Bile acid biotransformation rates of selected gram-positive and gram-negative intestinal anaerobic bacteria

Vol. 69, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

BILE ACID BIOTRANSFORMATION GRAM-NEGATIVE

RESEARCH COMMUNICATIONS

RATES OF SELECTED GRAM-POSITIVE

INTESTINAL

AND

ANAEROBIC BACTERIA

P. B. Hylemon and E. J. Stellwag Department of Microbiology Health Sciences Division Virginia Commonwealth University Richmond, Virginia 23298

Received

February

6,1976

SUMMARY: Treatment of whole cell suspensions of Eubacterium aerofaciens and Bacteroides fragilis with lysozyme resulted in a marked increase (>lOOfold) in the rates of biotransformation of cholate to 7-ketodeoxycholate (7-KD) in the former but only a 2-fold increase in the latter bacterium. In B-. fraqilis the total activity of both NAD-dependent 7-a-hydroxysteroid dehydrogenase (7-a-OHSDH) and bile salt hydrolase (BSH) increase markedly during the stationary growth phase. Both enzymes were found in the spheroplast lysate and the Triton-soluble washed membrane fractions but only BSH was found in the spheroplast medium. INTRODUCTION: are

obligate

anaerobes

respectively,

free

for

bile

into

carbon

of bile

fraqilis

strains

bacteria

salt

There

is

regulation clear

which

regulating

the

In this mations

the

of man

strains

account,

enzymes

in the

the

in

treated

on the cellular

bacterial

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

(3-6).

bacteria

cells

are

g.

(7,R).

location

anaerobic

the cells

rates

of selected

was shown to effect 1088

bile

of a Gram-positive

(G-) (B. - fraqilis)intestinal

of growth

group

at

carbons

c1 and D hydroxyls

intestinal

these

group

at either

7-a-hydroxyl

the

and nor

important

is in

biotransformation.

we report

and lysozyme

only

and

include

hydroxyl

groups

to both

conjugated

reactions

of the

information

acid

stage

group

to oxidize

rates

of bile

The major

the hydroxyl

3-keto

features

and Gram-negative

-B. fraqilis

the

oxidize

physiological

communication,

by whole

faciens)

that

acid

flora

to transform

dehydroxylation

of published

of the bile

able

of metabolites.

reported

a paucity

bacterial

and -E. aerofaciens

of man are

conjugates,

as reduce

intestinal

and 8% (1,2).

a variety

are

99% of the B. - fragilis

20-25%

7 and dehydrogenases

3,7 or 12 as well

Copyright All rights

about intestinal

acids

than

of which

The normal

hydrolysis

it

Greater

anaerobic the

level

acid

transfor-

(G+) (E. -~ aerobacterium.

In

of two such enzymes

Vol. 69, No. 4, 1976

MATERIALS

AND METHODS:

10852,

-B. fragilis

kindly

donated

Institute

BIOCHEMICAL

ssp. by J.

(VPI)

bacteria

were

Holdeman

and Moore

Glucose

(10)

in either

Growth

with

a Bausch

Preparation centrifugation early Tris-HCl

lysozyme

lysozyme.

yielded

ed by Bell

et al. for

lysozyme

maximal

NaOH and protein Approximately England

Boston,

(Amersham-Searle,

removed with

tography Eneroth

Arlington

volume

at selected

concentrated

extracted

Mass.)

and lysozyme

0.5 mM (total

(13).

into

cells

were

cell

of reaction

et al.

were

(0

products by 20)

Radiolabeled

were

1.0

spotted

and separated products

ml

acid

in solvent

were

located

1089

50 ~g/ml describ-

with

100 ug/ml in 1 N

(40 Ci/M)(New

14 acid individually

Samples

Silica

Ci/M)

to whole

(1.0

ml)

acidified

gel

cell of

were to pH 1.0

ethylacetate.

systems by using

(52

concentration

redistilled

onto

(2-MB)

procedures

to a final

was 5 ml).

with

50 mM

two and one por-

solubolized

up to 6 h, immediately

intervals

the

(12).

was added

suspensions

mixture

by

prepared

incubated

suspensions

Ill.)

Heights,

culture

during

thetaiotaomicron

or deoxycholic-carboxyl-C

treated

time

culture

divided

ssp.

to Lowry

HCl and extracted

radiolabeled plates

according

the

10 mM 2-mercaptoethanol

3.5 X lo6 dpm of cholic-carboxyl-C14

Nuclear,

suspensions

of the

previously

harvested

in 30 min at 25 C using

Samples

measured

a 3 liter

were

The E. - aerofaciens

3 h at 37 C.

B. - fragilis

by measuring

in 20 ml of freshly

fragilis

spheroplast

(11).

(4 C) from

suspensions B 2

cholate.

Cells

0.5 M sucrose,

igith

Extract-

as described

suspensions:

suspended

containing The cell

EDTA.

with

were

by

in Peptone-Yeast

medium

of

20 at 660 nm.

cell

15 min

phase

(pH 7.6)

and 1 mM sodium treated

g for

growth

buffer

treated

cultures

CO2 as described

was determined

and Lomb Spectronic

at 13,700

stationary

growth

were

Polytechnic

Stock

and 0.5 mM sodium

defined

of bacteria

of lysozyme

Laboratory.

was cultured

3 PM hemin

chemically

or PYG medium.

absorbancy

tion

containing

NCTC

VP1 J4-26

of the Virginia

medium under

-E. aerofaciens

thetaiotaomicron

and -E. aerofaciens

Anaerobe

in chopped-meat

RESEARCH COMMUNICATIONS

ssp.

and W. E. C. Moore

University,

(9).

(PYG) medium

of -B. fragilis

ATCC 25285

L. Johnson

maintained

was cultured

Strains

fragilis

and State

AND BIOPHYSICAL

The

thin-layer

chroma-

described

by

a Packard

radio-

Vol. 69, No. 4, 1976

chromatogram bile

scanner.

acids

lation

were

Individual

scrapped

spectrometry.

acid

acid

fragilis: taken

were

20 mM potassium suspending

phosphate

sucrose,

treated

with

10,000

g for

yielded lysozyme

1 mM sodium

French

Pressure

fuged

at 105,000

"spheroplast

Cell

by homogenization

(2% vol/vol) at 105,000 washed

g for

of Nair

et al.

(14)

to the method

were

to recover

and BSH activity are

spheroplast

in

reported

into

and glutamate

of Phibbs

approximately the

lysed

as total

once

International

for

fraction

fraction

BSH activity

were

was again 1 h with

Triton

initial

X-100

centrifuged

"Triton-soluble for

7-c(-OHSDH

according

this

respectively.

(1-U.)

as

centri-

was again

Using

centri-

in 20 ml PP buffer

(GDH) activity

(15).

at

designated

was assayed

sediment,

1090

were

an automatic

was assayed

dehydrogenase

fraction.

through

designated

fraction

Units

at

as "spheroplast

fluid

treated

60% and 70% of the

spheroplast

culture

spheroplasts

The homogenate

and Bernlohr

was 0.5 M in

suspensions

were washed

membrane

(7).

by

20 ml of 20 mM PP buffer

supernatant

membranes

Each spheroplast

prepared

which

designated

The lysed

60 min and the supernatant

previously

were

Cell

fluid

sediment

culture

1 h at 30 C, centrifuged

diluted

homogenizer.

fraction".

one 6 liter

of cell

of cells.

lb/in2.

The Triton-treated

according able

One liter

supernatant were

ssp.

once at 25 C in 200 ml of

approximately

30 min and the

as described

method

for

The sedimented

at 4 C.

from

Spheroplasts

EDTA.

30 min at 4 C and the

g for

membrane

activity

sodium

(4 C) at 8,000

in a tissue

at 105,000

and washed

EDTA and 20 mM 2-ME and passed

lysate".

to keto-bile

in 20 ml of 20 mM PP buffer

spheroplasts

g for

scintil-

of B. fragilis

in methods,

(pH 7.0).

20 min at 20 C and the

containing

and keto-

by liquid

acid

fractions

2.2 g wet weight

(50 ug/ml)

The sedimented

ities

of cells

about

medium".

fuged

stages

20 mM in 2-ME and 1 mM in

absorbance

acids

point.

(PP) buffer

8 g wet weight

of each bile

as indicated

growth

to bile and counted

in spheroplast

harvested,

at different

vials

conversion

each time

RESEARCH COMMUNICATIONS

corresponding

scintillation

enzyme activities

Cells

sample

into

for

AND BIOPHYSICAL

spots

The percent

was determined Bile

1.6

BIOCHEMICAL

to the

was assayed

procedure,

amount

we

of 7-a-OHSDH

Enzyme activ-

of enzyme activity

per

Vol. 69, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

60

0

Fig. 1. Rates of conversion of cholate to 7-ketodeoxycholate in whole cell suspensions (01, spheroplasts (A), resuspended spheroplast co), resuspended sphercplasts plus 0.65 I.U. of exogenously added NAD-dependent 7-a-hydroxysteroid dehydrogenase (0) and "spheroplast medium" (m) of B. fragilis ssp. thetaiotaThe initial cell suspension contained 3.28 mq of whole cell omicron NCTC 10852. protein per ml.

RESULTS: to 7-KD in 6-8% in

24-36

72 h.

of cholate cells

Growing

h, whereas The data

showed

faciens

over

verted

rapidly

a 2-fold

course

(>lOO-fold)

of conversion

lysozyme

treated

Figures

taken

derived

from

the

cells

taken

and "spheroplast

points

indicated

(Fig.

rates

(12-KL) ,

whereas

to 12-KL

only

about

of transformation

of cholate

in cell

In contrast,

of conversion

cholate

The lysozyme

2).

(Fig. total

converted

of conversion occurred

of 6 h (Fiq.

3) and 7-c(-OHSDH

membrane" at time

transformation

suspensions

1 show the

70-80%

and spheroplasts.

in the rate

the rate

3 and 4 shows

of BSH (Fig.

cells

of deoxycholate

cell

(G+) cultures

in Figure

to 12-ketolithocholate

the rate

washed

presented

cholate

(G-1 converted

E aerofaciens -A

increase

a time

increased

fractions

of B. - fragilis

to 7-KD in B. - fraqilis

No detectable

edly

cultures

treated

to 7-KD.

suspensions

of E . aero

deoxycholate lysozyme

of cholate increased

was con-

treatment

to 7-KD. only

2-fold

markHowever,

in the

same

2).

units

of enzyme

at different 4) activity lysate"

fractions

by numbered

arrows

1091

activity

growth

in

stages.

increased

in

derived during

The total the from

the

spheroplast units

"Triton-soluble culture

stationary

samples peroid

Vol. 69, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

HOURS

Fig. 2. Rates of conversion of selected bile acid to keto-bile whole cell suspensions (*,A) and lysozyme treated cell suspensions The initial cell suspension contained of E. aerofaciens VP1 J4-26. whole cell protein per ml.

acid

in

(O,A)

4.5 mg

1.6 1.0 0. a cE 0.6 8 :

a

IO

12

I4

I6

HOURS Fig.

3. Regulation and NADP-dependent SSP. fragilis ATCC 25285 national units (I.U.) of soluble washed membrane" was recorded as culture angles)

of growth.

In contrast,

(Figs.

3 and 4).

(Fig.

3) in these

(Fig.4)

and cellular location of bile salt hydrolase (BSH) (triglutamate dehydrogenase (GDH) (squares) in B. fragilis Symbols indicate tog1 intercultured in PYG medium. enzyme activity in "spheroplast lysate" (A,O),"Triton(A, 0) and "spheroplast medium" (A,a). Cell growth absorbancy at 660 nm (a).

Moreover,

the

total

units

BSH activity

same spheroplast

of GDH remained was detected

preparations

and GDH activities.

1092

relatively

in the

in contrast

constant

"spheroplast to both

medium'

7-ly-QHSDH

Vol. 69, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1.6 I.0 0.8 : s z

0.6 0.4-

0.

Fig. 4. dehydrogenase (GDH) (squares) Symbols indicate plast lysate" medium" (l),m).

by intestinal

anaerobic

permeability

rate

block treated

mation

by the

may play in the

We also

bacteria

14

act

16

Burman

fact

rapidly

regulation

is

can be detected

that

et al.

(16)

a more important

unclear. associated in the

acid

transformation

communication

show that

cell

wall

transform also

role

than

G+ bacteria

the

stationary

Large

amounts

as a significant of transformation

in

This

observation

at a markedly

suggests

that

is reduced

G- intestinal

in cholate

transfor-

of man. the activities growth

medium"

1093

of both

phase.

of 7-a-OHSDH

the membrane

"spheroplast

cholate

the

the peptidoglycan

to cholate.

data

in -B. fragilis

with

that

These

tract

act rates

reported

block

Gf bacteria

to G- bacteria.

during

of bile

in this of the

as a permeability

show that

to be tightly

reported

rates

as shown by the greater

gastrointestinal

increased

on the

layer

to cholate cells.

as compared

bacteria

12 HOURS

data

peptidoglycan

in --E. coli

supported

IO

The selected

lysozyme-sensitive

layer

8

Regulation and cellular location of NAD-dependent l-a-hydroxysteroid (7-a-OHSDH) (hexagons) and NADP-dependent glutamate dehydrogenase in k fragilis ssp. fragilis ATCC 25285 cultured in PYG medium. total international units (1-U.) of enzyme activity in "sphero(O,lJ), "Triton-soluble washed membrane" (a,a) and "spheroplast Cell growth was recorded as culture absorbancy at 660 nm (0).

DISCUSSION:

lysozyme

I

7-a-OHSDH

The mechanism

and BSH activities

fraction suggesting

and in addition, a "periplasmic"

and BSH

of enzyme are shown BSH activity location

Vol. 69, No. 4, 1976 of this that

enzyme.

The intracellular,

any spheroplast

lysis

ificance yet

BIOCHEMICAL

of these

clear,

bacterial

different

we suggest cytoplasmic

ACKNOWLEDGEMENTS: National Cancer Institute. technical assistance.

would

RESEARCH COMMUNICATIONS

GDH was used as a marker be detected.

locations

that

AND BIOPHYSICAL

of bile

the transformation

Although, acid

enzyme

the physiological

transforming

reactions

to assure

may occur

enzymes

signis not

in or on the

membrane. This

work was supported by grant No. CA17747-01 from the We also wish to thank Becky Fricke for her excellent

REFERENCES: 1. 2. 3. 4. 5.

6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16.

Darsar, B. S., Shiner, M. and McLoed, G. M. Gastroenterology 56:71-79 (1969). Moore, W. E. C. and Holdeman, L. Appl. Microbial. 27:961-979 (1974). Aries, V. and Hill, M. J. Biochim. Biophys. Acta 2=:526-534 (1970). Aries, V. and Hill, M. J. Biochim. Biophys. Acta 202:534-543 (1970). Midtvedt, T. and Norman, A. Acta Pathol. MicrobiorScand. (1967). -71:629-638 Lewis, R. and Gorbach, S. Arch. Intern. Med. 130:545-549 (1972). Hylemon, P. B. and Sherrod, J. A. J. Bacteriorl12:418-424 (1975). MacDonald, I. A., Williams, C. N., Mahony, D. E.Td Christie, W. M. Biochim. Biophys. Acta 384:14-24 (1975). L. Vxnd W. E. C. Moore (ed.) 1973 Anaerobe laboratory Manual, Holdeman, 2nd ed. Virginia Polytechnic Institute and State University, Blacksburg. Hylemon, P. B. and Phibbs, P. V. Biochem. Biophys. Res. Commun. 60:88-95 (1974). Bell, G. R., LeGall, J. and Peck, H. D. J. Bacterial. -120:994-99T(1974). Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. J. Biol. Chem. 193:265-275 (1951). Eneroth, P. J. Lipid Res. 4:11-16 (1963). Nair, P. P., Gordon, N. and Rebach, J. J. Biol. Chem. -242:7-11 (1967). Phibbs, P. V. and Bernlohr, R. N. J. Bacterial. 106:375-385 (1971). Burman, L. G., Nordstrom, K. and Bloom, G. D. J. Bacterial. 112:13641374 (1972).

1094