FEMSMicrobiologyLetters94 (1992) 121-126 © 1992Federationof European MicrobiologicalSocieties0378-1097/92/$05.00 Publishedby Elsevier
Characterization of an extracellular factor that stimulates bile salt hydrolase activity in Lactobacillus sp. strain 100-100 Scott G. Lundeen ~ and Dwayne C. Savage Department of Microbiology. Universityof Tennessee. Knoxville, Tennessee, USA
Received2 April 1992 Revisionreceived t0 April 1992 Accepted 10April iq92
Key words: Lactobacillus; Bile salt hydrolase
Bile salt hydrolase activity in Lactobaciilus sp. strain 100-100 is strictly intraccUular, The strain produces an ¢xtracellular factor that stimulates the intracclhlar hydrolase activity. The factor is inducible by conjugated bile salts, has an apparent molecular mass over 12 kDa but less than 25 kDa, is stable in air, and resistant to pronase and heat. It is partially extractable into organic solvents and inactivated by a sulhydryl group inhibitor, We postulate that the factor functions by a novel mechanism to facilitate entry of conjugated bile salts into the bacterial cells.
Bile salt hydrolase (BSH), the en~me that dcconjugatcs bile salts, is produced by bacteria of many species in the gastrointestinal tract . The mechanisms regulating the activity have not been studied ;.n detail in any bacterium. Until recently, the only reported factor regulating the enzyme was growth phase in Bacteroides fragilis  and Lactobacillus sp. strain 100-100 [3l. Hydrolase activity increases more than 300- and 70-fold, respectively, in cultures of these organisms when they reach stationary phase. BSH activity, which is strictly intracellular in strain 100-100, is also regulated by conjugated bile salts . The activity detected in whole cells increases as much as six-fold when conjugated, but not free, bile salts are added to a stationary phase culture of the strain. However, the salts do not induce the enzymes or a membrane transport system. Rather, they induce an extracellular factor that stimulates hydrolase activity in whole cells . This factor is partially characterized in this paper.
Correspondence to: D.C. Savage,Departmentof Microbiology,
Universityof Tennessee, Knoxville,TN 37996,USA. t Presentaddress: Department of Biochemistry,Universityof
Wisconsin-Madison,420 Henry Mall, Madison, WI 53706, USA.
122 3. MATERIALS AND METHODS
3.1. Strains and storage The lactobacillus strains used in this study (Table 5) were stored in MRS broth (Difco Laboratories, Detroit, MI)with 15% glycerol. They were grown in MRS broth in an anaerobic chamber (Coy Laboratory Products Inc., Ann Arbor, MI) with an atmosphere of 92% N2, 5% CO 2, and 3% H 2. 3.2. BSH assay Bile salt hydrolase activity was assayed anaerobically in whole cells by the radiochemicat method with ~4C-taurocholicacid (NEN DuPont, Boston, MA) as previously described . 3.3. Preparation of *SUP Supernatant solutions with the capacity to stimulate BSH activity (*SUP) were prepared from cultures of Lactobacillus sp. strain 100-100 grown an,~erobically in 40 ml of MRS broth (0.05% inocuIum) for 20-24 h at 370C. The culture was split into two aliquots. Taurocholic acid (TCA; Sigma Chemical Co., St. Louis, MO) was added to 0.4 mM to one aliquot; no bile salt was added to the other. Both cultures were then incubated for 30 min at 37°C. Thereafter, they were removed from the anaerobic chamber and centrifuged at 5000 × g for 10 min. The supernatant solutions from cultures containing TCA (* SUP) and those cultures without the bile salts (SUP) were removed from the centrifuge tubes and stored at 4°C. 3.4. Assay for *SUP activity The capacity of the extracellular factor in *SUP to stimulate BSH activity involved treating the solution with various agents under conditions described below. These solutions were then used to suspend cell pellets from stationary phase cultures of strain 100-100 grown in medium free of bile salts. These cell suspensions were diluted 1/10 in 0.2 M sodium acetate (pH 4.2) and assayed, in duplicate, for BSH activity. Controls of cells suspended in untreated *SUP and SUP were included in each assay. Unless otherwise indicated, experiments were repeated at least
three times. Hydrolase activity varied from experiment to experiment, but the relative proportion of BSH activity in cells suspended in *SUP, SUP, and the treated supernatant solutions, remained constant.
3.5. Treatment of the *SUP with t'arious physical and chemical agents 3.5.1. Aerobic conditions. The *SUP was removed from the anaerobic chamber and air was bubbled through it for 5 min. The solution was returned to the chamber and immediately assayed for its ability to stimulate BSH activity in strain 100-100. 3.5.2 Heat. The *SUP was heated to 55°C and 90°C for 10 min or autoclaved (121°C, 21 psi for 15 min). Thereafter the supernatant ~olutions were assayed for their capacity to stimulate BSH activity. 3.5.3. UItrafiltration. The *SUP was filtered through membranes with molecular mass exclusion limits of 10, 30 and 100 kDa (Omega series; Pharmacia LKB, Piscataway, NJ) in an Amicon (Danvers, MA) stirred ceil. Both the filtrate and retentate were then assayed for BSH-stimulating activity. 3.5.4. Dialysis. The supernatant solutions (5 ml) were dialyzed against two changes of 2 I of PG buffer (50 mM NaPO4 [pH 7.0] with 10% glycerol) at 4°C, in dialysis tubing having molecular mass exclusion limits of 1, 6-8, 12-14, and 25 kDa (Spectrum Medical Industries, Inc., Los Angeles, CA). The retentate was then assayed for its capacity to stimulate BSH activity. 3.5.5. Protease. The effect of pronase E (protease type XIV; Sigma) on *SUP activity was tested. The pH of this supernatant solution was adjusted to 6.0 with 0.5 M dibasic sodium phosphate. Pronase was added to *SUP to a final concentration of 1.2 U/ml. This solution and controls of *SUP (pH 6.0) and untreated *SUP and SUP (pH 4.3) were incubated for 1 h at 37"C. The samples were then heated to 95°C to inactivate the protease, cooled to 37°C, and assayed for BSH-stimulating activity. 3.5.6. Suifhydryl group inhibitor. The sulfhydryl group inhibitor, N-ethylmaleimide (NEM; Sigma) was dissolved in PG buffer (pH 7.0) and added to
123 *SUP to final concentrations of 1 mM and 10 raM. The solutions were incubated for 4 h at room temperature then dialyzed against two changes of 2 I of PG buffer (pH 7.0). They were then asayed for BSH-stimulating activity. 3.5.7. Extractions with organic soh'ents. The supernatant solutions were extracted with equal volumes of chloroform, ethyl acetate or hexadecane. The solutions were mixed; the organic phases were removed, put into 2-ml screw cap tubes, and dried in a Speed Vac (Savant Instruments, Inc., Farmington, NY). The residue remaining after the samples were dried was suspended in 2 ml of PG buffer (pH 7.(I). These solutions were assayed for BSH-stimulating activity.
3.6. Effect of TCA and cholic acid on BSH actil'ity TCA and cholic acid (CA) were assayed for their capacity directly to stimulate BSH activity in whole cells of strain 100-100. These compounds were added to 0.4 mM to supernatant solutions from cultures of strain 100-100 grown in medium free of bile salts (SUP). The solutions were immediately assayed for BSH-stimulating activity. 3.7. Effect of *SUP from strain 100-100 on BSH actit'ity in other Lactobacilli Cultures of 26 strains of intestinal iactobaciUi (Table 5) were grown overnight. A portion of each culture was centrifuged to pellet the cells. The cell pellets were thee suspended to their original volume in *SUP 'tom strain 100-100. BSH activity was assayed in these cell suspensions and the cells from the original overnight cultures of the strains. 3.8. TCA binding assay The ability of the extracellular compound in *SUP to bind bile acids was assayed. *SUP (5 ml) and SUP (5 ml) were placed in dialysis tubing with molecular mass exclusion limits of 6-8 kDa, and mixed with 1 mM 14C-TCA (specific activity of 0.1 ~Ci//~mol TCA). Samples were removed from both solutions at 0, 20, 40, 60, 80, 100, 120, 150, 180, 210, 270, 420 and 720 min after the start of the dialysis. 10 mi of scintillation cocktail (Formula 989; NEN DuPont) was added to each
sample and the amount of TCA remaining in the dialysis tubing was quantkated by liquid scintillation counting in a Beckman LS7000 (Beckman Instruments, Fulterton, CA).
4. I. Treatment of *SUP with physical and chemical agents Several physical and chemical treatments were used to characterize the extracellular factor in * SUP. 4.1.I. Aerobiosis. BSH activity in strain 100-100 grown aerobically does not increase in response to conjugated bile salts (unpublished data). Therefore, the sensitivity of the extracellular factor to air was examined. *SUP activity was not irreversibly affected by air. BSH activity in cells suspended in untreated *SUP and SUP was 150.3 and 70.1 nmol CA formed/min/mi cell suspension, respectively; the activity in cells suspended in the *SUP treated with air, was 169.5 nmol CA formed/min/mi cell suspension. 4.1.2. Heating. * SUP activity was unaffected by heating to 55°C and 90°C for 10 rain. Autoclaving also did not effect its capacity to stimulate hydrolase activity. BSH activity in cells suspended in untreated * SUP and SUP was 55.2 and 19.9 nmol CA formed/min/ml cell suspension, respectively. The activity in cells suspended in the autoclaved *SUP was 58.8 nmol CA formed/min/ml of cell suspension. 4.1.3. Uitra~itration. *SUP activity was retained by all membranes used; nominal activity was detected in the filtrate (Table 1). 4.1.4. Dialysis. The extracellular factor in * SUP was retained by membranes with molecular mass exclusion limits up to 12-14 kDa (Table 2). It was only partially retained in the dialysis bag with the exclusion limit of 25 kDa (Table 2). 4.1.5. Protease treatment. *SUP activity was not reduced in solutions treated with pronase compared to the untreated controls (Table 3). Pronase alone had no hydrolase activity. Samples from the supernatant solutions treated with the enzyme were analyzed by SDS-PAGE. The peptide patterns indicated that the pronase was ae-
124 Table i
Effect of ultrafiltration on the extracellular effector of BSH activity in Lactobacilh~s sp. strain 100-100 ,,t,
The effect of protease treatment on the extracellular factor that enhances bile salt hydrolase activity in Lactobacillus sp. strain 100-100 ;,.b
Supernatant solution ~'
*SUP SUP *SUP * SUP * SUP
Molecular mass exch:sion limit (kDa)
BSH activity d
10 30 I IX)
104.0 ~ 55.1 " 99.0 117.2 98.4
63.5 59.1 52.4
" The supernatant solution from cultures of Lactobacillus sp. strain 100-10(i were filtered through ultrafiltration membranes. h The experiments were repeated three times. The activity varied from experiment to experiment, but the proportions remained constant. *SUP, supernatant solutions from cultures incubated in medium containing bile saIts; SUP, supernatant solutions from cultures incubated in medium free of bile salts. d Bile salt hydrolase activity assayed in lactobacillus cells suspended in the supernatant solutions and expressed as nmol cholic acid formed/min/ml cell suspension. " BSH activity in cells suspended in unfiltered *SUP and SUP.
tire, and degraded proteins in the supernatant solutions. 4.1.6. Suifliydryi group inhibitor. * SUP activity was inhibited by NEM. The hydrolase activity in cells suspended in *SUP and SUP was 55.2 and 19.9, respectively. The activity in cells suspended
Supernatant solution ¢
BSH activity d
* SUP SUP * SUP * SUP
4.3 6.0 c 6.0 ~ 6.0"
76.9 47.7 82.4 81.0
~' Pronase E was used at a concentration of 1.2 U / m l of culture supernatant solution. h Experiments repeated twice. The activity varied from experiment to experiment, but the proportions remained constant. ¢.d See footnotes Table 1, ~' The pit was adjusted with 0.5 M dibasic sodium phosphate, r None detected.
in the *SUP treated with 1 mM and 10 mM NEM was reduced to levels equivalent to cells suspended in SUP, 21.0 and 22.1, respectively. 4. I. 7. Organ& extraction. The extracellular factor in *SUP partially separated into chloroform and ethylacetate (Table 4); however, some activity remained in the aqueous phase (Table 4). The hexadexane could not be dried and was not assayed for activity. However, virtually all of the *SUP activity remained in the aqueous phase after this extraction.
Table 2 Effect of dialysis of *SUP on the extracellular factor that stimulates bile salt hydrolase activity in Lactobacillus sp. strain 100-100 ~,.b
Supernatant solution '
*SUP SUP *SUP *SUP * SUP
none none chloroform ethyl acetate hexadecane
Supernatant solution "
Molecular mass exclusion limit (kDa) -
BSH activity d
*SUP SUP *SUP *SUP *SUP
1 6-8 12-14
40.9 17.9 41,7 39.5 37.7
*SUP SUP *SUP
50,9 19.8 30.7
" SUP was dialyzed against two changes of 21 of PG buffer (pH 7.0), then assayed for its ability to stimulate BSH activity in strain 100-100. h.~.d See footnotes Table I.
Extraction of *SUP from Lactobacillus sp. strain 100-100 with organic solvents a.b BSH activity d Organic phase
Aqueous phase ~
25.6 24.4 ND f
33.6 12.8 24.5 23.7 34.9
Equal volumes of the supernatant solutions and solvents were mixed, the organic layer was removed, dried, and the residue suspended in PG buffer (pH 7.0). bx,~f See footnote: Table 1. The supel~~aat solution remaining after the organic extraction. f Not determined.
4.2, Effect of TCA and CA on BSH actit'ity in strain 100-I00
Neither T C A nor C A affected BSH activity in cells of strain 100-100. The hydrolase activities in cells suspended in * S U P and SUP were 119.3 and 39.9 nmol CA f o r m e d / m i n / m l cell suspension, respectively. The activities in the SUP with T C A and CA were 43.0 and 46.0 nmol CA f o r m e d / m i n / m l cell suspension, respectively.
8, ~g + ., ~.
~. o .+ [ ~, o
P'- ,m L Table 5 Effect of *SUP from Lactobacilhessp, strain 100-100 on the BSH activity in other lactobacilli [4,51 '' Strain
100-100 100-I 100-5 100-10 100-12 100-14 100-16 100-20 100-21 100-22 100-23 100-27 100-28 100-30 100-31 100-32 100-33 100-34 t00-37 168S 207 220 313 DI 11 D287 R! C7
Rat stomach Mouse stomach Mouse stomach Human feces Human feces Mouse stomach Mouse stomach Mouse stomach Human feces Human feces Swine feces Human feces Swine feces Chickencrop Chicken crop Chickencrop Swine feces Chickencrop Mouse stomach Swine feces Chicken crop Chicken crop Rat intestines Calf feces Calf feces Mouse stomach Chicken crop
BSH activity ~ Uwn
74.1 ND ~ ND" 52.4 47.6 0.4 I.I !60.1 2.9 ND ~' 22,2 4,4 81.8 6.6 123, I 17.8 25.1 59.7 59.9 12,1 ND" 35.6 ND " 3.8 9.6 8.4 53.8
197.0 8.8 7.1 48.9 52.4 7.1 10.8 222,0 1I. l 13.7 15,9 16.9 58.8 22,9 51.6
22.8 25,6 32.1 145.5 18.5 15,3 13.2 10.7 16.7 20.9 22.7 48,7
" Cells from overnight cultures of these strains were suspended in the *SUP from strain L00-100 and assayed for BSH activity. b Animal and site that the organisms were isolated from, ¢ Bile salt hydrolase activity assayed in cells suspended in either their own supernatant solution or the *SUP from strain 100.100. Expressed as nmol eholic acid formed/min/ ml cell suspension. *SUP had residual BSH activity of approximately 10 nmol cholic acid formed/min/ml supernatant solution. ¢ Not detectable.
Fig. I. Taurochotic ac;d binding a~ay. L'tC-TCA (I raM) was
added to the supernatant solutions from cultures of strain 100-10(I incubated with (+ SUP) and without (SUP) bile salts. These solutions were then placed in dialysis tubing and dialyzed against I I of PG buffer (pH 7,0). Samples were removed at various times and the amount of TCA remaining in the dialysis tubing wa~.quantitated by liquid scintillation counting. The data are plotted as the mean of three experiments.
4.3+ Effect of *SUP from strain 100,I00 oil BSH actit'ity #1 other Lactobacilli Hydrolase activity varied greatly in the lactGbacillus strains tested: it was undetected in some and high in others (Table 5), The * S U P from strain 100-100 stimulated the BSH activity in only two of these strains, Lactobaciflus, sp strain 10020 and L. acidophih+s strain 100-37 (Table 5).
4.4. TCA binding assay I+C-TCA diffused from dialysis tubing containing * S U P and SUP at the same rate (Fig. 1). The comnound with the capacity to stimulate BSH activity in strain 100-100 was retained in the dialysis tubing with * SUP.
5. D I S C U S S I O N We have found that BSH activity in Lactobacillus sp. strain 100-100 is regulated by growth phase and conjugated bile salts . BSH activity increases more than 70-fold when cultures of this strain enter stationary phase . Likewise, the activity in whole cells ir~creases as much as six-fold within 20 min after conjugated bile salts are added to stationary phase cultures. However, the bile salts are not inducing the enzymes or a mere-
brahe transport system . Rather, an inducible extracetlular factor that regulates BSH activity is present in the spent medium of cultures incubated with bile salts in the medium (*SUP). This factor has the capacity to stimulate hydrolase activity two- to three-fold in cells suspended in *SUP. The effect is immediate; the activity does not continue to increase with longer incubation in the supernatant solution. The capacity to respond to and produce such a compound was not limited to strain 100-100. The BSH activity in two other lactobacillus strains increased when they were suspended in *SUP from strain 100-100. Moreover, +SUP prepared from cultures of these two strains, strain 100-20 and 100-37, stimulated hydrolase activity in strain 100-100 (data not shown). BSH activity in strains with low activity also increased when they were suspended in *SUP. This was due, however, to low levels of BSH activity in the * SUP. Interestingly, the hydrolase activity decreased in several strains when they were suspended in * SUP from strain 100-100. This finding may indicate that the BSH's in these strains are extracellular, a hypothesis yet to be examined. The extraceIIular factor in *SUP is quite stable. It is resistant to autoclaving and proteases can be stored for at least six months at 4°C without significant loss of activity, and is resistant to pH's as low as 2.0 and as high as 12.0. However, NEM inhibits its capacity to stimulate BSH activity. Therefore, sulfhydryl groups may be important in its function. In the experiments performed to test. to that hypothesis, however, residual NEM remaining in the *SUP could have inhibited transport of the bile salts into the lactobacilli. Transport of bile salts into hepatocytes is sensitive to sulfhydryl group inhibitors including NEM . This hypothesis seems unlikely; the concentration of NEM remaining in the solution after its dialysis is quite low (unpublished results). Moreover, if NEM had affected a transporter, then the apparent hydrolase activity would be likely to drop to levels below those of the controis. BSH activity decreased only to background levels (i.e. levels of cells suspended in SUP) in cells suspended in the NEM-treated *SUP. Therefore, the simplest hypothesis is that the
NEM altered the factor in *SUP that enhances BSH activity. The extracellular factor is a lipid or has a lipid moiety since it extracted into organic solvents. Moreover, it appears to form large aggregates. Bite acids fit this description (i.e. they are lipids and form micelles). However, neither TC~ nor CA directly stimulated BSH activity. Therefore, bite acids can be ruled out as the factor in *SUP. That TCA does not directly affect BSH activity is further supported by the experiments designed to assess the capacity of *SUP and SUP to bind naC-TCA. * SUP activity was retained by the dialysis tubing used in the experiments, while less than 10 nM TCA remained. This concentration of TCA is far below that needed to stimulate BSH activity in strain 100-100. Several hypotheses can be proposed to explain the function of *SUP. One is that a factor or factors in it affects the proportions or activity of the four BSH isozymes. However, all four hydrolases can be isolated in roughly the same proportions from cells incubated with and without bile salts (Lundeen and Savage, submitted), in addition, purified BSH A and B incubated with *SUP were unaffected in activity (unpublished data). The possibility that *SUP is affecting BSH C or D cannot yet be ruled out, however, since these isozymes have yet to be examined for how * SUP affects them in purified form. Therefore, the mechanism of action of the extracellular factor will require further study. Moreover, more study will be required before its function in the gastrointestinal tract is understood. REFERENCES  Midvedl, T, (1974) Am. J. CLin. Nutr, 27, 1341-1347,  Hylemon, P.B. and Stellwag, E.J. (1976) Biochem. Biophys. Res. Commun. 69, 1088-1094.  Lundeen, S.G. and Savage, D.C. (1990) J. Baeteriol. 172, 417t-4177.  Lin, J.H-C, and Savage, D.C. (I984) FEMS MicrobioL Lett, 24, 67-71.  Lin, J,H-C. and Savage, D.C, (1984) Appl. Environ, Microbiol. 49, 1004-1006.  Feighner, S.D. and Daskevicz, M.P. (1987) Appl, Environ. Microbiol. 53, 331-336.  Blumrich, M. and Petzinger, E. (1990) Biochim, Biophys. Acta. 1029, !-t2.