Vol. 31, No. 2INFECTION AND IMMUNITY, Feb. 1981, p. 798-8070019-9567/81/020798-10$02.00/0

Effect of Bile Acid Derivatives on Taurine Biosynthesis andExtracellular Slime Production in Encapsulated

Staphylococcus aureus S-7tTOSHICHIKA OHTOMO,' 2 KOSAKU YOSHIDA,' AND C. L. SAN CLEMENTE2*

Department ofMicrobiology and Public Health, Michigan State University, East Lansing, Michigan 48824,2and Department ofMicrobiology, St. Marianna University School ofMedicine, 2095, Sugao, Takatu-Ku,

Kawasaki, Kanagawa-Ken, 213, Japan'

Various bile acids were added to cultures of encapsulated strains of Staphylo-coccus aureus growing in serum-soft agar medium of brain heart infusion broth.We examined effects of these compounds on cellular characteristics such asgrowth type, cell volume index, clumping factor reaction, slime yield, taurinecontent, and L-(-)-cysteic acid decarboxylase activity. Upon addition to themedium of either taurochenodeoxycholic acid, taurocholic acid (25 to 50 ,ug/ml),or cholic acid (10 to 25 jig/ml), the colonial morphology of taurine-positive cells(strain S-7) was altered from the diffuse to the compact type in serum-soft agar.Also, the titer of the clumping factor reaction increased, while the cell volumeindex and slime yield were markedly decreased. Tauro-bile acids, includingtaurocholic acid, taurochenodeoxycholic acid, taurodehydrocholic acid, and tau-rodeoxycholic acid (50 ,ug/ml) inhibited the synthesis of taurine and resulted indecreased L-(-)-cysteic acid decarboxylase activity. Among all of the derivativescholic acid itself was found to inhibit slime production and L-(-)-cysteic aciddecarboxylase activity to the greatest extent. Glyco-bile acid derivatives andtaurolicholic acid (50 to 100 ,tg/ml) had no effect on L-(-)-cysteic acid decarbox-ylase activity. Compounds such as glycodeoxycholic acid (50 to 100lg/ml) hadno effect upon any of the cellular characteristics tested. No effect was observedupon addition of any of these compounds to cultures of the taurine-negativestrain (T-26-B). We did find a correlation between the inhibition of taurinebiosynthesis and decreased slime production. Electron micrographs indicated thatthis encapsulated strain was converted to an unencapsulated state in the presenceof bile acids.

Previous work has shown that nontoxic bileacids (27) may alter both metabolic character-istics and colonial morphologies of Staphylococ-cus aureus (16). The conjugation of taurine orglycine to bile acids present on the cell surfacemay cause this alteration. In the case of S.aureus M, taurine on the cell surface polysac-charide fraction is linked through a carboxylgroup of N-acetyl-D-aminogalacturonic acid res-idue of the surface polysaccharide (12, 13). Tau-rine has been detected in a wide range of cap-sular materials from encapsulated strains of S.aureus (20).

Cultures of S. aureus strains in serum-softagar (SSA) have been regarded as encapsulatedwhen growing diffusely and unencapsulatedwhen growing compactly (1, 34). Also, we noteda cell surface substance (alkali-stable polysac-charide) in staphylococcal strains which corre-

t.Journal article no. 9099 from the Michigan AgriculturalExperiment Station.

lated with compact colony formation either inserum or fibrinogen soft agar medium (32, 33).This culture system provides a method for ob-taining information on various cell surface prop-erties (10, 22, 30, 33).

In the present study, we investigated a possi-ble correlation between bile acids and variouscell surface properties and the possible controlof taurine biosynthesis in an encapsulated strainof S. aureus by using the SSA system. We haveshown that when cholic acid (CA) (10 to 25 ,g/ml) and tauro-bile acid derivatives (50 ,ug/ml)are added to the medium, colony morphology isconverted from diffuse to compact in SSA witha frequency of 50 to 100%, whereas glyco-bileacid derivatives (50 to 100 ,tg/ml) were lesseffective. The increased frequency of conversionfrom diffuse to compact growth type in SSA wasaccompanied by changes in the clumping factorreaction, cell volume index, slime yield, taurinecontent, and L-(-)-cysteic acid decarboxylase(LCAD) activity. Bile acid derivatives in the

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BILE ACIDS AND TAURINE SYNTHESIS IN S. AUREUS 799

concentrations used were not inhibitory togrowth.

MATERIALS AND METHODS

Bacterial strains. Taurine-positive (Tau') S. au-reus S-7 and a taurine-negative (Tau-) mutant strain,T-26-B, (both strains encapsulated) were used. Theorigin, properties, and the various biochemical char-acteristics of these strains have been described else-where (21, 29). Both strains were positive for coagu-lase, phosphatase, deoxyribonuclease, and mannitolfermentation. They were negative for clumping factorreaction, produced diffuse growth in SSA (7), and werephage nontypable.Source of chemicals. Taurine (2-amino ethane-

sulfonic acid), taurochenodeoxycholic acid (sodiumsalt) (TCDCA), taurocholic acid (TA), taurodeoxy-cholic acid (TDCA), taurodehydrocholic acid, taurol-icholic acid, glycodeoxycholic acid, glycholic acid, gly-cochenodeoxycholic acid, and CA were purchasedfrom Sigma Chemical So. (St. Louis, Mo.) and were atleast 98% pure. L-Cysteic acid (monohydro-L-amino-flsulphopropionic acid) was obtained from the UnitedStates Biochemical Co., Ltd. (Cleveland, Ohio). Be-fore use, all of the above chemicals were sterilizedby membrane filtration (pore size, 0.45 nm; Milli-pore Corp., Bedford, Mass.) after dissolution in 0.3M tris(hydroxymethyl)aminomethane-hydrochloridebuffer (pH 7.7) at 70°C for 15 min.Procedure for the conversion experiments in

SSA. The proportion in which the strains S-7 and T-26-B changed to compact from diffuse colony mor-phology in SSA (conversion amount) was determinedby the methods of Yoshida et al. (31). Various concen-trations of bile acid solutions were prepared as above;0.1 ml of the appropriate solution, together with 0.1ml of normal rabbit serum and 0.1 ml of a 10-7 or 10'dilution of an overnight brain heart infusion culturegrown at 37°C, were all combined with 10 ml of brainheart infusion soft agar (0.15% agar; Difco Laborato-ries, Detroit, Mich.) and incubated at 37°C for 24 h.Identical cultures without bile acids were used ascontrols. Observations of colony morphology were re-corded (Fig. la and b).

Preparation and quantitation of crude slimefraction. The slime layer fraction was prepared bythe method of Ekstedt and Bernhard (6), except forthe following modification. The organisms were grownat 370C for 16 h without shaking in 300 ml of dialyzed(against 5 volumes of distilled water overnight at 8°C)staphylococcus medium no. 110 (Difco) supplementedwith various concentrations of bile acids. The orga-nisms were then centrifuged at 10,000 x g for 15 minat 4°C. Five grams (wet weight) of cells was suspendedin potassium phthalate-sodium hydroxide buffer (pH4.5) containing glass beads (30- to 35-mm outer diam-eter) and blended in a Vortex mixer (Scientific Indus-tries, Queens Village, N.Y.) for 15 min at 4°C, and thecell debris was separated by means of a sintered glassfilter. The filtrate was centrifuged at 8,000 x g for 20min at 40C. The supernatant was dialyzed againstdistilled water for 48 h, lyophilized, and weighed.

Determination of cell yield, relative clumpingfactor reaction, and cell volume index. Approxi-

mately 109 cells of strain S-7 were inoculated into 100ml of the medium described above and grown at 37°Cfor 12 h without shaking. The cells were harvested bycentrifugation at 7,000 x g at 4°C, lyophilized, andweighed. The relative clumping factor titer was de-fined as the highest dilution oforganisms which causedclumping, as described by Duthie (5). The cell volumeindex of the organisms was determined by the methodsof Wiley (28) and Yoshida et al. (34). Cells wereharvested after 16 h of growth at 37°C in plates of thedescribed medium. Five milliliters of the cell suspen-sion in sterile saline was placed in Hopkins tubes andcentrifuged at 3,000 x g at 4°C for 30 min. Readingswere taken with vernier calipers, and the height of thecolumn ofthe packed cells was recorded in millimeters.Also, the number of colony-forming units was esti-mated from plates made from the packed cells whichhad been shaken with glass beads (10- to 20-mm outerdiameter) in sterile 25-ml flasks. Cell volume indexwas calculated by using the following formula: [packedcell volume (milliliters)/colony-forming unit] x 10"'.

Quantitation of taurine in whole cells and inthe slime fraction. Taurine content was determinedby the dinitrophenol method of Pentx et al. (24).Bacterial cells were grown in the liquid medium de-scribed above at 37°C for 14 h, harvested and washedwith distilled water. The total taurine concentrationof the crude slime fraction was determined as follows.Five milligrams ofcrude slime material was hydrolyzedby treatment with 6 N HCl at 105°C for 12 h in asealed ampoule. Thereafter, liquid was removed byrepeated evaporation over P205. These hydrolysateswere mixed with 1.0 g of Dowex 50 (50-100 mesh, H+form) in potassium phthalate-sodium hydroxide buffer(pH 4.5) and allowed to stand at room temperature for5 min with occasional agitation followed by filtrationthrough Whatman no. 40 filter paper. The yellowderivative produced by the reaction between 1-fluoro-2,4-dinitrobenzene and taurine was measured at 355nm by the method of Pentx et al. (24). The valuesobtained were compared with a standard curve.

Extraction of crude L-cysteic acid decarbox-ylase from packed cells of strain S-7. A cell-freeextract was prepared by suspending frozen cells in 4 to5 volumes of 0.075 M phosphate buffer (pH 7.4).Lysostaphin (Schwartz/Mann, Orangeburg, N.Y.) wasadded to this suspension at a final concentration of200 ug/ml; the suspension was then kept at 37°C for2 h, centrifuged for 15 min at 15,000 x g, and dialyzedagainst 0.075 M phosphate buffer (pH 7.4) at 4°Covernight. The crude cell-free extract usually con-tained 25 to 30 mg of protein per ml. To this extract,whose pH was maintained at 7.4 with 0.075 M phos-phate buffer, solid ammonium sulfate was addedslowly to 70% saturation. The turbid mixture was thencentrifuged at 2,000 x g for 15 min, and the superna-tant fluid was discarded. The pellet was redissolved in1 volume of 0.1 M phosphate buffer (pH 7.4) anddialyzed overnight against 0.03 M potassium phos-phate buffer (pH 7.4) at 4°C. This fraction containedabout 5 to 7 mg of protein per ml and was stored at-300C.

Preparation of crude LCAD from the cell-freeculture broth of strain S-7. Solid ammonium sulfatewas added to the cold (40C) culture broth. The initial

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800 OHTOMO, YOSHIDA, AND SAN CLEMENTE

1- - -,..-!..lFIG. 1. Colonial morphologies of S. aureus S-7 in SSA containing (a) TCDCA and (b) TA in decreasing

concentration extending from 50 to 0 pg/ml in decrements of ten from left to right.

precipitate at 40% saturation was discarded, and thatforming after 20 min at 70%o saturation was centrifugedat 10,000 x g for 15 min, suspended in 0.03 M potas-sium phosphate buffer (pH 7.4), and dialyzed againstthe same buffer at 40C for 24 h. This fraction containedabout 5 to 6 mg of protein per ml and was stored at-30°C.Enzyme assays. LCAD activity was determined

manometrically by the method of Blaschko (3) whichwas based on C02 evolution from L-(-)-cysteic acid(mole per mole). The crude enzyme sample containing9.4 mg of total protein (ca. 3 mg of enzyme) wassuspended in 2.0 ml of 0.075 M potassium phosphatebuffer (pH 7.4). The reaction was initiated by theaddition of neutralized L-(-)-cysteic acid in 3.0 ml of0.01 M phosphate buffer, pH 7.4. The center wellcontained 0.3 ml of 1 N H2SO,. In each case, a controlwas employed in which distilled water was substituted

for L-(-)-cysteic acid. All experiments were carriedout at 37°C under N2. The enzyme activity was cal-culated as microliters of C02 formed per milligram ofcrude enzyme per hour. The resulting L-(-)-cysteicaaid is expressed as a quotient: q(N2/CO2). By multi-plying the quotients by 5.58 (see Table 2) the amountof taurine formed can be calculated as micrograms permilligram of crude enzyme. Protein was determinedby the method of Lowry et al. (14) with bovine serumalbumin as the protein standard.

Electronmicroscopy study. Cells of S. aureuswere harvested by centrifugation at 15,000 x g at 4°Cfor 20 min. They were fixed initially with 7% glutaral-dehyde in 0.1 M phosphate buffer (pH 1.0) at 4°C for16 h and then fixed with 0.1% osmic acid at roomtemperature for 5 h by the method of Ohtomo et al.(19). The fixed material was first dehydrated withethanol and acetone and then embedded in Epon 812

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BILE ACIDS AND TAURINE SYNTHESIS IN S. AUREUS 801

by the regular procedure. Thin sections were preparedwith the LKB microtome, mounted on carbon-coatedgrids, and stained with aqueous uranyl acetate. Thesesections were observed in a Philips 300 transmissionelectron microscope operating at 80 kV.

RESULTSMorphological changes in serum-soft

agar caused by bile acid derivatives. Thecolonial morphologies of strains S-7 (Tau', dif-fuse type) and T-26-B (Tau-, diffuse type) inSSA supplemented with various concentrationsof bile acid derivatives were investigated. Con-

version from diffuse to compact growth wasobserved when strain S-7 was grown in SSAsupplemented with CA, TA, and TCDCA (10 to50 ,tg/ml of medium). No spontaneous mutationor lysogenic conversion was observed when 108organisms of strain S-7 were tested as shown inFig. la and b. The percent conversion (Table 1)was 40, 80, and iOO% when TCDCA, TA, andCA, respectively, were added at a final concen-tration of 25 ,ug/ml of medium. Differences wereobserved among the five tauro-bile acids at var-ious concentrations of 50 to 100 ,ug/ml, the con-version frequency was only 5 to 6%. However,

TABLE 1. Comparative effects of bile acid derivatives in SSA on an encapsulated strain of S. aureusconverting from diffuse to compact growth

Strain S-7 (Tau') Strain T-26-B

Compound Supplement No. of colonies (10-8) % (Tau-)(nlgml) % Conversion'~Diffuse Compact sionb

TCDCA 0.0 24 + 6 0 ±0 0 0.0

TA

2550

0.02550

Taurodehydrocholic acid

TDCA

0.02550

0.02550

13 ± 30±0

32 ± 76± 10±0

30 ± 529 ± 624 ± 3

39 ± 436 ± 619 ± 3

9 ± 229 + 4

0±027 ± 436 ± 5

0±00±04± 1

0±02± 116 ± 3

40.9 ± 2.3100.0 ± 1.0

081.8 ± 1.4100.0 ± 2.0

00

11.1 ± 1.5

05.2 ± 0.8

45.7 ± 2.3

0.00.0

0.00.00.0

0.00.00.0

0.00.00.0

Taurolicholic acid

Glycholic acid

Glycochenodeoxycholic acid

Glycodeoxycholic acid

CA

Taurine

0.050100

0.050100

0.050100

0.050100

0.01025

0.050100

27 ± 423 ± 820 ± 9

38 ± 432 ± 636 ± 7

31 ± 328 ± 430 ± 2

34 ± 432 ± 635 ± 9

29 ± 32± 10±0

37 ± 635 ± 839 ± 5

0±00±00±0

0±00±00±0

0±00±02 ± 1

0±02 ± 02 ± 1

0±028 ± 433 ± 4

0±00±00±0

000

000

006.6 ± 0.4

005.7 ± 0.7

093.3 ± 0.2100 ±0.1

000

a The colony counts are the mean ± standard error of the mean of four experiments.b Average of four trials.

0.00.00.0

0.00.00.0

0.00.00.0

0.00.00.0

0.00.00.0

0.00.00.0

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802 OHTOMO, YOSHIDA, AND SAN CLEMENTE

CA was found to be most active, with a conver-sion frequency of 93% even at a level of 10 ,ug/ml.Effects of various bile acid derivatives

upon growth yield, clumping factor reac-tion, and cell volume index. To determinethe biological characteristics which accompanythe morphological change, we examined the ef-fects of various bile acid derivatives on the cellsurface properties. The cell yield, clumping fac-tor titer, and cell volume index were all deter-mined after stationary growth for 16 h in liquidbrain heart infusion medium containing the bileacid derivatives (Fig. 2 and 3). Six of the bileacids had no effect on cell yield. However, in thepresence of TCDCA, TA, TDCA, and CA, thecell yield stabilized, and the cell volume indexdecreased markedly as the concentration of bileacid added was increased. These results indicatethat the amount of slime material was reduced.

1:102TCDCA

1:512

1:64

X- 1: ''

c~~~~~~~~~~~~~~~

C~~~DC.251

ffi . TLA

.U _i_i

In addition, the clumping factor titer of thosecells cultivated with either TCDCA,TDCA, TA,or CA was significantly higher than those grownwith other bile acid derivatives. For comparison,Fig. 3 shows the effect of taurine at variousconcentrations as a control. The changes ob-served in the clumping factor titer and cell vol-ume index are in good agreement with the con-version frequencies found in Table 1 for thecorresponding bile acids.Quantitation of slime material of cells

grown in the presence of various bile acidderivatives. Six bile acids had no effect on theamount of slime produced (data not shown).However, slime yield was somewhat reducedwhen cells were grown in the presence of TAand CA (Fig. 4). Slime yield in the absence ofbile acids was approximately 97 ± 3.2 mg/g (dryweight) of cells as opposed to 94 ± 4.9, 83 ± 4.5,63 ± 3.6, 63 + 3.1, and 40 ± 4.2 mg/g (dry weight)

l 25

TCA20 15i

is-10to

5

:25

TDCA

2 wo-15.1x I16 E

LU10

10 A.j

UU25

GCA

20.15

I1510

-10

BILE ACID [Mg/mlIFIG. 2. Three effects of different concentrations of various individual bile acids on the encapsulated strain

of S. aureus S- 7. TCA, Taurocholic acid.

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BILE ACIDS AND TAURINE SYNTHESIS IN S. AUREUS

1:1024

BILE ACID [pg/miFIG. 3. Three effects of different concentrations of individual bile acids as well as of taurine alone on the

encapsulated strain of S. aureus S- 7.

FIG. 4. Effect of various bile acids on the yield ofcell surface slime by an encapsulated strain of S.aureus (S-7): 0, glycholic acid; *, TDCA; A, TCDCA;A, TA; and 0i, CA. Average offour trials.

of cells when 50 ,ug of glycholic acid, TDCA,TCDCA, TA, and CA, respectively, were addedper ml of medium (Fig. 3). The observed de-crease in the amount of slime produced corre-lated with the change in conversion frequency,clumping factor titer, and cell volume indexnoted earlier. Also (not illustrated), when the

same five bile acids were used at twice theconcentration per ml medium (100 jig), the slimeyields respectively were 85 ± 4.7, 74 ± 3.7, 42 ±3.6, 30 ± 2.7, and 18 ± 2.2 mg/g (dry weight) ofcells.

Effect of bile acid derivatives on taurinebiosynthesis. Taurine biosynthesis was par-tially inhibited by TDCA and almost completelyinhibited by either CA, TA, or TCDCA (Fig. 5).Neither taurolicholic acid, glycholic acid, gly-cochenodeoxycholic acid, nor glycodeoxycholicacid had any significant effect on taurine biosyn-thesis (data not shown). At individual concen-trations of 200 and 300 ,tg/ml of medium, TAand TCDCA each had little additional effect ontaurine biosynthesis beyond that observed at100 ,ug/ml of medium.Effect of various bile acid derivatives on

LCAD activity. Taruine has been shown to beformed through the decarboxylation of cysteicacid (3). The effect of various bile acid deriva-tives on intracellular and extracellular LCADactivity was examined (Table 2 and Fig. 6). LessLCAD activity was observed in extracts pre-pared from intracellular material than was ob-served in extracts of extracellular material (Ta-ble 2). Supplementation with CA at 10 ,ug/ml ofmedium reduced the LCAD activity more thanTCDCA, TA, and TDCA each alone did at 50,ug/ml and more than taurolicholic acid did at100 ,tg/ml of medium. However, no reduction in

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804 OHTOMO, YOSHIDA, AND SAN CLEMENTE

activity was observed with the glyco-bile acidsglycholic acid, glycochenodeoxycholic acid, andglycodeoxycholic acid. LCAD activity was foundto decrease directly with the taurine content ofthe whole cells. Among the bile acids, LCADactivity was inhibited most by and in proportionto the concentration of CA and TA (Fig. 6).Ultrastructure of strain S-7 of S. aureus.

When the culture was supplemented with CA at25 ,ug/ml of medium and incubated at 37°C for14 h, slime material was not seen on the cellsurface (Fig. 7B). However, slime formation wasobserved in cells grown in the control withoutCA (Fig. 7A).

DISCUSSIONAccording to Yoshida (29) a strain of S. aureus

is classified as encapsulated if it grows diffusely

AlI I

46

.E2

5 10 20 30 40 50 100Bile acids(pg/ml of medium)

FIG. 5. Effect of various bile acids on taurine bio-synthesis on the cell surface ofan encapsulated strainof S. aureus (S- 7). TCA, taurocholic acid.

in SSA, is phage nontypable, is clumping factornegative, and possesses a high cell volume index.Conversely, a strain of S. aureus is classified asunencapsulated when it grows compactly inSSA, is phage typable, is positive for clumpingfactor reaction, and possesses a low cell volumeindex. Also, electron microscopic observation ofthe strain of S. aureus used in this study indi-cates that there is a large production of slimematerial (Fig. 7A). An overall change after ex-

posure to these bile acids was the conversioninto an unencapsulated strain (Fig. 7B). Obser-vation with electron microscopy was preferredto staining with India ink preparations whichcould be invalidated by artifacts.

In this study, a Tau' strain of S. aureus (S-7)was shown to convert from diffuse to compactgrowth in SSA when it was grown in the pres-ence of various bile acids. Similar conversionswere not observed with a Tau- strain (T-26-B),

FIG. 6. Effect of various concentrations of TA onthe LCAD activity of an encapsulated strain of S.aureus (S-7): 0, control; 0, 10 ,ug/ml; A, 25 ,ig/ml;A, 50 ,ug/ml; l, 100 ,ug/ml; and D, CA, 10 ,ug/ml.

TABLE 2. Effect of various bile acids on LCAD of an encapsulated strain of S. aureus (S- 7) "

Supple

Cysteic acid q(N2/IC02) (tdI Of jug of taurine formed/(60Supple- CO2/60 min/g of crude en- min/g of crude enzyme)Compound ment (tLg/ zyme) min/gofcrude_enzyme)mnl)

Intracellular Extracellular Intracellular ExtracellularTCDCA 50 0.3 ± 0.09 1.0 ± 0.3 1.6 ± 0.2 5.5 ± 0.4TCA 50 0.2 ± 0.03 0.6 ± 0.01 1.1 ± 0.1 3.3 ± 0.6TDCA 50 0.8 ± 0.1 1.9 ± 0.4 4.4 ± 0.3 10.6 ± 0.9Taurodehydrocholic acid 50 0.9 ± 0.12 3.1 ± 0.9 5.0 ± 0.6 17.2 ± 0.6Taurolicholic acid 100 1.1 ± 0.4 3.4 ± 0.3 6.1 ± 0.2 18.9 ± 0.8Glycholic acid 100 1.4 ± 0.7 3.7 ± 0.9 7.8 ± 0.9 20.6 ± 0.5Glycochenodeoxycholic acid 100 1.6 ± 0.5 3.9 ± 0.4 8.3 ± 0.2 21.7 ± 0.9Glycodeoxycholic acid 100 1.7 ± 0.6 3.6 ± 0.3 9.4 ± 0.1 20.0 ± 0.7CA 10 0.1 ± 0.08 0.3 ± 0.01 0.6 ± 0.08 1.8 ± 0.8Control 0.0 1.9 ± 0.4 3.8 ± 0.83 10.6 ± 0.9 21.2 ± 0.9

" The results are the mean ± standard error of the mean of five experiments.

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BILE ACIDS AND TAURINE SYNTHESIS IN S. AUREUS 805

A'w_

4

.

6 'I

4-^I

.1 ..P,'-; t, .i.e v

4 * i 1>~~~~~~~~ vc.

.I .

Is Ie .P. V - . .%II,

I &

I*B4.

..I

B

FIG. 7. Electromicrographs of S. aureus S-7 (Tau+) grown in (A) brain heart infusion agar medium at37°C for 14 h and in (B) the same medium supplemented with CA (25 pg/ml of medium). Bars, 0.) sum.

which suggests that neither the biosynthesis oftaurine nor of slime is effected by any one of thebile acids. Lack of slime in the cell-free mediaafter incubation indicates that slime producedby the Tau- mutant adheres to the cell surfaceand does not slough off in the presence of bileacids.

It is well known that bile acids exert threepossible types of action on bacteria, namely,

bacteriostatic, bactericidal, or bacteriolytic (27).Apparently, no relationship exists between thechemical structure of the bile acid and its bac-teriostatic activity against gram-positive orga-nisms. The ability to reduce the surface tensionof growth media has been investigated for aseries of bile acids closely related in molecularstructure (26). Bile acids may be anionic, cati-onic, or nonionic depending upon the nature of

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806 OHTOMO, YOSHIDA, AND SAN CLEMENTE

the charge carried by the hydrophobic portionof the molecule (4). The acid derivatives of thebile acids are generally conjugated to glycine ortaurine via a peptide bond (9). By virtue of theirsurface active properties, these water-solubleconjugates solubilize lipophilic substances in thegut to form micelles (4). Among the severalpossible structural alterations which may occur,the most widely studied has been the enzymatichydrolysis of the peptide bond by bacteria (2,18). In our experiment, the frequency of conver-sion from diffuse to compact growth in SSA,while in the presence of the CA and tauro-bileacid compounds (10 to 50 ,ug/ml of medium) wasgreater than that observed with the glyco-bileacid derivatives. Although TA and TCDCA,among the bile acids tested, were the most in-hibitory to slime production, slime accumulatedfrom the inability of the microorganisms to de-grade these acids during cultivation (17). Con-jugation of the various bile acids with taurine inthe slime fraction may have protected themagainst degradation. However, the control ofslime production by chemical substances doesnot appear to have been studied. Our resultsindicate that CA, which is not conjugated totaurine or glycine, is more inhibitory to slimeproduction and taurine biosynthesis than are theother tauro-bile acids (Table 1, Fig. 6). Thesefindings suggest a possible interaction ofCA andtaurine on the cell surface. Possibly, TA effec-tively inhibits taurine biosynthesis and slimeproduction by being hydrolyzed in the mediumwith the release of taurine which is in fact theinhibitory substance.During the morphological conversion (diffuse

to compact in SSA) of strain S-7 (Tau') in thepresence of the CA and tauro-bile acid deriva-tives, a remarkable inhibition of taurine biosyn-thesis was observed (Table 2, Fig. 5). It appearsthat the decrease in slime production by theencapsulated strain was closely related to theconversion from diffuse to compact growth(SSA) as well as the decreased LCAD activity(Fig. 6). However, a direct correlation betweenslime production and taurine biosynthesis couldnot be made. Except for an encapsulated strainof S. aureus under freeze-drying treatment (22),the change in colonial morphology observedhere has not been reported previously.The metabolic pathway of taurine formation

in these microorganisms is unknown. Cysteicacid and cysteine sulfonic acid decarboxylaseactivities have been found in the livers of severalanimals, including dogs, rabbits, rats, and guineapigs (3), but as yet the required enzymes are notavailable (15). Also, methylated taurine deriva-tives which may have been precursors of cholinesulfate have been obtained from red algae (15).

S-sulfocysteine is one of several metabolic prod-ucts found after injection of cysteine into the rat(25). This amino acid has also been implicatedin the biosynthesis of cysteine in various micro-organisms (8). Kelly and Weed (11) and Liau etal. (13) found taurine to be a unique constituentof the cell wall or cell surface of the microorga-nisms. However, no one has observed the pres-ence of taurine in the cell wall of S. aureus (23).We have shown that a crude enzyme preparationof cysteic acid decarboxylase can catalyze theformation of taurine in vitro. Ammonium sulfatefractions of extracellular material exhibitedhigher levels ofLCAD activity than intracellularpreparations (Table 2). From these experimentswe suggest that encapsulated S. aureus orga-nisms produce taurine extracellularly. Recentfindings (20; T. Ohtomo, Y. Usui, J. Narikawa,K. Yoshida, H. Iizuka, and C. L. San Clemente,Abstr. Annu. Meet. Am. Soc. Micribiol. 1979,K100, p. 162) suggest that large amounts of L-(-)-cysteic acid (determined by gas chromatog-raphy and mass spectrometry) contained in theslime fraction probably act as an intermediate intaurine production in the encapsulated strain ofS. aureus.

In conclusion, it appears that bile acidsnegatively affect taurine biosynthesis and slimeproduction. Further investigation of the possibleinvolvement of other metabolic pathways withinthe slime fraction is needed. It may be possibleto demonstrate in vitro that ['4C]CA and ['4C]-TA act as binding sites on the surface or areincorporated into the slime fraction. Details ofthe mechanisms of this phenomenon are underinvestigation.

ACKNOWLEDGMENTSWe acknowledge the excellent technical assistance given by

Sy Adler, Richard Rinzler, and Larry Forney.

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