Yonago Acta medica 2006;49:71–75

Aerobic and Anaerobic Biotransformation of Bile Acids byEscherichia coli (III)Yoshio Ogura, Tsuyoshi Takei, Nobuo Yamaga, Kazuo Yamada and Kiyohisa Uchida
Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503 JapanThe oxidation/reduction reactions of bile acids by Escherichia coli (E. coli) K-12 were
examined in both Davis and brain-heart infusion (BHI) media under aerobic and anaerobic conditions. The pH in the Davis medium changed by almost the same amount,around pH 6.5–7.0 in both aerobic and anaerobic cultures, but the pH in the BHI medium was different in both cultures, that is, about pH 9.0 in the aerobic culture but only
about 6.5 in the anaerobic culture. The growth curve of E. coli in the Davis medium
showed a similar pattern in both conditions. Cholic acid (CA) was oxidized to 3α12αdihydroxy-7-oxo-5β-cholanoic acid (3α12α7=O) in both cultures, but the reaction in theanaerobic culture was somewhat slower than that in the aerobic culture. On the other
hand, reduction of 3α12α7=O to CA did not occur in the aerobic culture, but about 10%
reduction was observed in the anaerobic culture after 4 days. These data suggest thatthe oxidation/reduction reaction of E. coli was oxidative in aerobic culture but reductive in anaerobic culture and these characteristics were not due to the changes in the pHof the medium. The reactions of CA and glycocholic acid to crude 7α-HSDH preparedfrom E. coli were examined and it was found that both free and conjugated CA as a substrate for the 7α-HSDH showed similar Km values.
Key words: Escherichia coli; cholic acid; glycocholic acid; 3α,12α-dihydroxy-7-oxo-5β-cholanoicacid; in vitro biotransformation

bile acid to CA, whereas in anaerobic culture
about 50% of CA was oxidized and about 50% of
7-oxo-bile acid was reduced. A similar result wasalso obtained with chenodeoxycholic acid. Therefore, we speculated that the oxidation/reduction
reaction of E. coli attained different equilibrium
values depending on the presence or absence ofoxygen. In other words, the reaction equilibrium
shifted to the oxidative side in aerobic culture but
was almost equally balanced in anaerobic culture.
In previous experiments with brain-heart

Escherichia coli (E. coli) possesses 7α-hydroxysteroid dehydrogenase (7α-HSDH) and oxidizes
7α-hydroxyl bile acids to the corresponding
7-oxo-bile acids, or reduces 7-oxo-bile acids to thecorresponding 7α-hydroxyl bile acids (Aries andHill, 1970; Haslewood and Haslwood, 1976; Macdonald et al., 1973). In a previous paper (Ogura etal., 2003), we reported that E. coli in aerobic culture transformed a large proportion of cholic acid
(CA) to 3α,12α-dihydroxy-7-oxo-5β-cholanoic
acid (3α12α7=O) but hardly transformed 7-oxo-

Abbreviations: BHI, brain-heart infusion; CA, cholic acid; GCA, glycocholic acid; GLC, gas-liquid chromatography;
7α-HSDH, 7α-hydroxysteroid dehydrogenase; Me-DMES, methyl ester dimethylethylsilyl ether; PHP GEL, piperidinohydroxypropyl dextran gel; 3α12α7=O, 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid; ββ, 7β,12β-dihydroxy-5β-cholanoic
acid

71

Y. Ogura et al.

Shimadzu, Kyoto, Japan, and dimethylethylsilylimidazole from Tokyo Kasei Kogyo, Tokyo, Japan. The other reagents and solvents of analyticalgrade were obtained from Wako Pure Chemical
Industries, Osaka, Japan. If not otherwise stated,
the solvents were distilled once before use.

infusion (BHI) medium, the pH of the cultured
medium increased to about 9 in aerobic culture
but decreased to around 6.5 in anaerobic culture(Ogura et al., 2003). Since it has been reported
that the reaction of 7α-HSDH is oxidative in alkaline conditions but reductive in acidic conditions(Groh et al., 1993; Macdonald et al., 1983), we
suspected our previous results were dependent on
the difference of pH of the medium rather thanon the difference between aerobic and anaerobic
conditions.
Meanwhile, we found that when E. coli wascultured in the Davis medium (Davis and Mingioli, 1950) containing 0.2% glucose, the pH of
the medium did not differ significantly between
aerobic and anaerobic conditions. Therefore, we
examined the effect of E. coli on bile acids inboth aerobic and anaerobic cultures in the Davis
medium and compared it with that in the BHI medium.
In addition, E. coli dehydrogenates taurineor glycine-conjugated CA to the corresponding
taurine- or glycine-conjugated 3α12α7=O without deconjugation, but the conversion rates forconjugated CA were lower than for free CA (Ogura
et al., 2003). In the present experiments, we analyzed the effect of concentrations of conjugatedand free CA on crude 7α-HSDH prepared from E.
coli to clarify the difference mentioned above.

Bacteriological procedures
E. coli K-12 was obtained from the American
Type Culture Collection (Manassas, VA). The
BHI medium (Becton Dickinson, Sparks, MD)
and the Davis medium (pH 7.2) were used for theculture experiments. The Davis medium contains
0.7% K 2 HPO 4, 0.2% KH 2 PO 4, 0.047% sodium
citrate, 0.005% MgSO4, 0.1% (NH4)2SO4 and 0.2%
glucose. The pH was adjusted to 7.2. E. coli
was precultured aerobically in the BHI medium
at 37˚C for 1 day. Aliquots of the precultured E.
coli (3 × 107 in 10 µL medium) was added eachto 2 mL of the fresh BHI medium, or to the fresh
Davis medium, containing 1 mM of bile acids andcultured at 37˚C for 4 days. The aerobic culture
was performed in the open air with an incubator(PCI-100; Iuchi, Osaka, Japan) adjusted at 37˚C,and the anaerobic culture was performed with theuse of a gas-pack (BBL Gas Pak Pouch System;
Becton Dickinson). Viable cells of E. coli in both
media were counted by colony formation on anagar plate after seeding the diluted culture medium.

Materials and Methods
Analytical methods
Chemicals

Bile acids were analyzed according to the method
reported previously (Ogura et al., 2003). A portion of the medium cultured, 100 µL, was heatedwith 8 volumes of ethanol at 80˚C for 10 min andthen ﬁltered through a glass microﬁber ﬁlter (GF/
C; Whatman, Maidstone, United Kingdom). The
ﬁltrate was evaporated to dryness under a streamof nitrogen and the residue was acidified withdiluted hydrochloric acid. The bile acids were extracted with ethylether, converted to methyl ester
dimethylethylsilyl ether (Me-DMES) derivatives

CA was purchased from Sigma Chemical, St.
Louis, MO. 3α ,12α-Dihyd roxy-7-oxo-5β cholanoic acid (3α12α7= O) was prepared byoxidation of CA with N-bromosuccinimide (Fieser and Rajagopalan, 1949). 7β,12β-Dihydroxy5β-cholanoic acid (ββ), an internal standard foranalysis by capillary gas-liquid chromatography(GLC), was synthesized as described previously(Arimoto et al., 1982). Piperidinohydroxypropyl dextran gel (PHP GEL) was purchased from72

Bile acid biotransformation by E. coli

both Davis and BHI media, which showed different pH after cultures in aerobic and anaerobic
conditions.
Changes in the pH of the BHI and Davis
media cultured with E. coli are given in Fig. 1.
The pH in the Davis medium changed almost bya similar value under both aerobic and anaerobic
conditions; it decreased slightly to pH 6.5 on theﬁrst day of incubation and remained almost constant or very slightly increased thereafter, while
the pH in the BHI medium markedly increased
to about pH 9.0 in the aerobic condition but decreased to about pH 6.5 in the anaerobic condition. The changes in the Davis medium were
almost the same to the change in the BHI medium
in the anaerobic condition.
Figure 2 shows changes in viable cell counts
during aerobic and anaerobic cultures of E. coli
K-12 in the Davis medium. Although the viable
cell counts in the anaerobic culture were higher
than those in the aerobic culture, they changed
almost by the same amount in both conditions.
Figure 3 shows the time course of changes in
7α-dehydrogenation of CA and 7α-hydrogenationof 3α12α7=O by E. coli in the Davis and BHI
media. In the Davis medium, CA was oxidized
to 3α12α7=O in both aerobic and anaerobic cultures, but the reaction in anaerobic culture wassomewhat slower than that in aerobic culture. On�� ���
�� ��

��

� ��
� ��
� ��
�� ��
��

��

��

��

� �����

����������
���������������

�����������������

�������������

���������������

Fig. 1. Changes in pH of the Davis medium and thebrain-heart infusion (BHI) medium during aerobic andanaerobic cultures of Escherichia coli K-12.

and quantiﬁed by gas-liquid chromatography witha Hicap CBP-1 capillary column (25 m × 0.25
mm i.d.; Shimadzu). Conversions were expressed
as % of bile acids converted in the culture medium.
Enzyme activity of cell-free 7α -HSDH
E. coli K-12 was cultured aerobically for one day
in the BHI medium. Crude 7α-HSDH was prepared according to the method of Macdonald etal. (1973). The enzyme activity was assayed by
the method of Macdonald et al. (1974, 1975) witha slight modification. Briefly, the reaction was
performed at 37˚C for 10 min in 0.18 M glycineNaOH buffer (pH 9.5) containing 7 mM NAD+, 0
to 4 mM bile acid and 45 µL of the crude enzyme
preparation in a ﬁnal volume of 1 mL.

����������
� ���

�����

��� �

Results and Discussion

�� �

��� �

Major conversions of bile acids by intestinal bacteria include deconjugation, oxidation of hydroxygroups at C-3, C-7 and/or C-12 positions, and 7α/β-dehydroxylation. E. coli possesses 7α-HSDH
for the oxidation, but has neither deconjugation
nor dehydroxylation activity. In the present study,
we examined the effect of E. coli on bile acids in

��

��

��

��

��

� �����

����������
�������

���������

Fig. 2. Changes in viable cell counts during aerobic andanaerobic cultures of Escherichia coli K-12 in the Davis
medium.

73

Y. Ogura et al.

A

Davis medium��������������

��������� ����� � ���������
�%�

���

����������

��
���
��
���

��

��

��

��

��
���
��
���

� �����

��������������

B���� � ���������
�%�

���

��

����������

����������

���

���
��
���

��

��

��

��

��

� �����

����������

BHI medium
�

���

�%�

��

� �����

����������

�����������������

���������

��������� �

���

�%�

��
���
��
���

��

��

��

��

� �����

����������
�����������������

���������

Fig. 3. Conversion of cholic acid (CA) and 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid (3α12α7=O) by Escherichia
coli K-12 in the Davis medium and brain-heart infusion (BHI) medium.

the other hand, the reduction of 3α12α7=O to CA
did not occur in aerobic culture, but about 10% reduction was observed in anaerobic culture after 4
days. In the BHI medium, the oxidation of CA in
aerobic culture was much higher than that in anaerobic culture and the reduction of 3α12α7=O,
though it was not observed in aerobic culture, occurred obviously in anaerobic culture.
These changes in the Davis medium wereless marked than those in the BHI medium, butnevertheless showed the same characteristics for
CA oxidation and 7-oxo-bile acid reduction, suggesting that this 7α-HSDH reaction was oxidative
in aerobic culture but reductive in anaerobic culture.
Therefore, these data indicate that the oxidation/reduction reaction of 7α-HSDH is not affected by extracellular pH. In addition, it has beenreported that the intercellular pH of E. coli ismaintained at around 7.6 in a wide range of extracellular pH (Padan et al., 1976; Shimamoto et al.,1994). Therefore, we conclude that the 7α-HSDH
activity of E. coli is not affected by changes in pHof the culture medium.
NAD+ is a cofactor required for the oxidative
activity of 7α-HSDH. The content of NAD+ in
E. coli was reported to increase in aerobic culture(Leonardo et al., 1996). This would explain theshift to the oxidative side in the transformation ofbile acids by 7α-HSDH.
In our previous paper, we found that conjugated CA was dehydrogenated by E. coli, but toa lesser extent than free CA (Ogura et al., 2003).
The reactions of CA and glycocholic acid (GCA)
by the crude enzyme preparation were then examined in vitro. As shown in Fig. 4, CA and GCA

74

Bile acid biotransformation by E. coli

showed almost the same reactions, suggesting thatboth free and conjugated CA have similar afﬁnities for the enzyme. This result coincides with thedata demonstrated by Haslewood and Haslewood(1976). Therefore, the data with the crude enzyme preparation suggest that the lower reaction
for conjugated CA is due to a slower penetration
rate of conjugated CA into living cells than freeCA.
In conclusion, the oxidation/reduction reaction of E. coli was oxidative in aerobic culture but
rather reductive in anaerobic culture. These results were not due to the changes in the pH of themedium. In addition, the transport step throughthe cell membrane will be one of the rate limits
for the transformation of bile acids in whole cells.

����������������

�����������������������
������� �
��������������������������
����� �

������ �

�
���

��

��

��

��

��

��

��

��

� ������

��

��

�

��

��

� ����

���������
�����

Fig. 4. Dehydrogenation of cholic acid (CA) and glycocholic acid (GCA) in vitro by 7α-hydroxysteroid dehydrogenase prepared from Escherichia coli K-12.

Acknowledgments: This work was supported by a Grantin-Aid for Scientific Research from the Yakult BioScience Foundation (2005).

Hydroxysteroid dehydrogenase from Escherichia
coli B: preliminary studies. Biochim Biophys Acta
1973;309:243–253.
9 Macdonald IA, Williams CN, Mahony DE. A 3αand 7α-hydroxysteroid dehydrogenase assay for
conjugated dihydroxy-bile acid mixtures. Anal Biochem 1974;57:127–136.
10 M a c d o n a ld I A , Wi l l i a m s C N, M a ho ny DE ,Christie WM. NAD- and NADP-dependent 7αhydroxysteroid dehydrogenases from Bacteroides
fragilis. Biochim Biophys Acta 1975;384:12–24.
11 Macdona ld I A, Bok ken heuser V D, Winter J,
McLernon AM, Mosbach EH. Degradation of steroids in the human gut. J Lipid Res 1983;24:675–700.
12 Ogura Y, Yamaga N, Kido Y, Katayama R, Yamada
K, Uchida K. Aerobic and anaerobic biotransformation of bile acids by Eschirichia coli (I). Bioscience
Microﬂora 2003;22:133–137.
13 Padan E, Zilberstein D, Rottenberg H. The proton
electro-chemical gradient in Escherichia coli cells.
Eur J Biochem 1976;63:533–541.
14 Shimamoto T, Inaba K, Thelen P, Ishikawa T,
Goldberg EB, Tsuda M, Tsuchiya T. The NhaB Na+/H+ antiporter is essential for intracellular pH regulation under alkaline conditions in Escherichia coli. J
Biochem 1994;116:285–290.

References
1 Aries V, Hill MJ. Degradation of steroids by intestinal bacteria II. Enzymes catalysing the oxidoreduction of the 3α-, 7α- and 12α-hydroxyl groups in cholic acid, and the dehydroxylation of the 7-hydroxyl
group. Biochim Biophys Acta 1970;202:535–543.
2 A r i moto K , Ad a ch i K , Ya m aga N. 7β,12 β Dihydroxy-5-cholan-24-oic acid as an internal standard for quantitative determination of bile acids bygas chromatography. Steroids 1982;39:631–641.
3 Davis BD, Mingioli ES. Mutants of Escherichia coli
requiring methionine or vitamin B12 . J Bacteriol
1950;60:17–28.
4 Fieser LF, Rajagopalan S. Selective oxidation with
N-bromosuccinimide. I. Cholic acid. J Am Chem
Soc 1949;71:3935–3938.
5 Groh H, Schade K, Hörhold-Schubert C. Steroid
metabolism with intestinal microorganisms. J Basic
Microbiol 1993;33:59–72.
6 Haslewood ES, Haslewood GAD. The speciﬁcity ofa 7α-hydroxysteroid dehydrogenase from Escherichia coli. Biochem J 1976;157:207–210.
7 Leonardo MR, Dailly Y, Clark DP. Role of NAD in
regulating the adhE gene of Escherichia coli. J Bacteriol 1996;178:6013–6018.
8 Macdonald IA, Williams CN, Mahony DE. 7α-

Received January 11, 2006; accepted February 24, 2006
Corresponding author: Yoshio Ogura, PhD

75