JOURNAL OF BACrERIOLOGY, Nov. 1992, p. 7217-7220 0021-9193/92/227217-04$02.00/0 Copyright X) 1992, American Society for Microbiology
Vol. 174, No. 22
Multiple Forms of Bile Salt Hydrolase from Lactobacillus sp. Strain 100-100 SCOTT G. LUNDEENt AND DWAYNE C. SAVAGE* Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996 Received 20 April 1992/Accepted 5 September 1992
Four isozymes of bile salt hydrolase (BSH) have been purified from the cytosol of cells of LactobaciUus sp. strain 100-100. The four proteins were designated BSH A, B, C, and D. They eluted from anion-exchange high-pressure liquid chromatography columns at 0.15, 0.18, 0.21, and 0.25 M NaCl, respectively. They are catalyticaly similar, except that the V__ of BSH D is about 10-fold lower than those of the other three isozymes. All four proteins consist of one or two polypeptides. The peptides have molecular weights of 42,000 and 38,000 and are designated a and 1, respectively. The approximate native molecular weights of BSH A, B, C, and D are 115,000, 105,000, 95,000, and 80,000, respectively. The native proteins are probably trimers; the four isozymes are the array of possible subunit combinations a3, ad2h1 alP2, and 133 for A, B, C, and D, respectively. The two subunits are antigenically distinct. Polyclonal antibodies raised against BSH A (all a peptide) react in Western blots (immunoblots) only with proteins containing the a peptide; such antibodies raised against BSH D (all 13 peptide) react only with proteins containing the I% peptide. The amino acid compositions of the two peptides differ. This is the first report of a bacterium that makes four BSH isozymes.
Bile acids are synthesized from cholesterol in the liver, conjugated to either glycine or taurine, and then released into the intestines, where they facilitate fat absorption by the epithelial cells (7). These acids are deconjugated, dehydroxylated, dehydrogenated, and desulfated in the intestines by microbial enzymes (8). The capacity to deconjugate bile acids (hydrolyze the amide bond between the steroid nucleus and amino acid) is widespread among members of the autochthonous gastrointestinal microflora (3, 6, 14). The reaction is catalyzed by the enzyme bile salt hydrolase
(BSH) (6, 14).
BSH has been purified from three organisms, Bacteroides fragilis (17), Clostridium perfringens (5, 15), and Lactobacillus sp. strain 100-100 (12). The BSHs of these three organisms differ in many respects: native and subunit molecular weights, subunit composition, kinetic properties, pH optima, and regulation of the enzymatic activity (5, 10, 12, 17). B. fragilis and C. perfringens each produce one enzyme with the activity. By contrast, two forms of this hydrolase with apparent native molecular weights of about 115,000 and 105,000 have been purified from Lactobacillus sp. strain 100-100 (12). One subunit, with an apparent molecular weight (Mr) of 42,000, is common to both (12). The two isozymes of BSH are very similar with respect to substrate specificities, kinetic properties, and pH optima (12). We now report findings from further work with the BSHs of strain 100-100. This work has revealed that, including the two previously reported (12), the organism produces four proteins with BSH activity. The four isozymes are trimers consisting of two different subunits that associate in four possible combinations.
Detroit, Mich.) with 15% glycerol. Cultures of strain 100-100 were grown overnight in MRS broth with an atmosphere of 90% N2 and 10% CO2. BSH assay. BSH activity was determined by the previously described radiochemical assay (4). Purification of BSH. Enzymes with BSH activity were purified from the cytosol of cells of strain 100-100 grown overnight in 4 liters of MRS broth by a previously outlined procedure (12), with one change. The proteins were eluted from the DEAE-high-pressure liquid chromatography (HPLC) column with a 0.125 to 0.30 M NaCl gradient over 125 min with a flow rate of 0.8 ml/min. Protein concentrations were determined by the Bradford assay (2). Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), with gels of 10% acrylamide, was used to analyze the proteins (11). Nondenaturing polyacrylamide gels of 7.5% acrylamide with buffers free of SDS and 2-mercaptoethanol were used to separate proteins in their native conformations. The polyacrylamide gels were stained with silver by the method of Oakley et al. (16). Densitometry scans of the gels were carried out with an LKB laser scanning densitometer (Pharmacia LKB, Piscataway, N.J.). Polyclonal antibodies against BSH A and BSH D. Specific polyclonal antibodies against purified BSH A and BSH D were raised in adult male New Zealand White rabbits (Myrtle Rabbitry, Thompson Station, Tenn.). Preimmune serum was obtained from the rabbits before immunization with bleeding from the ear artery (1). The rabbits were immunized with 100 pg of either BSH A or BSH D emulsified in Freund's complete adjuvant (Sigma Chemical Co., St. Louis, Mo.). Each animal received an intramuscular injection in a hind leg. Booster immunizations, with approximately 75 ,g of the appropriate enzyme emulsified in Freund's incomplete adjuvant (Sigma), were given 6 weeks later. The immune serum was obtained by bleeding the rabbits from the ear artery 10 to 14 days after the boosters. Both preimmune and immune sera were prepared from 30 to
MATERIALS AND METHODS Strains and storage. Lactobacillus sp. strain 100-100 was maintained at -80°C in MRS broth (Difco Laboratories,
40 ml of whole blood as follows. The blood was incubated for 2 to 3 h at room temperature and then overnight at 4°C. The clots were removed, and the remaining serum was centri-
Corresponding author. t Present address: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706. *
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fuged at 3,800 x g to remove any cells. The sera were aliquoted and stored at -80°C. Western blot (immunoblot) analysis. SDS-PAGE and nondenaturing PAGE were used to separate proteins that were then electroblotted (19) to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, Mass.). The proteins were transferred from SDS-polyacrylamide gels for at least 6 h at 30 V with EB buffer (192 mM glycine, 25 mM Tris HCl [pH 8.3], 20% methanol). Proteins separated by nondenaturing PAGE were transferred for 6 h at 60 V with EB buffer that was free of methanol. After the transfer, the membranes were soaked in phosphate-buffered saline (PBS) with 3% gelatin for at least 4 h at room temperature and then washed three times in PBS (pH 7.0). The antisera against purified BSH A or BSH D were diluted in PBS with 1% bovine serum albumin (BSA) (fraction V; Sigma). The PVDF membranes, with the blotted proteins, were soaked in these solutions of dilute antisera for at least 4 h at 4°C and then washed again three times in PBS. The secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Sigma), was diluted 1/5,000 in PBS with 1% BSA. The membranes were soaked in this solution for 4 h at 4°C and again washed three times in PBS. The horseradish peroxidase-conjugated antibodies, bound to the rabbit immunoglobulin G, were located on the membranes with 3,3-diaminobenzidine (DAB; Sigma). The membranes were soaked in color development solution (0.1 M citrate, 0.1 M ammonium acetate [pH 5.0], 0.7 ml of p-cresol, 10 mg of DAB, 66 ,ul of 30% H202) for 30 min. Color development was stopped by rinsing the membranes in distilled water. pH optima and kinetic analysis. The pH optima and kinetic properties of the four hydrolases were determined as previously described (12). Amino acid composition analysis and peptide sequencing. The amino acid composition and the N-terminal amino acid sequence were determined from proteins blotted onto PVDF membranes. SDS-PAGE was used to separate the peptides, which were then transferred to the membranes and stained with either Coomassie brilliant blue (0.1% in methanol) or amido black (0.1% in water). The appropriate protein bands were removed with a razor blade. The polypeptides analyzed for amino acid composition were hydrolyzed with 6 N HCI (vapor phase) and derivatized with phenyl-isothiocyanate. These derivatized amino acids were separated by HPLC with a Waters PICO-TAG reversephase column. Microsequencing of proteins blotted on PVDF membranes was done as previously described (13). The amino-terminal residues were sequenced with an Applied Biosystems model 477A pulsed-liquid-phase protein sequencer. RESULTS
Purification of four isozymes of BSH. Four proteins with hydrolase activity were purified from the cytosol of cells of strain 100-100. The salt gradient used to elute the proteins from the anion-exchange HPLC column was extended from that previously used (0.125 to 0.30 M NaCl instead of 0.1 to 0.2 M) (12). The time for the gradient was also increased from 75 to 125 min. Neither the slope of the gradient nor the resolution of the proteins eluting from the column was altered by these changes. Hydrolase activity eluted in four sets of fractions. The proteins in these fractions were designated BSH A, BSH B, BSH C, and BSH D. BSH A and B eluted at 0.15 and 0.18 M NaCl, as previously described (12).
B.
A.
1 2 3 4 5
1 2 3 4 5 97,400
-
67,000
-
43.000
-
29,000
-
232.000
154.000
.4- a
67.000
FIG. 1. Electrophoretic analysis of purified BSH A, B, C, and D from Lactobacillus sp. strain 100-100 separated in 7.5% nondenaturing polyacrylamide gels (A) and SDS-10% polyacrylamide gels (B). Both gels are stained with silver (16). Lanes (same order in both gels): 1, molecular weight standards; 2, BSH A; 3, BSH B; 4, BSH C; 5, BSH D.
The newly discovered BSH C and BSH D eluted at 0.21 and 0.25 M NaCl, respectively. The proteins in the fractions migrated to positions in nondenaturing PAGE gels indicating Mrs of approximately 115,000, 105,000, 95,000, and 80,000 for BSH A, B, C, and D, respectively (Fig. 1A). When the same fractions were run in SDS-PAGE, the proteins were all found to contain at least one of two polypeptides. These peptides, designated a and 1, had Mrs of 42,000 and 38,000, respectively (Fig. 1B). BSH A had only the a peptide, BSH B and C had both, and BSH D had only the 1 peptide (Fig. 1B). When the peptides in these gels were analyzed with a scanning densitometer, the ratios of a to ,1 in BSH A, B, C, and D were 1:0, 2:1, 1:2, and 0:1, respectively. The other peptides in the gels varied in amount in each purification and are believed not to be subunits of the hydrolases but rather to be proteins that copurified with the hydrolases. Western blot analysis of the four BSH isozymes. The four purified hydrolase isozymes were examined in Western blots developed with specific polyclonal antibodies, raised in New Zealand White rabbits, against BSH A (all a subunit) and BSH D (all 13 subunit). Preimmune serum from these rabbits had high titers of anti-a-peptide antibodies but no detectable anti-13-peptide antibodies. The anti-BSH A immune serum was diluted 1/7,500 and 1/40,000 for Western blots of proteins separated by nondenaturing PAGE and SDS-PAGE, respectively. Western blots of the purified hydrolases separated by nondenaturing PAGE, along with freshly prepared crude cell extracts of strain 100-100, showed that only those proteins with the a peptide (BSH A, B, and C) reacted with these antibodies (Fig. 2A); the antibodies did not cross-react with BSH D (Fig. 2A). a peptides in BSH A, B, and C strongly reacted with the anti-BSH A antibodies in Western blots of the hydrolases separated by SDS-PAGE (Fig. 2B); the antibodies only weakly cross-reacted with the 1 peptide in BSH B, C, and D (Fig. 2B). The anti-BSH D immune serum was diluted 1/2,000 and
BSH ISOZYMES IN LACTOBACILLUS SP. STRAIN 100-100
VOL. 174, 1992
A.
B. 1
2
3 4 5
1
2
3 4
TABLE 1. Amino acid compositions of the a and 1 peptides of the BSHs from Lactobacilus sp. strain 100-100 % of total amino acid composition in: from a peptide BSH:
Amino acid(s)
Asp + Asn Glu + Gln Ser Gly His Arg Thr
~ ~~~~~~~~ "
C
Ala Pro
Tyr Val Met Ile Leu Phe Lys
FIG. 2. Western blot analysis of purified BSH A, B, C, and D from LactobaciUlus sp. strain 100-100, separated in 7.5% nondenaturing polyacrylamide gels (A) and SDS-10% polyacrylamide gels (B). The proteins were probed with anti-BSH A polyclonal antibodies. (A) Lanes: 1, crude cell extract; 2, BSH A; 3, BSH B; 4, BSH C; 5, BSH D. Arrows indicate the positions of the hydrolases that react with the antibodies. (B) Lanes: 1, BSH A; 2, BSH B; 3, BSH C; 4, BSH D.
1/1,000 for Western blots of proteins separated by nondenaturing PAGE and SDS-PAGE, respectively. These antibodies reacted only with native proteins having the i peptide (BSH B, C, and D) (Fig. 3A); they did not react with the native BSH A. They reacted with both the a and 1 peptides, however, in blots of the hydrolases separated by SDS-PAGE (Fig. 3B). This result was not unexpected, since the preim-
B.
A.
1 2 3 4
1 2 3 4
7219
1 peptide BSH: from
A
B
B
D
17.38 11.41 4.71 8.56 3.42 2.04 5.13 5.51 7.32 5.25 7.05 0.00 5.05 10.22 3.97 2.97
17.41 11.65 4.67 8.34 3.40 2.23 5.00 5.43 7.10 5.29 6.70 0.00 5.18 10.26 4.08 3.28
8.98 7.63 7.53 11.83 1.63 2.77 5.52 8.47 7.05 4.87 6.09 0.86 6.13 8.45 4.09 7.10
8.74 8.41 7.38 9.36 1.91 3.09 7.22 7.53 8.42 3.84 7.61 1.22 6.26 9.54 4.61 3.94
a Cys and Trp cannot be
quantitated after acid hydrolysis.
mune serum had high titers of anti-a-peptide antibodies and the anti-1-peptide immune serum was not sufficiently diluted to eliminate these antibodies. Unlike the preimmune serum, however, the anti-BSH D immune serum had antibodies that reacted with the 13 peptide (Fig. 3B). pH optima and kinetic analysis of BSHs. The pH optima of the four BSHs were approximately the same, between 4.2 and 4.5. The Vm,s for BSH A, B, C, and D were 17, 53, 24, and 2.4 ,umol of cholic acid formed per min per mg of protein, respectively. Their Kms were 0.76, 0.95, 0.45, and 0.37 mM, respectively, for taurocholic acid. Amino acid composition of the a and 13 peptides. The amino acid compositions of the a and 1 peptides were determined from proteins blotted onto PVDF membranes (Table 1). The a peptides from BSH A and B were similar to one another. Interestingly, these peptides had no methionine residues. The 1 peptides from BSH B and D were also similar to each other. However, the amino acid composition of the a peptide differed from that of the 1 peptide. N-terminal amino acid sequence of the a peptide of BSH A. The sequence of the first 25 amino acids from the amino terminus of the a peptide of BSH A blotted on PVDF membranes was determined. The sequence was Gly-Thr-
Ser-Ile-Val-Tyr-Ser-Ser-Asn-Asn-His-His-Tyr-Phe-Gly-Arg-
Asn-Leu-Asp-Leu-Gln-Ile-Ser-Phe-Gly. DISCUSSION 4--
B
a
4-
4-- D
FIG. 3. Western blot analysis of purified BSH from Lactobacillus sp. strain 100-100, separated in 7.5% nondenaturing polyacrylamide gels (A) and SDS-10% polyacrylamide gels (B). Proteins were incubated with anti-BSH D polyclonal antibodies. Lanes (same in both blots): 1, BSH A; 2, BSH B; 3, BSH C; 4, BSH D.
We previously demonstrated that two proteins with BSH activity, BSH A and B, could be purified from cell extracts of Lactobacillus sp. strain 100-100 (12). Soon after publishing those findings, we discovered that three proteins reacted with anti-BSH A polyclonal antibodies in Western blots of nondenaturing polyacrylamide gels of proteins in crude cell extracts from the strain. Two of the proteins corresponded to BSH A and B. The third had an Mr lower than those of both BSH A and B and did not correspond to any known hydrolase of the strain. Four proteins with hydrolase activity were ultimately purified from strain 100-100. The four proteins were designated BSH A, B, C, and D. They were
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purified from the soluble portion of the cell extract. Little BSH activity could be detected in the spent medium from cultures and membrane fractions from the organism. When nondenaturing PAGE was used to analyze the proteins, each migrated as a single band. Hydrolase A had the highest Mr, followed by BSH B, C, and D. When SDS-PAGE was used to separate the peptides of the hydrolases, the proteins all had at least one of two peptides, designated a and P. Hydrolase A had only the a peptide; hydrolases B and C had both; hydrolase D had only the peptide. The apparent molecular weights of the native proteins and their peptide subunits, along with the densitometry data, suggest that the hydrolases are trimers consisting of the a and 13 peptides. The isozymes are the four possible combinations of these two peptides (a3, a2P13, a"12, and 13). Isozymes have also been described for another enzyme involved in bile acid transformations. Two isozymes of the 7a-hydroxysteroid dehydrogenase have been found in several strains of B. fragilis (9). These isozymes have different cofactor requirements. One is NAD dependent; the other is NADP dependent. The a and P peptides of the BSHs from strain 100-100 were not antigenically related. Moreover, analysis of the amino acid compositions of the two peptides provided evidence that they are not structurally related. We did not expect that their amino acid compositions would be the same, since they have different molecular weights. However, the peptide, but not the a peptide, has methionine residues. Moreover, other amino acids were present in different concentrations in the two peptides. Finally, the amino-terminal sequence of the a peptide of the BSHs from strain 100-100 has no sequence similarities to that of the hydrolase from C. perfringens (5). This finding supports the evidence that hydrolases from these two strains are antigenically distinct (12). This is the first report that any bacterium makes four BSH isozymes. Three of the four hydrolases, BSH A, B, and C, have similar catalytic properties. They differ only in native molecular weight, subunit composition, and behavior in anion-exchange chromatography. Each of these three enzymes has the a subunit. BSH D, however, had very low enzymatic activity and only had the peptide. This finding suggests that the a peptide is the main catalytic subunit in these enzymes or that conformational changes occur when the a and peptides interact. Such changes could increase the activity of one or both peptides. Lactobacilli have been shown to be the predominant producers of BSH activity in the mouse gut (18). Whether other lactobacilli synthesize BSH isozymes is unknown. In fact, most studies of their hydrolase activity have been performed with whole cells. Therefore, an understanding of the structural, enzymatic, and regulatory properties of the hydrolases from lactobacilli will be important to the understanding of the role of these organisms in bile acid transformations in the gastrointestinal tract. REFERENCES 1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. Wiley Interscience, New York.
2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248254. 3. Dickinson, A. B., B. E. Gustafsson, and A. Norman. 1971. Determination of bile acid conversion potencies of intestinal bacteria by screening in vitro and subsequent establishment in germ free rats. Acta Pathol. Microbiol. Scand. Sect. B 79:691698. 4. Feighner, S. D., and M. P. Dashkevicz. 1987. Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency, and bacterial cholyltaurine hydrolase activity. Appl. Environ. Microbiol. 53:331-336. 5. Gopal-Srivastava, R., and P. B. Hylemon. 1988. Purification and characterization of bile salt hydrolase from Clostridium perfringens. J. Lipid Res. 29:1079-1085. 6. Hayakawa, S. 1973. Microbial transformation of bile acids. Adv. Lipid Res. 11:143-192. 7. Hofinann, A. F., and H. S. Mehjian. 1973. Bile acids and the intestinal absorption of fat and electrolytes in health and disease, p. 103-152. In P. P. Nair and D. Kritchevsky (ed.), The bile acids: chemistry, physiology, and metabolism, vol. II. Physiology and metabolism. Plenum Press, New York. 8. Hylemon, P. B., and T. J. Glass. 1983. Biotransformation of bile acids and cholesterol by the intestinal microflora, p. 189-213. In D. J. Hentges (ed.), Human intestinal microflora in health and disease. Academic Press, Inc., New York. 9. Hylemon, P. B., and J. A. Sherrod. 1975. Multiple forms of 7-a-hydroxysteroid dehydrogenase in selected strains of Bacteroides fragilis. J. Bacteriol. 122:418-424. 10. Hylemon, P. B., and E. J. Stellwag. 1976. Bile acid biotransformation rates of selected gram-positive and gram-negative intestinal anaerobic bacteria. Biochem. Biophys. Res. Commun. 69:1088-1094. 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
227:680-685. 12. Lundeen, S. G., and D. C. Savage. 1990. Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100. J. Bacteriol. 172:4171-4177. 13. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035-10038. 14. Midvedt, T. 1974. Microbial bile acid transformations. Am. J. Clin. Nutr. 27:1341-1347. 15. Nair, P. P., M. Gordon, and J. Reback. 1967. The enzymatic cleavage of the carbon nitrogen bond in 3a,7a,12a-tri-hydroxy5p-cholan-24-oylglycine. J. Biol. Chem. 242:7-11. 16. Oakley, B. R., D. R. Kirsch, and N. R. Morris. 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105:361-363. 17. Stellwag, E. J., and P. B. Hylemon. 1976. Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis. Biochim. Biophys. Acta 452:165-176. 18. Tannock, G. W., M. P. Dashkevicz, and S. D. Feighner. 1989. Lactobacilli and bile salt hydrolase in the murine intestinal tract. Appl. Environ. Microbiol. 55:1848-1851. 19. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.
Vol. 174, No. 22
Multiple Forms of Bile Salt Hydrolase from Lactobacillus sp. Strain 100-100 SCOTT G. LUNDEENt AND DWAYNE C. SAVAGE* Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996 Received 20 April 1992/Accepted 5 September 1992
Four isozymes of bile salt hydrolase (BSH) have been purified from the cytosol of cells of LactobaciUus sp. strain 100-100. The four proteins were designated BSH A, B, C, and D. They eluted from anion-exchange high-pressure liquid chromatography columns at 0.15, 0.18, 0.21, and 0.25 M NaCl, respectively. They are catalyticaly similar, except that the V__ of BSH D is about 10-fold lower than those of the other three isozymes. All four proteins consist of one or two polypeptides. The peptides have molecular weights of 42,000 and 38,000 and are designated a and 1, respectively. The approximate native molecular weights of BSH A, B, C, and D are 115,000, 105,000, 95,000, and 80,000, respectively. The native proteins are probably trimers; the four isozymes are the array of possible subunit combinations a3, ad2h1 alP2, and 133 for A, B, C, and D, respectively. The two subunits are antigenically distinct. Polyclonal antibodies raised against BSH A (all a peptide) react in Western blots (immunoblots) only with proteins containing the a peptide; such antibodies raised against BSH D (all 13 peptide) react only with proteins containing the I% peptide. The amino acid compositions of the two peptides differ. This is the first report of a bacterium that makes four BSH isozymes.
Bile acids are synthesized from cholesterol in the liver, conjugated to either glycine or taurine, and then released into the intestines, where they facilitate fat absorption by the epithelial cells (7). These acids are deconjugated, dehydroxylated, dehydrogenated, and desulfated in the intestines by microbial enzymes (8). The capacity to deconjugate bile acids (hydrolyze the amide bond between the steroid nucleus and amino acid) is widespread among members of the autochthonous gastrointestinal microflora (3, 6, 14). The reaction is catalyzed by the enzyme bile salt hydrolase
(BSH) (6, 14).
BSH has been purified from three organisms, Bacteroides fragilis (17), Clostridium perfringens (5, 15), and Lactobacillus sp. strain 100-100 (12). The BSHs of these three organisms differ in many respects: native and subunit molecular weights, subunit composition, kinetic properties, pH optima, and regulation of the enzymatic activity (5, 10, 12, 17). B. fragilis and C. perfringens each produce one enzyme with the activity. By contrast, two forms of this hydrolase with apparent native molecular weights of about 115,000 and 105,000 have been purified from Lactobacillus sp. strain 100-100 (12). One subunit, with an apparent molecular weight (Mr) of 42,000, is common to both (12). The two isozymes of BSH are very similar with respect to substrate specificities, kinetic properties, and pH optima (12). We now report findings from further work with the BSHs of strain 100-100. This work has revealed that, including the two previously reported (12), the organism produces four proteins with BSH activity. The four isozymes are trimers consisting of two different subunits that associate in four possible combinations.
Detroit, Mich.) with 15% glycerol. Cultures of strain 100-100 were grown overnight in MRS broth with an atmosphere of 90% N2 and 10% CO2. BSH assay. BSH activity was determined by the previously described radiochemical assay (4). Purification of BSH. Enzymes with BSH activity were purified from the cytosol of cells of strain 100-100 grown overnight in 4 liters of MRS broth by a previously outlined procedure (12), with one change. The proteins were eluted from the DEAE-high-pressure liquid chromatography (HPLC) column with a 0.125 to 0.30 M NaCl gradient over 125 min with a flow rate of 0.8 ml/min. Protein concentrations were determined by the Bradford assay (2). Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), with gels of 10% acrylamide, was used to analyze the proteins (11). Nondenaturing polyacrylamide gels of 7.5% acrylamide with buffers free of SDS and 2-mercaptoethanol were used to separate proteins in their native conformations. The polyacrylamide gels were stained with silver by the method of Oakley et al. (16). Densitometry scans of the gels were carried out with an LKB laser scanning densitometer (Pharmacia LKB, Piscataway, N.J.). Polyclonal antibodies against BSH A and BSH D. Specific polyclonal antibodies against purified BSH A and BSH D were raised in adult male New Zealand White rabbits (Myrtle Rabbitry, Thompson Station, Tenn.). Preimmune serum was obtained from the rabbits before immunization with bleeding from the ear artery (1). The rabbits were immunized with 100 pg of either BSH A or BSH D emulsified in Freund's complete adjuvant (Sigma Chemical Co., St. Louis, Mo.). Each animal received an intramuscular injection in a hind leg. Booster immunizations, with approximately 75 ,g of the appropriate enzyme emulsified in Freund's incomplete adjuvant (Sigma), were given 6 weeks later. The immune serum was obtained by bleeding the rabbits from the ear artery 10 to 14 days after the boosters. Both preimmune and immune sera were prepared from 30 to
MATERIALS AND METHODS Strains and storage. Lactobacillus sp. strain 100-100 was maintained at -80°C in MRS broth (Difco Laboratories,
40 ml of whole blood as follows. The blood was incubated for 2 to 3 h at room temperature and then overnight at 4°C. The clots were removed, and the remaining serum was centri-
Corresponding author. t Present address: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706. *
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fuged at 3,800 x g to remove any cells. The sera were aliquoted and stored at -80°C. Western blot (immunoblot) analysis. SDS-PAGE and nondenaturing PAGE were used to separate proteins that were then electroblotted (19) to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, Mass.). The proteins were transferred from SDS-polyacrylamide gels for at least 6 h at 30 V with EB buffer (192 mM glycine, 25 mM Tris HCl [pH 8.3], 20% methanol). Proteins separated by nondenaturing PAGE were transferred for 6 h at 60 V with EB buffer that was free of methanol. After the transfer, the membranes were soaked in phosphate-buffered saline (PBS) with 3% gelatin for at least 4 h at room temperature and then washed three times in PBS (pH 7.0). The antisera against purified BSH A or BSH D were diluted in PBS with 1% bovine serum albumin (BSA) (fraction V; Sigma). The PVDF membranes, with the blotted proteins, were soaked in these solutions of dilute antisera for at least 4 h at 4°C and then washed again three times in PBS. The secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Sigma), was diluted 1/5,000 in PBS with 1% BSA. The membranes were soaked in this solution for 4 h at 4°C and again washed three times in PBS. The horseradish peroxidase-conjugated antibodies, bound to the rabbit immunoglobulin G, were located on the membranes with 3,3-diaminobenzidine (DAB; Sigma). The membranes were soaked in color development solution (0.1 M citrate, 0.1 M ammonium acetate [pH 5.0], 0.7 ml of p-cresol, 10 mg of DAB, 66 ,ul of 30% H202) for 30 min. Color development was stopped by rinsing the membranes in distilled water. pH optima and kinetic analysis. The pH optima and kinetic properties of the four hydrolases were determined as previously described (12). Amino acid composition analysis and peptide sequencing. The amino acid composition and the N-terminal amino acid sequence were determined from proteins blotted onto PVDF membranes. SDS-PAGE was used to separate the peptides, which were then transferred to the membranes and stained with either Coomassie brilliant blue (0.1% in methanol) or amido black (0.1% in water). The appropriate protein bands were removed with a razor blade. The polypeptides analyzed for amino acid composition were hydrolyzed with 6 N HCI (vapor phase) and derivatized with phenyl-isothiocyanate. These derivatized amino acids were separated by HPLC with a Waters PICO-TAG reversephase column. Microsequencing of proteins blotted on PVDF membranes was done as previously described (13). The amino-terminal residues were sequenced with an Applied Biosystems model 477A pulsed-liquid-phase protein sequencer. RESULTS
Purification of four isozymes of BSH. Four proteins with hydrolase activity were purified from the cytosol of cells of strain 100-100. The salt gradient used to elute the proteins from the anion-exchange HPLC column was extended from that previously used (0.125 to 0.30 M NaCl instead of 0.1 to 0.2 M) (12). The time for the gradient was also increased from 75 to 125 min. Neither the slope of the gradient nor the resolution of the proteins eluting from the column was altered by these changes. Hydrolase activity eluted in four sets of fractions. The proteins in these fractions were designated BSH A, BSH B, BSH C, and BSH D. BSH A and B eluted at 0.15 and 0.18 M NaCl, as previously described (12).
B.
A.
1 2 3 4 5
1 2 3 4 5 97,400
-
67,000
-
43.000
-
29,000
-
232.000
154.000
.4- a
67.000
FIG. 1. Electrophoretic analysis of purified BSH A, B, C, and D from Lactobacillus sp. strain 100-100 separated in 7.5% nondenaturing polyacrylamide gels (A) and SDS-10% polyacrylamide gels (B). Both gels are stained with silver (16). Lanes (same order in both gels): 1, molecular weight standards; 2, BSH A; 3, BSH B; 4, BSH C; 5, BSH D.
The newly discovered BSH C and BSH D eluted at 0.21 and 0.25 M NaCl, respectively. The proteins in the fractions migrated to positions in nondenaturing PAGE gels indicating Mrs of approximately 115,000, 105,000, 95,000, and 80,000 for BSH A, B, C, and D, respectively (Fig. 1A). When the same fractions were run in SDS-PAGE, the proteins were all found to contain at least one of two polypeptides. These peptides, designated a and 1, had Mrs of 42,000 and 38,000, respectively (Fig. 1B). BSH A had only the a peptide, BSH B and C had both, and BSH D had only the 1 peptide (Fig. 1B). When the peptides in these gels were analyzed with a scanning densitometer, the ratios of a to ,1 in BSH A, B, C, and D were 1:0, 2:1, 1:2, and 0:1, respectively. The other peptides in the gels varied in amount in each purification and are believed not to be subunits of the hydrolases but rather to be proteins that copurified with the hydrolases. Western blot analysis of the four BSH isozymes. The four purified hydrolase isozymes were examined in Western blots developed with specific polyclonal antibodies, raised in New Zealand White rabbits, against BSH A (all a subunit) and BSH D (all 13 subunit). Preimmune serum from these rabbits had high titers of anti-a-peptide antibodies but no detectable anti-13-peptide antibodies. The anti-BSH A immune serum was diluted 1/7,500 and 1/40,000 for Western blots of proteins separated by nondenaturing PAGE and SDS-PAGE, respectively. Western blots of the purified hydrolases separated by nondenaturing PAGE, along with freshly prepared crude cell extracts of strain 100-100, showed that only those proteins with the a peptide (BSH A, B, and C) reacted with these antibodies (Fig. 2A); the antibodies did not cross-react with BSH D (Fig. 2A). a peptides in BSH A, B, and C strongly reacted with the anti-BSH A antibodies in Western blots of the hydrolases separated by SDS-PAGE (Fig. 2B); the antibodies only weakly cross-reacted with the 1 peptide in BSH B, C, and D (Fig. 2B). The anti-BSH D immune serum was diluted 1/2,000 and
BSH ISOZYMES IN LACTOBACILLUS SP. STRAIN 100-100
VOL. 174, 1992
A.
B. 1
2
3 4 5
1
2
3 4
TABLE 1. Amino acid compositions of the a and 1 peptides of the BSHs from Lactobacilus sp. strain 100-100 % of total amino acid composition in: from a peptide BSH:
Amino acid(s)
Asp + Asn Glu + Gln Ser Gly His Arg Thr
~ ~~~~~~~~ "
C
Ala Pro
Tyr Val Met Ile Leu Phe Lys
FIG. 2. Western blot analysis of purified BSH A, B, C, and D from LactobaciUlus sp. strain 100-100, separated in 7.5% nondenaturing polyacrylamide gels (A) and SDS-10% polyacrylamide gels (B). The proteins were probed with anti-BSH A polyclonal antibodies. (A) Lanes: 1, crude cell extract; 2, BSH A; 3, BSH B; 4, BSH C; 5, BSH D. Arrows indicate the positions of the hydrolases that react with the antibodies. (B) Lanes: 1, BSH A; 2, BSH B; 3, BSH C; 4, BSH D.
1/1,000 for Western blots of proteins separated by nondenaturing PAGE and SDS-PAGE, respectively. These antibodies reacted only with native proteins having the i peptide (BSH B, C, and D) (Fig. 3A); they did not react with the native BSH A. They reacted with both the a and 1 peptides, however, in blots of the hydrolases separated by SDS-PAGE (Fig. 3B). This result was not unexpected, since the preim-
B.
A.
1 2 3 4
1 2 3 4
7219
1 peptide BSH: from
A
B
B
D
17.38 11.41 4.71 8.56 3.42 2.04 5.13 5.51 7.32 5.25 7.05 0.00 5.05 10.22 3.97 2.97
17.41 11.65 4.67 8.34 3.40 2.23 5.00 5.43 7.10 5.29 6.70 0.00 5.18 10.26 4.08 3.28
8.98 7.63 7.53 11.83 1.63 2.77 5.52 8.47 7.05 4.87 6.09 0.86 6.13 8.45 4.09 7.10
8.74 8.41 7.38 9.36 1.91 3.09 7.22 7.53 8.42 3.84 7.61 1.22 6.26 9.54 4.61 3.94
a Cys and Trp cannot be
quantitated after acid hydrolysis.
mune serum had high titers of anti-a-peptide antibodies and the anti-1-peptide immune serum was not sufficiently diluted to eliminate these antibodies. Unlike the preimmune serum, however, the anti-BSH D immune serum had antibodies that reacted with the 13 peptide (Fig. 3B). pH optima and kinetic analysis of BSHs. The pH optima of the four BSHs were approximately the same, between 4.2 and 4.5. The Vm,s for BSH A, B, C, and D were 17, 53, 24, and 2.4 ,umol of cholic acid formed per min per mg of protein, respectively. Their Kms were 0.76, 0.95, 0.45, and 0.37 mM, respectively, for taurocholic acid. Amino acid composition of the a and 13 peptides. The amino acid compositions of the a and 1 peptides were determined from proteins blotted onto PVDF membranes (Table 1). The a peptides from BSH A and B were similar to one another. Interestingly, these peptides had no methionine residues. The 1 peptides from BSH B and D were also similar to each other. However, the amino acid composition of the a peptide differed from that of the 1 peptide. N-terminal amino acid sequence of the a peptide of BSH A. The sequence of the first 25 amino acids from the amino terminus of the a peptide of BSH A blotted on PVDF membranes was determined. The sequence was Gly-Thr-
Ser-Ile-Val-Tyr-Ser-Ser-Asn-Asn-His-His-Tyr-Phe-Gly-Arg-
Asn-Leu-Asp-Leu-Gln-Ile-Ser-Phe-Gly. DISCUSSION 4--
B
a
4-
4-- D
FIG. 3. Western blot analysis of purified BSH from Lactobacillus sp. strain 100-100, separated in 7.5% nondenaturing polyacrylamide gels (A) and SDS-10% polyacrylamide gels (B). Proteins were incubated with anti-BSH D polyclonal antibodies. Lanes (same in both blots): 1, BSH A; 2, BSH B; 3, BSH C; 4, BSH D.
We previously demonstrated that two proteins with BSH activity, BSH A and B, could be purified from cell extracts of Lactobacillus sp. strain 100-100 (12). Soon after publishing those findings, we discovered that three proteins reacted with anti-BSH A polyclonal antibodies in Western blots of nondenaturing polyacrylamide gels of proteins in crude cell extracts from the strain. Two of the proteins corresponded to BSH A and B. The third had an Mr lower than those of both BSH A and B and did not correspond to any known hydrolase of the strain. Four proteins with hydrolase activity were ultimately purified from strain 100-100. The four proteins were designated BSH A, B, C, and D. They were
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J. BACTIERIOL.
LUNDEEN AND SAVAGE
purified from the soluble portion of the cell extract. Little BSH activity could be detected in the spent medium from cultures and membrane fractions from the organism. When nondenaturing PAGE was used to analyze the proteins, each migrated as a single band. Hydrolase A had the highest Mr, followed by BSH B, C, and D. When SDS-PAGE was used to separate the peptides of the hydrolases, the proteins all had at least one of two peptides, designated a and P. Hydrolase A had only the a peptide; hydrolases B and C had both; hydrolase D had only the peptide. The apparent molecular weights of the native proteins and their peptide subunits, along with the densitometry data, suggest that the hydrolases are trimers consisting of the a and 13 peptides. The isozymes are the four possible combinations of these two peptides (a3, a2P13, a"12, and 13). Isozymes have also been described for another enzyme involved in bile acid transformations. Two isozymes of the 7a-hydroxysteroid dehydrogenase have been found in several strains of B. fragilis (9). These isozymes have different cofactor requirements. One is NAD dependent; the other is NADP dependent. The a and P peptides of the BSHs from strain 100-100 were not antigenically related. Moreover, analysis of the amino acid compositions of the two peptides provided evidence that they are not structurally related. We did not expect that their amino acid compositions would be the same, since they have different molecular weights. However, the peptide, but not the a peptide, has methionine residues. Moreover, other amino acids were present in different concentrations in the two peptides. Finally, the amino-terminal sequence of the a peptide of the BSHs from strain 100-100 has no sequence similarities to that of the hydrolase from C. perfringens (5). This finding supports the evidence that hydrolases from these two strains are antigenically distinct (12). This is the first report that any bacterium makes four BSH isozymes. Three of the four hydrolases, BSH A, B, and C, have similar catalytic properties. They differ only in native molecular weight, subunit composition, and behavior in anion-exchange chromatography. Each of these three enzymes has the a subunit. BSH D, however, had very low enzymatic activity and only had the peptide. This finding suggests that the a peptide is the main catalytic subunit in these enzymes or that conformational changes occur when the a and peptides interact. Such changes could increase the activity of one or both peptides. Lactobacilli have been shown to be the predominant producers of BSH activity in the mouse gut (18). Whether other lactobacilli synthesize BSH isozymes is unknown. In fact, most studies of their hydrolase activity have been performed with whole cells. Therefore, an understanding of the structural, enzymatic, and regulatory properties of the hydrolases from lactobacilli will be important to the understanding of the role of these organisms in bile acid transformations in the gastrointestinal tract. REFERENCES 1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. Wiley Interscience, New York.
2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248254. 3. Dickinson, A. B., B. E. Gustafsson, and A. Norman. 1971. Determination of bile acid conversion potencies of intestinal bacteria by screening in vitro and subsequent establishment in germ free rats. Acta Pathol. Microbiol. Scand. Sect. B 79:691698. 4. Feighner, S. D., and M. P. Dashkevicz. 1987. Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency, and bacterial cholyltaurine hydrolase activity. Appl. Environ. Microbiol. 53:331-336. 5. Gopal-Srivastava, R., and P. B. Hylemon. 1988. Purification and characterization of bile salt hydrolase from Clostridium perfringens. J. Lipid Res. 29:1079-1085. 6. Hayakawa, S. 1973. Microbial transformation of bile acids. Adv. Lipid Res. 11:143-192. 7. Hofinann, A. F., and H. S. Mehjian. 1973. Bile acids and the intestinal absorption of fat and electrolytes in health and disease, p. 103-152. In P. P. Nair and D. Kritchevsky (ed.), The bile acids: chemistry, physiology, and metabolism, vol. II. Physiology and metabolism. Plenum Press, New York. 8. Hylemon, P. B., and T. J. Glass. 1983. Biotransformation of bile acids and cholesterol by the intestinal microflora, p. 189-213. In D. J. Hentges (ed.), Human intestinal microflora in health and disease. Academic Press, Inc., New York. 9. Hylemon, P. B., and J. A. Sherrod. 1975. Multiple forms of 7-a-hydroxysteroid dehydrogenase in selected strains of Bacteroides fragilis. J. Bacteriol. 122:418-424. 10. Hylemon, P. B., and E. J. Stellwag. 1976. Bile acid biotransformation rates of selected gram-positive and gram-negative intestinal anaerobic bacteria. Biochem. Biophys. Res. Commun. 69:1088-1094. 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
227:680-685. 12. Lundeen, S. G., and D. C. Savage. 1990. Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100. J. Bacteriol. 172:4171-4177. 13. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035-10038. 14. Midvedt, T. 1974. Microbial bile acid transformations. Am. J. Clin. Nutr. 27:1341-1347. 15. Nair, P. P., M. Gordon, and J. Reback. 1967. The enzymatic cleavage of the carbon nitrogen bond in 3a,7a,12a-tri-hydroxy5p-cholan-24-oylglycine. J. Biol. Chem. 242:7-11. 16. Oakley, B. R., D. R. Kirsch, and N. R. Morris. 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105:361-363. 17. Stellwag, E. J., and P. B. Hylemon. 1976. Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis. Biochim. Biophys. Acta 452:165-176. 18. Tannock, G. W., M. P. Dashkevicz, and S. D. Feighner. 1989. Lactobacilli and bile salt hydrolase in the murine intestinal tract. Appl. Environ. Microbiol. 55:1848-1851. 19. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.
