pang. 1973. The genus bacillus. U.S. Department of Agriculture, Washington, D.C. 9. Guo, L. H., and R. Wu. 1983. Exonuclease III: use for DNA sequence analysis and in specific deletion of nucleotides. Methods Enzymol. 100:60-97. 10. Gurujeyalakshmi, G., and P. Oriel. 1989. Isolation of phenoldegrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase. Appl. Environ. Microbiol. 55:500-502. 11. Harayama, S., P. R. Lehrbach, and K. N. Timmis. 1984. Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2. J. Bacteriol. 160:251-255. 12. He, X. S., R. Q. Shen, and Z. J. Sheng. 1990. The construction of promoter probe vector pFDC4 and gene expressing vector pFDC11 with high transformation efficiency in Bacillus stearothermophilus. Acta Genet. Sin. 17:216-225. (In Chinese.) 13. Inouye, S., A. Nakazawa, and T. Nakazawa. 1981. Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS. J. Bacteriol. 148:
413-418. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant 3870355). We thank Q. Y. Zhou and S. F. Wang for providing the strain of thermophile FDTP-3, S. Harayama for providing E. coli ED2196(pPL392), and P. Dhaese for providing E. coli 514(pTG402) and plasmid pPGV5. REFERENCES 1. Adams, D., and D. W. Ribbons. 1988. The metabolism of aromatic compounds by thermophilic bacilli. Appl. Biochem. Biotechnol. 17:231-244. 2. Bayly, R. C., and M. G. Barbour. 1984. The degradation of aromatic compounds by the meta and gentisate pathway, p. 253-294. In D. T. Gibson (ed.), Microbial degradation of organic compounds. Marcel Dekker, Inc., New York. 3. Bayly, R. C., S. Dagley, and D. T. Gibson. 1966. The metabolism of cresols by species of Pseudomonas. Biochem. J. 101:293301. 4. Bayly, R. C., and G. J. Wigmore. 1973. Metabolism of phenol and cresols by mutants of Pseudomonas putida. J. Bacteriol. 113:1112-1120. 4a.Bethesda Research Laboratories, Inc. DNA detection system. Bethesda Research Laboratories, Inc., Gaithersburg, Md. 5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic
14. Inouye, S., A. Nakazawa, and T. Nakazawa. 1983. Molecular cloning of regulatory gene xylR and operator-promoter regions of the xyLABC and xylDEGF operons of the TOL plasmid. J. Bacteriol. 155:1192-1199. 15. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. A restriction map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 117:69-72. 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 17. Nakazawa, T., and T. Yokota. 1973. Benzoate metabolism in Pseudomonas putida(arvilla) mt-2: demonstration of two benzoate pathways. J. Bacteriol. 115:262-267. 18. Ramos, J. L., A. Stolz, W. Reineke, and K. N. Timmis. 1986. Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. Proc. Natl. Acad. Sci. USA 83:8467-8471. 19. Saito, H., and K.-I. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta. 72:619-629. 19a.Zhou, Q. Y., and S. F. Wang. Unpublished data. 20. Zukowski, M. M., D. F. Gaffney, D. Speck, M. Kauffmann, A. Findeli, A. Wisecup, and J.-P. Lecocq. 1983. Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc. Natl. Acad. Sci. USA 80:1101-1105.
0099-2240/92/082531-05$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 58, No. 8
Molecular Cloning and Mapping of Phenol Degradation Genes from Bacillus stearothermophilus FDTP-3 and Their Expression in Escherichia coli FU-MIN DONG, LI-LI WANG, CHANG-MEI WANG, JIE-PING CHENG, ZHI-QING HE, ZU-JIA SHENG, AND REN-QUAN SHEN*
Institute of Genetics, Fudan University, Shanghai 200433, People's Republic of China Received 18 December 1991/Accepted 13 May 1992
Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacilus stearothermophilus and were mapped by subcloning and by use of a TnS insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida. Phenolic compounds are hazardous pollutants. Utilization and degradation of phenolic compounds by mesophiles were reported early in the 1950s (7). The degradation pathways and regulatory mechanisms in metabolism of phenol, cresols, toluene, and xylenes in mesophiles have been extensively studied during the past 30 years, especially in pseudomonads (2-4, 13, 14, 18). The benzene ring of toluene or related compounds may be cleaved by either the meta or the ortho pathway through the intermediate compound catechol or substituted catechols (17). However, not as much information is available on thermophiles. Buswell and Twomey isolated a strain of Bacillus stearothermophilus capable of utilizing phenol and cresols (6). Adams and Ribbons isolated a strain of B. stearothermophilus which degraded both phenol and benzoic acid (1). Gurujeyalakshmi and Oriel reported the isolation of a phenol-degrading B. stearothermophilus strain and the partial characterization of its phenol hydroxylase (10). In this paper, we report the catabolic pathway of phenol degradation in a strain, FDTP-3, of B. stearothermophilus isolated from the effluent of the Nanjing Oil Refinery (19a) and the cloning and mapping of two genes relevant to the phenol degradation pathway.
Preparation of total bacterial DNA was performed as described by Saito and Miura (19). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs or Sino-American Biotechnology Company. Reaction conditions were as described by Maniatis et al. (16). Isolation of DNA fragments from the gel was done as described by Guo and Wu (9). Southern transfer and hybridization were performed as described by Maniatis et al. (16). The biotin labelling kit and the nick translation kit were purchased from Bethesda Research Laboratories, Inc. Labelling of probes with biotinyl-11-dUTP and detection of homology were done as recommended by the manufacturer (4a). Total DNA of donor strain FDTP-3, plasmid DNA of pFDA11, pPL392 carrying genexylE from Pseudomonas putida (11), and other controls digested with BamHI were used for Southern transfer. Transformation. Preparation of competent E. coli cells and plasmid transformation were performed as described by Maniatis et al. (16). Preparation of crude enzyme extract. Bacterial strains were grown overnight in liquid rich medium, supplemented with antibiotics when necessary, and pelleted. The pellet was suspended in 0.1 M phosphate buffer (pH 7.6) and pelleted once again. Fifteen grams (wet weight) was resuspended in
MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. Culture media and culture temperature. The rich medium (pH 7.2) contained 10 g of polypeptone, 5 g of yeast extract, and 5 g of NaCl per liter. The minimal medium (pH 7.2) contained 2 g of glucose, 0.5 g of K2HPO4, 1 g of MgSO4 7H20, 1 g of NH4Cl, 10 ,ug of CuSO4. 5H20, 50 ,g of CaCl2. 6H20, 70 ,ug of ZnSO4 7H20, 0.4 mg of nicotinamide, 0.4 mg of thiamine, and 2 ,ug of biotin per liter. Chloramphenicol (5 ,ug/ml), ampicillin (AMP) (50 ,ug/ml), and kanamycin (KAN) (3 ,ug/ml) (which was used instead of neomycin) were added when necessary. All Escherichia coli strains were cultured at 37°C, and strain FDTP-3 was cultured at 60°C. DNA manipulation. Plasmid DNA extraction was performed as described by Birnboim and Doly (5). *
6.5 ml of the same buffer and was sonicated with a VirSonic 300 sonicator for 5 min. After being centrifuged for 15 min at 23,000 x g, the supernatant was used as the crude enzyme extract.
Detection of catechol 2,3-dioxygenase. FDTP-3 was spread rich medium and incubated overnight. A 0.5 M catechol solution was sprayed onto the plate. After the yellow color of the colonies (as well as the surrounding medium) appeared, after incubation at 60°C for 5 min, the plate was eluted with a small amount of phosphate buffer (0.1 M, pH 7.6) and centrifuged. The supernatant was scanned with a Shimadzu UV-240 spectrophotometer. The yellowish compound 2-hydroxymuconic semialdehyde (HMS) gives a characteristic absorption peak at 375 nm. Identification of catechol as an immediate degradation product of phenol. The crude extract prepared from E. coli K514(pTG402), which possesses catechol 2,3-dioxygenase activity like that of the mesophile P. putida (20), was used to on
Corresponding author. 2531
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DONG ET AL. TABLE 1. Bacterial strains and plasmids
Strain or
Source, reference,
Characteristics
plasmid
Donated by Q. Y. Zhou
FDTP-3 E. coli TG1
or derivation
supE hsd-5 thi (lac-proAB) F' [traD36 proAB' lacIq lacZ AM15] supE44 hsdS20 (r- mB) recA13 ara-14 proA2 lacYl galK2 rpsL20 xyl-5 mtl-l F- his trp tsx spc nalA AlacX74 ara A(lac-proAB) rpsL thi (480 lacZ AM15) A(srl-recA)306::Tn1O(Tcr) F' [traD36 proAB+ lacIq lacZ AM15] A(lac proB)Valr Tn5 F' lac+ pro' hsrK hsmK derivative of C600
Laboratory stock
pPL392
Apr
pTG402 pPGV5 pBR322 pFDC11 pFDC111 pFDA10
xylE Apr Cm' Apr Nmr Cms
pFDA11 pFDA13
Same as pFDA10 Apr pheA pheB
pFDA16
Apr pheB
pFDA17 pFDA21
Apr pheB Apr pheA
pBR322 cloned with 16.4-kb fragment from Tol plasmid, donated by S. Harayama (11) Donated by P. Dhaese; 20 Donated by P. Dhaese Laboratory stock 12 This work pFDC111 cloned with 6.4-kb fragment of DNA from FDTP-3 (this work) This work pUC18 cloned with 6.4-kb fragment from pFDA11 (unpublished data) Subcloned from pFDA11 with pUC119 as vector (unpublished data) Deletion derivative of pFDA16 (unpublished data) Subcloned from pFDA13 with pUC18 as vector (unpublished data)
HB101 ED2196 MV1184
GmlapX K514
Apr Tcr Cmr Nmr Apr Cmr Nmr Apr Cmr Km pheA pheB
identify catechol, the enzymatic reaction product of phenol, by the characteristic absorption peak of HMS. Assay of enzyme thermostability. The crude extract with proper dilution in phosphate buffer (0.1 M, pH 7.6) was inactivated at different temperatures for 15 min. A 0.5 M catechol solution was added to a final concentration of 2.5 mM. The optical density measurements at 37 and 55°C without inactivation were taken as 100% for mesophilic and thermophilic enzymes, respectively. Therefore, the optical density at 375 nm read from the scanned curve was taken as a measure of the enzyme activity remaining. Transposon mutagenesis. Transposon Tn5 with a genetic marker for neomycin resistance was used. Since the four PstI sites on Tn5 were known (15) and there is one PstI site on the multiple cloning site of pFDA13, the insertion site of TnS in pFDA13 was analyzed according to the size of the fragments obtained by digesting the plasmid with PstI and PstI-BamHI. RESULTS
Identification of the bacterial strain FDTP-3. FDTP-3 is a gram-positive, spore-forming, starch-hydrolyzing, catalasepositive thermophile. Negative tests included the VogesProskauer reaction, growth on rich medium with 5% NaCl or 0.02% sodium azide, and growth on rich medium of pH 5.7. The optimum growth temperature was within the range 60 to 65°C. FDTP-3 was thus identified as B. stearothermophilus by the methods of Gordon et al. (8). Carbon source. FDTP-3 can use glucose as the sole carbon source on minimal medium supplemented with vitamins and
Laboratory stock 11 Laboratory stock
Laboratory stock Donated by P. Dhaese
microelements. Among phenol, toluene, benzene, o-cresol, p-cresol, and benzoic acid, only phenol (0.05%) was shown to be used by FDTP-3 as the sole carbon source on the same medium. Catechol 2,3-dioxygenase activity of FDTP-3. The presence of catechol 2,3-dioxygenase activity in FDTP-3 was detected by scanning the eluent from the plate as described above. The curve reveals the characteristic absorption maximum of HMS at 375 nm. When distilled water was used instead of catechol solution, the eluent did not show the characteristic absorption maximum. The same characteristic absorption maximum was observed for the eluent of E. coli ED2196 harboring the plasmid pPL392, which carries gene xylE of P. putida encoding catechol 2,3-dioxygenase (11), while the eluent of E. coli HB101 harboring pBR322 gave no absorption maximum at 375 nm. It is known that the enzyme catechol 2,3-dioxygenase converts catechol to HMS. Therefore, it was concluded that FDTP-3 possesses catechol 2,3-dioxygenase activity. It is known that the enzymes taking part in the degradation of hydrocarbons in P. putida are all inducible. Catechol 2,3-dioxygenase of FDTP-3 was found to be strongly induced by phenol or benzoic acid but not by benzene, toluene, p-cresol, or o-cresol (data not shown). It is interesting that benzoic acid cannot be used as the sole carbon source by FDTP-3. It is a gratuitous inducer. Cloning of phenol degradation genes from FDTP-3. Phenol degradation genes from FDTP-3 were cloned on a shuttle plasmid, pFDC111 (Fig. 1), by ligating the BamHI digest of pFDC111 and that of the total DNA from FDTP-3. pFDC111 was constructed from pFDC11 (12). E. coli TG1 transformed
VOL. 58, 1992
PHENOL DEGRADATION GENES FROM B. STEAROTHERMOPHILUS
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a° a
.X.t >
I
a
w Eco RI
DNA ligase, T4
I
0
40
60
Temp
FIG. 2. Comparison of the thermostability of catechol 2,3-dioxfrom B. stearothernophilus FDTP-3 with that of catechol 2,3-dioxygenase from P. putida. Enzyme activities of extracts of both E. coli K514(pTG402) (-) and E. coli MV1184(pFDA17) (a) were determined after inactivation at different temperatures for 15 min. ygenase
FIG. 1. Construction of the shuttle plasmid pFDC111 used for cloning the phenol degradation genes. pPGV5 (7.7 kb) was completely digested with EcoRI, and pFDC11 (5.5 kb) was partially digested with EcoRI. The ligation mixture was transformed into E. coli HB101. Apr Cmr transformants with fragments inserted at different EcoRI sites were selected. pFDC111 (8.2 kb) was identified by digesting the plasmid with XbaI. Ba, BamHI; Bg, BgIII; E, EcoRI; H, HindIII; P, PstI; SI, Sall; Su, Sau3A; X, XbaI; cat, chloramphenicol acetyltransferase.
with the ligation mixture was spread on a rich medium plate supplemented with AMP. After the rich medium plate was sprayed with catechol solution, five yellow colonies were obtained from among 1,558 transformants. Two of the yellow transformants harboring recombinant plasmids pFDA10 and pFDA11 were further identified on a plate containing AMP and KAN. Agarose gel electrophoresis of pFDA10 and pFDA11 digested with HindIII or BamHI gave exactly the same pattern (data not shown). A 6.4-kb insert was found in both plasmids. With the biotinylated pFDA11 DNA as the probe, Southern hybridization was carried out against pPL392 or the total DNA of the donor strain. A hybridization signal between the donor DNA and the probe pFDA11 was evident, but only a very weak signal between pFDAll and pPL392 was observed (data not shown) despite the fact that they carry a gene encoding an enzyme with the same catalytic function. The thermostability of catechol 2,3-dioxygenase encoded by pFDA17 (a derivative of pFDA13 [see Fig. 4]) was compared with that of the enzyme encoded by pTG402 (20) (Fig. 2). It was found that the former enzyme was much more thermostable than the latter; thus, the origin of the cloned gene was further confirmed. Restriction endonuclease analysis of plasmid pFDA13 showed that the 6.4-kb insert has four HindIII sites, three ClaI sites, two EcoRI sites, one EcoRV site, and one PvuII site. No BglII, PstI, or SalI site was found (see Fig. 4).
0le
3~~~~~~~
o~~~~~
1 9() w.iv
300 .
o( ig
(raw)
FIG. 3. Absorption spectrum curves showing the conversion of phenol into catechol by cloned genes from FDTP-3. All organisms were cultured on rich medium supplemented with AMP. (Line 1) Catechol solution (final concentration, 3 mM) was added to the extract of E. coli(pFDA13), and the mixture was incubated at 60°C for 15 min. (Line 2) Phenol (final concentration, 3 mM) was added to the extract of E. coli(pFDA21), and the mixture was incubated at 60°C for 45 min; next, the extract of E. coli(pTG402) was added, and the mixture was incubated at 37°C for 30 min. (Line 3) Phenol (final concentration, 3 mM) was added to the extract of E. coli(pFDA21), and the mixture was incubated at 60°C for 45 min.
2534
APPL. ENVIRON. MICROBIOL.
DONG ET AL. E
H
C
i.9I..
e
i*
H
I
H
C
I I
I
phA
ph& aU
H
i .1
I
P/EV C
1t t
E
e
1.
-|-4
pFDA13 E
E
pFDA21
e1
H
pFDA 16
pFDA 17
FIG. 4. Physical maps of the cloned fragments of recombinant plasmids containing phenol degradation genes and genetic map showing the location of the inserted TnS. pFDA13 was constructed by digesting the 6.4-kb insert from pFDA1l with BamHI and subcloning the fragment into the BamHI sites of pUC18. pFDA21 was obtained by digesting pFDA13 with EcoRI and subcloning the 3.7-kb fragment into the EcoRI site of pUC18. pFDA16 is a recombinant plasmid carrying the 2.75-kb fragment (from BamHI to the second HindIII site) from the 6.4-kb insert subcloned in pUC119 in a stepwise manner. pFDA17 (1.3 kb) was derived from pFDA16 by shortening the left terminal with the ExoIII-mung bean nuclease system. The sites of Tn5 insertion are indicated as follows: A, insertion within the genespheA andpheB; A, insertion outside both genes. Restriction cleavage sites: B, BamHI; E, EcoRI; C, ClaI; H, HindIII; P, PstI; EV, EcoRV; P/EV, PvuII and EcoRV very close to one another.
Catechol is the immediate degradation product of phenol in FDTP-3. Colonies of FDTP-3 grown on rich medium containing phenol were yellow. That phenol is degraded to HMS through catechol was proved by studying the cloned genes. pFDA21 is a recombinant plasmid subcloned from pFDA13 carrying a 3.7-kb fragment bordered by the two EcoRI sites (see Fig. 4). The crude extract (or resting cells) of E. coli MV1184(pFDA21) incubated with phenol at 60°C for 45 min gave no absorption maximum at 375 nm (Fig. 3, line 3). However, on addition of the crude extract (or resting cells) of E. coli K514(pTG402), which carries the subcloned xylE gene from P. putida, an absorption maximum at 375 nm did appear after incubation at 37°C for 30 min (Fig. 3, line 2). This suggests that pFDA21 does not carry the catechol 2,3-dioxygenase gene but carries a gene encoding phenol hydroxylase that converts phenol to catechol, which is then converted to HMS by the enzyme catechol 2,3-dioxygenase, which exists in the extract of E. coli K514(pTG402). Therefore, it was suggested that FDTP-3 degrades phenol via the meta pathway as in P. putida, although it is not necessarily the only pathway. Mapping of the genes on the 6.4-kb cloned fragment by subcloning. The colonies of E. coli MV1184 harboring the multicopy plasmid pFDA13 grown on rich medium supplemented with AMP and phenol were yellow. This suggests that the cloned 6.4-kb fragment carries at least two genes, pheA and pheB, coding for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. pFDA16 and pFDA21 are subclones of the 6.4-kb fragment (Fig. 4). E. coli MV1184 (pFDA16) grown on rich medium supplemented with AMP and phenol was not yellow. However, a yellow color appeared when MV1184(pFDA16) was grown on rich medium supplemented with AMP but without phenol and then sprayed with catechol solution. It was inferred that gene pheB, coding for catechol 2,3-dioxygenase, resides on pFDA16. Colonies (as well as the surrounding medium) of E. oH
phenol hydiroxylase pheA
OH
coli MV1184(pFDA21) were brown when streaked on rich medium supplemented with AMP and phenol. Furthermore, when E. coli MV1184(pFDA21) was inoculated on rich medium supplemented with AMP and phenol adjacent to an E. coli strain with only catechol 2,3-dioxygenase activity, such as E. coli MV1184(pFDA16), a yellow color appeared in the region of the latter. It is inferred that the brown is due to the accumulation of catechol (from its precursor, phenol), which is autooxidized to quinone substances in the presence of air, and that the yellow color is due to the conversion of catechol to HMS by the nearby strain with catechol 2,3dioxygenase activity. It is therefore inferred that genepheA coding for phenol hydroxylase resides on pFDA21. Transposon mutagenesis of the recombinant plasmid pFDA13. pFDA13 was used to transform the TnS-containing E. coli TnS Gmlo3A. Yellow transformants were selected from rich medium supplemented with AMP, KAN, and phenol and were spread on the same medium and incubated overnight. Bacterial mass was collected, and the plasmid DNA was extracted and used to transform E. coli TG1. White, yellow, and brown colonies were noticed on rich medium supplemented with AMP, KAN, and phenol. The white colonies were believed to be those with TnS inserted within gene pheA, the brown colonies were believed to be those with TnS inserted within gene pheB, and the yellow colonies were believed to be those with TnS inserted anywhere except in pheA or pheB. The nature of the insertion mutants was further confirmed by the following observations. That the brown colonies are those with TnS inserted within the pheB gene was confirmed by the fact that they remained white on rich medium supplemented with AMP and KAN but without phenol even after being sprayed with catechol solution, while those with TnS inserted within gene pheA turned yellow under the same conditions. Furthermore, when mutants with Tn5 inserted within genes pheA and pheB were streaked side by side, a yellow color ap-
catechol 2
tOH
l3-dioxygcnltse
tiohexB
FIG. 5. Metabolic pathway of phenol in strain FDTP-3.
OOH OH
VOL. 58, 1992
PHENOL DEGRADATION GENES FROM B. STEAROTHERMOPHILUS
peared in the region of the former. These observations were compatible with those obtained by subcloning. To determine the location of each insertion in pFDA13::Tn5 mutant plasmids, plasmid DNA from each derivative was isolated and analyzed by cleavage with the restriction enzymes PstI and PstI-BamHI. The insertion map is shown in Fig. 4, and the locations of the two genes were demonstrated. DISCUSSION The results reported here suggest that the metabolic pathway of phenol in the strain FDTP-3 is as shown in Fig. 5. Attempts to detect the enzyme catalyzing the reaction following catechol 2,3-dioxygenase activity were unsuccessful. Catechol 2,3-dioxygenase is functionally the same as the enzyme encoded by xylE in P. putida, although its thermostability is quite different and the homology between the two genes is rather limited. Three different classes of insertion mutation can be visualized directly by the color developed on the plate. The site of Tn5 insertion seems to be unevenly distributed, particularly in the pheA gene. No polar mutant has been found; thus, we are not in a position to suggest whether these genes are organized into an operon or to suggest the direction of transcription.
2535
Acids Res. 7:1513-1523. 6. Buswell, J. A., and D. G. Twomey. 1975. Utilization of phenol and cresols by Bacillus stearothermophilus, strain PH24. J. Gen. Microbiol. 87:377-379. 7. Dagley, S., and M. D. Patel. 1957. Oxidation of p-cresol and related compounds by a Pseudomonas. Biochem. J. 66:227-233. 8. Gordon, R. E., W. C. Hayness, and C. H.-N. Pang. 1973. The genus bacillus. U.S. Department of Agriculture, Washington, D.C. 9. Guo, L. H., and R. Wu. 1983. Exonuclease III: use for DNA sequence analysis and in specific deletion of nucleotides. Methods Enzymol. 100:60-97. 10. Gurujeyalakshmi, G., and P. Oriel. 1989. Isolation of phenoldegrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase. Appl. Environ. Microbiol. 55:500-502. 11. Harayama, S., P. R. Lehrbach, and K. N. Timmis. 1984. Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2. J. Bacteriol. 160:251-255. 12. He, X. S., R. Q. Shen, and Z. J. Sheng. 1990. The construction of promoter probe vector pFDC4 and gene expressing vector pFDC11 with high transformation efficiency in Bacillus stearothermophilus. Acta Genet. Sin. 17:216-225. (In Chinese.) 13. Inouye, S., A. Nakazawa, and T. Nakazawa. 1981. Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS. J. Bacteriol. 148:
413-418. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant 3870355). We thank Q. Y. Zhou and S. F. Wang for providing the strain of thermophile FDTP-3, S. Harayama for providing E. coli ED2196(pPL392), and P. Dhaese for providing E. coli 514(pTG402) and plasmid pPGV5. REFERENCES 1. Adams, D., and D. W. Ribbons. 1988. The metabolism of aromatic compounds by thermophilic bacilli. Appl. Biochem. Biotechnol. 17:231-244. 2. Bayly, R. C., and M. G. Barbour. 1984. The degradation of aromatic compounds by the meta and gentisate pathway, p. 253-294. In D. T. Gibson (ed.), Microbial degradation of organic compounds. Marcel Dekker, Inc., New York. 3. Bayly, R. C., S. Dagley, and D. T. Gibson. 1966. The metabolism of cresols by species of Pseudomonas. Biochem. J. 101:293301. 4. Bayly, R. C., and G. J. Wigmore. 1973. Metabolism of phenol and cresols by mutants of Pseudomonas putida. J. Bacteriol. 113:1112-1120. 4a.Bethesda Research Laboratories, Inc. DNA detection system. Bethesda Research Laboratories, Inc., Gaithersburg, Md. 5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic
14. Inouye, S., A. Nakazawa, and T. Nakazawa. 1983. Molecular cloning of regulatory gene xylR and operator-promoter regions of the xyLABC and xylDEGF operons of the TOL plasmid. J. Bacteriol. 155:1192-1199. 15. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. A restriction map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 117:69-72. 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 17. Nakazawa, T., and T. Yokota. 1973. Benzoate metabolism in Pseudomonas putida(arvilla) mt-2: demonstration of two benzoate pathways. J. Bacteriol. 115:262-267. 18. Ramos, J. L., A. Stolz, W. Reineke, and K. N. Timmis. 1986. Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. Proc. Natl. Acad. Sci. USA 83:8467-8471. 19. Saito, H., and K.-I. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta. 72:619-629. 19a.Zhou, Q. Y., and S. F. Wang. Unpublished data. 20. Zukowski, M. M., D. F. Gaffney, D. Speck, M. Kauffmann, A. Findeli, A. Wisecup, and J.-P. Lecocq. 1983. Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc. Natl. Acad. Sci. USA 80:1101-1105.
