APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p. 3437-3440
Vol. 58, No. 10
0099-2240/92/103437-04$02.00/0
Cloning of a Creatinase Gene from Pseudomonas putida in Escherichia coli by Using an Indicator Plate MING CHUNG CHANG,* CHUN CHIN CHANG, AND JINQ CHYI CHANG Department of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan, Republic of China Received 21 April 1992/Accepted 22 July 1992 A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants of Escherichia coli in combination with Proeus mirabiis cells grown on creatinase test plates were screened for creatinase activity; transformants were considered positive for creatinase activity if a red-pink zone appeared around the colonies. One creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was obsernred in recombinant E. coli by minicell analysis.
Various kinds of bacteria such asAlcaligenes (13),Arthrobacter (4), Flavobacterinum (14), Micrococcus (14), and Pseudomonas (1, 3, 6, 15) species produce creatinase (creatine amidinohydrolase [EC 3.5.3.3]), which catalyzes the hydrolysis of the creatine to sarcosine and urea. Because this enzyme is useful for clinical estimation of creatine contents in human serum and urine, increasing its yield in industrial production is desirable. Most microorganisms, however, inducibly produce the creatinase, and this is a drawback in commercial production because of the use of the expensive inducer. To resolve this problem, one approach would be to isolate constitutive or -hyperproductive mutants for improving production of the enzyme effectively. Another approach would be to clone the gene encoding creatinase in order to analyze the genetic information and modify expression of this gene by means of molecular biology. Creatinine-assimilating Pseudomonas putida NTU-8, which was a gift from W. H. Liu, University of National Taiwan, Taipei, Taiwan, Republic of China, was isolated from soil (10); is able to use creatinine as its carbon and nitrogen source when grown on creatinine medium consisting of 1% creatinine, 0.5% K2HPO4, 0.1% NaCl, and 0.1% MgSO4. 7H20 (pH 7.0); and has been found to produce inducibly high levels of the creatininase and creatinase (9), suggesting that these two enzymes could be a useful model for fundamental studies of protein expression and regulation as well as for applied research. Thus, we have used recombinant DNA techniques to clone the creatinase gene from P. putida and to analyze the genetic information. The P. putida NTU-8 genomic DNA library was constructed as follows. Pseudomonas chromosomal DNA was isolated by the method of Marmur (12) and partially cleaved with Sau3A, and fragments of 5 to 20 kb were isolated by using sucrose density gradient centrifugation. The DNA fragments were ligated with T4 DNA ligase to BamHIdigested plasmid pBR322, and the ligation mixture was used to transform competent Escherichia coli JA221 cells (2). After replica plating on Luria-Bertani (LB) agar medium (11) containing ampicillin and tetracycline, almost 90% of the clones were tetracycline sensitive (Tcs), indicating insertion of a foreign DNA fragment. A total of 20,000 such ampicillin*
Corresponding author.
resistant (Apr) Tcs transformants were kept in a collection. To isolate the gene encoding creatinase from this genomic library, a screening method may be performed as described by Koyama et al. (7). However, this screening method is both time-consuming and cumbersome, and it was desirable to develop an assay that would facilitate simultaneous screening of a large number of clones easily. An alternative strategy was thus adopted, i.e., replica plating of transformants on creatinase test plates containing 0.1% peptone, 0.1% glucose, 0.5% NaCl, 0.2% KH2PO4, 0.01% yeast extract, 0.03% thiamine, 1% creatine, 0.0012% phenol red, and 1.5% agar, pH 6.8, at a colony density of 30 colonies per plate. After incubation at 37°C for 24 h, when the colonies had reached at least 2 mm in diameter, Proteus mirabilis organisms (ATCC 7002; obtained from the Culture and Development Institute, Hsinchu, Taiwan, Republic of China), which were previously grown on nutrient agar (Difco 0001), were inoculated close to each colony and then incubated at 37°C for 24 h. Creatinase can hydrolyze creatine to sarcosine and urea. P. mirabilis possesses the enzyme urease (5), which can hydrolyze urea, releasing ammonia and producing a red-pink color change in the medium. Thus, the creatinase-positive clones have red-pink zones around them (Fig. 1). By using this approach, three creatinasepositive clones were isolated from ca. 5,000 transformants screened. The hybrid plasmids, designated pCR40, pCR50, and pCR60, were used in retransformation of E. coli JA221 to verify the presence of the creatinase gene on the plasmid. All of the transformants containing pCR50 or pCR60 were identified as having creatinase activity by using both screening methods mentioned above, but because most of the transformants containing pCR40 were unstable, only two hybrid plasmids, pCR50 and pCR60, were studied further. The DNA inserts of pCR50 and pCR60 were characterized by digestion with restriction enzymes, and detailed physical maps showed that the two plasmid inserts were identical except that the pCR50 insert (7.6 kbp) was 5 kb smaller than that of pCR60. To further localize the creatinase gene, a more detailed restriction was performed on plasmid pCR50, and the results of subcloning of various restriction fragments indicated that the creatinase gene was located on the 2.7-kb EcoRI-SalI fragment. This EcoRI-SalI fragment was subcloned into pUC18 and pUC19 (16), and the resultant plasmids were designated pCR5006 and pCR5007 (Fig. 2). Although the inserted DNA fragment in pCR5007 was oriented 3437
3438
NOTES
APPL. ENvIRON. MICROBIOL.
FIL. 1. Icentincation o0 creatinase-positive clones o0 E. coli by replica plating in the creatinase test plate. Arrowheads: wild-type strain, P. putida (A); P. putida plus P. mirabilis (B); E. coli JA221 (pBR322) (C); E. coli JA221 (pBR322) plus P. mirabilis (D); E. coli JA221 (pCR50) (E); E. coli JA221 (pCR50) plus P. mirabilis (F). P. putida and E. coli JA221 (pCR50) formed red-pink zones because of the presence of an indicator strain of P. mirabilis.
opposite to that in pCR5006 with respect to the lac promoter, both plasmids coded for active creatinase in E. coli JM109 (17) as determined by formation of red-pink zones on creatinase test plates. In addition, in E. coli JM109 harboring pCR5006 or pCR5007, nearly similar levels of creatinase activity were detected with and without isopropyl-3-D-thiogalactopyranoside in all cases (data not shown). From these results, it was concluded that transcription was not initiated at the lac promoter but that the promoter of the creatinase gene was cloned together with the gene and was functional in E. coli. To confirm that the cloned DNA fragment was derived from P. putida NTU-8, we performed Southern blot hybridization (11) with the cloned 2.7-kb EcoRI-SalI fragment as a probe (Fig. 3). The probe showed strong hybridization with the EcoRI-SalI fragment obtained from plasmid pCR50, which was used a positive control (lane 1). An identical 2.7-kb band was also visible in the genomic EcoRI-SalI digest (lane 2), and no cross hybridization with E. coli DNA was detected (lanes 3 and 4). The cloned gene product was examined by using the E. coli minicell system, which permits specific labeling of plasmid-encoded proteins. The minicell-producing strain E. coli P678-54 (F- thr leu thi supE lacYfhuA gal mal xyl mtl) was transformed with plasmid pCR50 or pCR5006, and minicells were isolated, labeled with [35S]methionine, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Lejeune et al. (8). A unique 50-kDa polypeptide was expressed in the strain carrying pCR50 or pCR5006 (Fig. 4, lanes 2 and 4). The inability to produce the 50-kDa polypeptide was observed in the strain
1 kb
Creatinase Vector Activity
H~~
E
Uuu *
.
.
..
o
v
U.
pCRSO
_e0 cn
I
(Bam HI)
(-)
pUC18
(+)
pBR322
(+)
pUC18
(-)
pUC18
(+)
pUC18
(+)
pUC18
(+)
pUC19
Sac I
I~~~~~~
Sac I m
pCR5002
-m
EcoR I pCR5003 1+ EcoR I
pBR322
cn
Sac I
pCR5004 I -
(+)
0.4 .,,,
a4
pCR5001
EcoR I
I
Sma I I
EcoR I
Pst I
pCR5005 I Sal I
EcoR I
pCR5006 EcoR I
pCR5007
)
I
Sal I
I~~~~~
FIG. 2. Subclones of pCR50 containing the cloned creatinase
gene.
Creatinase activity was determined
on
the creatinase test plates.
VOL. 58, 1992
NOTES
A A2 bp
3
, BM
1 2 3 4
23130
6557-
023
1
2
3439
3 4
;
-
*
ZE_1
FIG. 3. Southern hybridization analysis with the 2.7-kb EcoRISall fragment as a probe against complete EcoRI and Sall digests of genomic DNA from P. putida and E. coli. (A) Photograph of a 0.8% agarose gel stained with ethidium bromide. (B) Autoradiogram of DNA transferred to nitrocellulose and hybridized with the 32p_ labeled EcoRI-Sall fragment. Lanes: M, molecular size markers (lambda-HindIII digest); 1, 0.1 ,ug of EcoRI-SalI fragment from pCR50; 2 to 4, EcoRI and SalI digests of P. putida, E. coli JA221, and E. coli JM109 DNA, respectively. Each genomic digest contained 3 ,ug of DNA.
containing pUC18 (lane 3). We conclude that pCR50 or pCR5006 confers the creatinase phenotype upon E. coli by specifying the synthesis of 50-kDa creatinase protein. The effects of creatinine or creatine on creatinase production were investigated in basal media of P. putida NTU-8 and E. coli JA221 (pCR50). For P. putida, creatinase production occurred only in the medium containing creatinine or creatine. However, the creatinase activity of E. coli JA221 (pCR50) was 4 x 10-2 U/ml of culture at the early stationary phase (measured by the method of Koyama et al. [7]; 1 U of creatinase was defined as the amount of enzyme which liberates 1 ,umol of urea) in LB medium containing 50 jig of ampicillin per ml in the absence of creatinine or creatine. The production of creatinase activity was shown to be constitutive in E. coli JA221 (pCR50) but creatine inducible in the parent organism, P. putida. This result suggests that a regulatory region which is functional in P. putida is not recognized in the E. coli transformant. Molecular analysis of the cloned 2.7-kb fragment will enable us to define and characterize the P. putida creatinase and its regulatory function. Cloning in E. coli of the creatinase enzyme gene from P. putida and from Flavobacterium sp. strain U-188 has been reported by Hoeffken et al. (3) and Koyama et al. (7), respectively. In the former study, positive clones containing the P. putida creatinase gene were screened by using an immunochemical method, while in the latter, the Flavobacterium creatinase gene was cloned by testing whether each transformant expresses the creatinase activity after being culturing in LB medium as mentioned above. To supplement these two cloning strategies, we report the cloning of the creatinase gene of P. putida NTU-8 by using a conventional plate assay. The results indicate that this screening plate
FIG. 4. Minicell labeling of plasmid-encoded proteins. Fluorography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysates of [35S]methionine-labeled proteins expressed from E. coli P678-54 transformed with plasmids pCR50 (lane 2), pUC18 (lane 3), and pCR5006 (lane 4). Molecular mass markers were as follows (lane 1, from top to bottom): phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; a-lactalbumin, 14.4 kDa. The arrow indicates the creatinase.
assay is a rapid, simple, and unequivocal method for screening large numbers of colonies for the detection of creatinasepositive clones in E. coli. It is possible that a similar methodology may be applicable to screen some other enzyme-positive clones which could be detected directly from plating by combining with a suitable indicator strain. REFERENCES 1. Appleyard, G., and D. D. Woods. 1956. The pathway of creatine catabolism by Pseudomonas ovalis. J. Gen. Microbiol. 14:351356. 2. Beggs, J. D. 1978. Transformation of yeast by a replicating hybrid plasmid. Nature (London) 275:104-108. 3. Hoeffken, H. W., S. H. Knof, P. A. Bartlett, R. Huber, H. Moellering, and G. Schumacher. 1988. Crystal structure determination, refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida. J. Mol. Biol. 204:417-433. 4. Kaplan, A., and D. Naugler. 1974. Creatinine hydrolase and creatine amidinohydrolase. I. Presence in cell-free extracts of Arthrobacter ureafaciens. Mol. Cell. Biochem. 3:9-15. 5. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., W. M. Janda, H. M. Sommers, and W. C. Winn, Jr. 1988. Color atlas and textbook of diagnostic microbiology, 3rd ed. Lippincott, Phila-
delphia. 6. Kopper, P. H., and H. H. Beard. 1947. Creatinase activity of a strain of Pseudomonas. Arch. Biochem. 15:195-199. 7. Koyama, Y., S. Kitao, H. Yamamoto-Otake, M. Suzuki, and E. Nakano. 1990. Cloning and expression of the creatinase gene from Flavobacterium sp. U-188 in Escherichia coli. Agric. Biol. Chem. 54:1453-1457. 8. Lejeune, A., V. Dartois, and C. Colson. 1988. Characterization and expression in Escherichia coli of an endoglucanase gene of Pseudomonas fluorescens subsp. cellulosa. Biochim. Biophys. Acta 950:204-214. 9. Liu, W. H., J. Y. Ho, and L. Y. Jang. 1986. Production of a clinical analysis enzyme, creatininase, by Pseudomonas putida NTU-8. J. Chin. Agric. Chem. Soc. 24:184-191. 10. Liu, W. H., J. Y. Ho, and S. T. Shu. 1986. Isolation and identification of a creatininase-producing bacterium. J. Chin. Agric. Chem. Soc. 24:175-183. 11. Maniatis, T., E. F. Fritsch, and J. Sambroolk 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 12. Marmur, J. 1961. A procedure for the isolation of deoxyribo-
3440
NOTES
nucleic acid from microorganisms. J. Mol. Biol. 3:208-218. 13. Matsuda, Y., N. Wakamatsu, Y. Inouye, S. Uede, Y. Hashimoto, K. Asano, and S. Nakamura. 1986. Purification and characterization of creatine amidinohydrolase of Alcaligenes origin. Chem. Pharm. Bull. 34:2155-2160. 14. Suzuki, M., and N. Saito. 1976. German patent 2,614,114. 15. Tsuru, D., I. Oka, and T. Yoshimoto. 1976. Creatinine decomposing enzymes in Pseudomonas putida. Agric. Biol. Chem.
APPL. ENvIRON. MICROBIOL.
40:1011-1018. 16. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268. 17. 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. 58, No. 10
0099-2240/92/103437-04$02.00/0
Cloning of a Creatinase Gene from Pseudomonas putida in Escherichia coli by Using an Indicator Plate MING CHUNG CHANG,* CHUN CHIN CHANG, AND JINQ CHYI CHANG Department of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan, Republic of China Received 21 April 1992/Accepted 22 July 1992 A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants of Escherichia coli in combination with Proeus mirabiis cells grown on creatinase test plates were screened for creatinase activity; transformants were considered positive for creatinase activity if a red-pink zone appeared around the colonies. One creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was obsernred in recombinant E. coli by minicell analysis.
Various kinds of bacteria such asAlcaligenes (13),Arthrobacter (4), Flavobacterinum (14), Micrococcus (14), and Pseudomonas (1, 3, 6, 15) species produce creatinase (creatine amidinohydrolase [EC 3.5.3.3]), which catalyzes the hydrolysis of the creatine to sarcosine and urea. Because this enzyme is useful for clinical estimation of creatine contents in human serum and urine, increasing its yield in industrial production is desirable. Most microorganisms, however, inducibly produce the creatinase, and this is a drawback in commercial production because of the use of the expensive inducer. To resolve this problem, one approach would be to isolate constitutive or -hyperproductive mutants for improving production of the enzyme effectively. Another approach would be to clone the gene encoding creatinase in order to analyze the genetic information and modify expression of this gene by means of molecular biology. Creatinine-assimilating Pseudomonas putida NTU-8, which was a gift from W. H. Liu, University of National Taiwan, Taipei, Taiwan, Republic of China, was isolated from soil (10); is able to use creatinine as its carbon and nitrogen source when grown on creatinine medium consisting of 1% creatinine, 0.5% K2HPO4, 0.1% NaCl, and 0.1% MgSO4. 7H20 (pH 7.0); and has been found to produce inducibly high levels of the creatininase and creatinase (9), suggesting that these two enzymes could be a useful model for fundamental studies of protein expression and regulation as well as for applied research. Thus, we have used recombinant DNA techniques to clone the creatinase gene from P. putida and to analyze the genetic information. The P. putida NTU-8 genomic DNA library was constructed as follows. Pseudomonas chromosomal DNA was isolated by the method of Marmur (12) and partially cleaved with Sau3A, and fragments of 5 to 20 kb were isolated by using sucrose density gradient centrifugation. The DNA fragments were ligated with T4 DNA ligase to BamHIdigested plasmid pBR322, and the ligation mixture was used to transform competent Escherichia coli JA221 cells (2). After replica plating on Luria-Bertani (LB) agar medium (11) containing ampicillin and tetracycline, almost 90% of the clones were tetracycline sensitive (Tcs), indicating insertion of a foreign DNA fragment. A total of 20,000 such ampicillin*
Corresponding author.
resistant (Apr) Tcs transformants were kept in a collection. To isolate the gene encoding creatinase from this genomic library, a screening method may be performed as described by Koyama et al. (7). However, this screening method is both time-consuming and cumbersome, and it was desirable to develop an assay that would facilitate simultaneous screening of a large number of clones easily. An alternative strategy was thus adopted, i.e., replica plating of transformants on creatinase test plates containing 0.1% peptone, 0.1% glucose, 0.5% NaCl, 0.2% KH2PO4, 0.01% yeast extract, 0.03% thiamine, 1% creatine, 0.0012% phenol red, and 1.5% agar, pH 6.8, at a colony density of 30 colonies per plate. After incubation at 37°C for 24 h, when the colonies had reached at least 2 mm in diameter, Proteus mirabilis organisms (ATCC 7002; obtained from the Culture and Development Institute, Hsinchu, Taiwan, Republic of China), which were previously grown on nutrient agar (Difco 0001), were inoculated close to each colony and then incubated at 37°C for 24 h. Creatinase can hydrolyze creatine to sarcosine and urea. P. mirabilis possesses the enzyme urease (5), which can hydrolyze urea, releasing ammonia and producing a red-pink color change in the medium. Thus, the creatinase-positive clones have red-pink zones around them (Fig. 1). By using this approach, three creatinasepositive clones were isolated from ca. 5,000 transformants screened. The hybrid plasmids, designated pCR40, pCR50, and pCR60, were used in retransformation of E. coli JA221 to verify the presence of the creatinase gene on the plasmid. All of the transformants containing pCR50 or pCR60 were identified as having creatinase activity by using both screening methods mentioned above, but because most of the transformants containing pCR40 were unstable, only two hybrid plasmids, pCR50 and pCR60, were studied further. The DNA inserts of pCR50 and pCR60 were characterized by digestion with restriction enzymes, and detailed physical maps showed that the two plasmid inserts were identical except that the pCR50 insert (7.6 kbp) was 5 kb smaller than that of pCR60. To further localize the creatinase gene, a more detailed restriction was performed on plasmid pCR50, and the results of subcloning of various restriction fragments indicated that the creatinase gene was located on the 2.7-kb EcoRI-SalI fragment. This EcoRI-SalI fragment was subcloned into pUC18 and pUC19 (16), and the resultant plasmids were designated pCR5006 and pCR5007 (Fig. 2). Although the inserted DNA fragment in pCR5007 was oriented 3437
3438
NOTES
APPL. ENvIRON. MICROBIOL.
FIL. 1. Icentincation o0 creatinase-positive clones o0 E. coli by replica plating in the creatinase test plate. Arrowheads: wild-type strain, P. putida (A); P. putida plus P. mirabilis (B); E. coli JA221 (pBR322) (C); E. coli JA221 (pBR322) plus P. mirabilis (D); E. coli JA221 (pCR50) (E); E. coli JA221 (pCR50) plus P. mirabilis (F). P. putida and E. coli JA221 (pCR50) formed red-pink zones because of the presence of an indicator strain of P. mirabilis.
opposite to that in pCR5006 with respect to the lac promoter, both plasmids coded for active creatinase in E. coli JM109 (17) as determined by formation of red-pink zones on creatinase test plates. In addition, in E. coli JM109 harboring pCR5006 or pCR5007, nearly similar levels of creatinase activity were detected with and without isopropyl-3-D-thiogalactopyranoside in all cases (data not shown). From these results, it was concluded that transcription was not initiated at the lac promoter but that the promoter of the creatinase gene was cloned together with the gene and was functional in E. coli. To confirm that the cloned DNA fragment was derived from P. putida NTU-8, we performed Southern blot hybridization (11) with the cloned 2.7-kb EcoRI-SalI fragment as a probe (Fig. 3). The probe showed strong hybridization with the EcoRI-SalI fragment obtained from plasmid pCR50, which was used a positive control (lane 1). An identical 2.7-kb band was also visible in the genomic EcoRI-SalI digest (lane 2), and no cross hybridization with E. coli DNA was detected (lanes 3 and 4). The cloned gene product was examined by using the E. coli minicell system, which permits specific labeling of plasmid-encoded proteins. The minicell-producing strain E. coli P678-54 (F- thr leu thi supE lacYfhuA gal mal xyl mtl) was transformed with plasmid pCR50 or pCR5006, and minicells were isolated, labeled with [35S]methionine, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Lejeune et al. (8). A unique 50-kDa polypeptide was expressed in the strain carrying pCR50 or pCR5006 (Fig. 4, lanes 2 and 4). The inability to produce the 50-kDa polypeptide was observed in the strain
1 kb
Creatinase Vector Activity
H~~
E
Uuu *
.
.
..
o
v
U.
pCRSO
_e0 cn
I
(Bam HI)
(-)
pUC18
(+)
pBR322
(+)
pUC18
(-)
pUC18
(+)
pUC18
(+)
pUC18
(+)
pUC19
Sac I
I~~~~~~
Sac I m
pCR5002
-m
EcoR I pCR5003 1+ EcoR I
pBR322
cn
Sac I
pCR5004 I -
(+)
0.4 .,,,
a4
pCR5001
EcoR I
I
Sma I I
EcoR I
Pst I
pCR5005 I Sal I
EcoR I
pCR5006 EcoR I
pCR5007
)
I
Sal I
I~~~~~
FIG. 2. Subclones of pCR50 containing the cloned creatinase
gene.
Creatinase activity was determined
on
the creatinase test plates.
VOL. 58, 1992
NOTES
A A2 bp
3
, BM
1 2 3 4
23130
6557-
023
1
2
3439
3 4
;
-
*
ZE_1
FIG. 3. Southern hybridization analysis with the 2.7-kb EcoRISall fragment as a probe against complete EcoRI and Sall digests of genomic DNA from P. putida and E. coli. (A) Photograph of a 0.8% agarose gel stained with ethidium bromide. (B) Autoradiogram of DNA transferred to nitrocellulose and hybridized with the 32p_ labeled EcoRI-Sall fragment. Lanes: M, molecular size markers (lambda-HindIII digest); 1, 0.1 ,ug of EcoRI-SalI fragment from pCR50; 2 to 4, EcoRI and SalI digests of P. putida, E. coli JA221, and E. coli JM109 DNA, respectively. Each genomic digest contained 3 ,ug of DNA.
containing pUC18 (lane 3). We conclude that pCR50 or pCR5006 confers the creatinase phenotype upon E. coli by specifying the synthesis of 50-kDa creatinase protein. The effects of creatinine or creatine on creatinase production were investigated in basal media of P. putida NTU-8 and E. coli JA221 (pCR50). For P. putida, creatinase production occurred only in the medium containing creatinine or creatine. However, the creatinase activity of E. coli JA221 (pCR50) was 4 x 10-2 U/ml of culture at the early stationary phase (measured by the method of Koyama et al. [7]; 1 U of creatinase was defined as the amount of enzyme which liberates 1 ,umol of urea) in LB medium containing 50 jig of ampicillin per ml in the absence of creatinine or creatine. The production of creatinase activity was shown to be constitutive in E. coli JA221 (pCR50) but creatine inducible in the parent organism, P. putida. This result suggests that a regulatory region which is functional in P. putida is not recognized in the E. coli transformant. Molecular analysis of the cloned 2.7-kb fragment will enable us to define and characterize the P. putida creatinase and its regulatory function. Cloning in E. coli of the creatinase enzyme gene from P. putida and from Flavobacterium sp. strain U-188 has been reported by Hoeffken et al. (3) and Koyama et al. (7), respectively. In the former study, positive clones containing the P. putida creatinase gene were screened by using an immunochemical method, while in the latter, the Flavobacterium creatinase gene was cloned by testing whether each transformant expresses the creatinase activity after being culturing in LB medium as mentioned above. To supplement these two cloning strategies, we report the cloning of the creatinase gene of P. putida NTU-8 by using a conventional plate assay. The results indicate that this screening plate
FIG. 4. Minicell labeling of plasmid-encoded proteins. Fluorography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysates of [35S]methionine-labeled proteins expressed from E. coli P678-54 transformed with plasmids pCR50 (lane 2), pUC18 (lane 3), and pCR5006 (lane 4). Molecular mass markers were as follows (lane 1, from top to bottom): phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; a-lactalbumin, 14.4 kDa. The arrow indicates the creatinase.
assay is a rapid, simple, and unequivocal method for screening large numbers of colonies for the detection of creatinasepositive clones in E. coli. It is possible that a similar methodology may be applicable to screen some other enzyme-positive clones which could be detected directly from plating by combining with a suitable indicator strain. REFERENCES 1. Appleyard, G., and D. D. Woods. 1956. The pathway of creatine catabolism by Pseudomonas ovalis. J. Gen. Microbiol. 14:351356. 2. Beggs, J. D. 1978. Transformation of yeast by a replicating hybrid plasmid. Nature (London) 275:104-108. 3. Hoeffken, H. W., S. H. Knof, P. A. Bartlett, R. Huber, H. Moellering, and G. Schumacher. 1988. Crystal structure determination, refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida. J. Mol. Biol. 204:417-433. 4. Kaplan, A., and D. Naugler. 1974. Creatinine hydrolase and creatine amidinohydrolase. I. Presence in cell-free extracts of Arthrobacter ureafaciens. Mol. Cell. Biochem. 3:9-15. 5. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., W. M. Janda, H. M. Sommers, and W. C. Winn, Jr. 1988. Color atlas and textbook of diagnostic microbiology, 3rd ed. Lippincott, Phila-
delphia. 6. Kopper, P. H., and H. H. Beard. 1947. Creatinase activity of a strain of Pseudomonas. Arch. Biochem. 15:195-199. 7. Koyama, Y., S. Kitao, H. Yamamoto-Otake, M. Suzuki, and E. Nakano. 1990. Cloning and expression of the creatinase gene from Flavobacterium sp. U-188 in Escherichia coli. Agric. Biol. Chem. 54:1453-1457. 8. Lejeune, A., V. Dartois, and C. Colson. 1988. Characterization and expression in Escherichia coli of an endoglucanase gene of Pseudomonas fluorescens subsp. cellulosa. Biochim. Biophys. Acta 950:204-214. 9. Liu, W. H., J. Y. Ho, and L. Y. Jang. 1986. Production of a clinical analysis enzyme, creatininase, by Pseudomonas putida NTU-8. J. Chin. Agric. Chem. Soc. 24:184-191. 10. Liu, W. H., J. Y. Ho, and S. T. Shu. 1986. Isolation and identification of a creatininase-producing bacterium. J. Chin. Agric. Chem. Soc. 24:175-183. 11. Maniatis, T., E. F. Fritsch, and J. Sambroolk 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 12. Marmur, J. 1961. A procedure for the isolation of deoxyribo-
3440
NOTES
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