Gerze, 10X (1991) 121-125 8 1991 Elsevier Science Publishers
GENE
121
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06136
Bacillus subtilis inositol dehydrogenase-encoding Escherichia coli (Recombinant
DNA;
~11; prophage;
transformation;
gene (idh): sequence and expression in
inositol
catabolism;
iol class of genes)
Yasutaro Fujita, Katsuhiro Shindo, Yasuhiko Miwa and Ken-ichi Yoshida Department of Biotechnology, Fukuyama Universit!‘,Fukuyama 729-02 (Japan) Received by A. Nakazawa: 4 July 1991 Revised/Accepted: 13 August/l4 August 1991 Received at publishers: 3 September 1991
SUMMARY
The Bacillus subtilis inositol dehydrogenase (Idh)-encoding gene (idh) was cloned in the B. subtilis temperate phage, ~11, and then in Escherichiu coli plasmids (pBR322 and pUC 118). The nucleotide sequence of the idh gene, which consists of 344 codons and whose product has an M, of 38 351, was determined. E. coli, bearing pIOLOSd15, in which expression of the idh gene is under the control of the iuc promoter of pUCl18, overproduced an active Idh to approx. 20% of total protein upon addition of isopropyl-/?-D-thiogalactopyranoside. This overproduced enzyme cross-reacted with an anti-Idh antibody, and exhibited the same M, and substrate specificity as those of the B. subtilis enzyme.
INTRODUCTION
A variety of microorganisms, including B. subtilis, can utilize myo-inositol as their sole carbon source. Idh (WZJJOinositol 2-dehydrogenase; EC 1.1.1.18) converts m_voinositol to 2-keto-myo-inositol (2-inosose). This enzyme is an inducible enzyme responsible for the first step of inositol catabolism, as first verified in Aerobacter aerogenes (Berman and Magasanik, 1966). In B. subtilis Idh is also induced
Correspotzdence of Engineering, Fukuyama-shi,
to; Dr. Y. Fujita, Fukuyama Hiroshima
Tel.(Xl-X49)36-2111; Abbreviations: pair(s);
of Biotechnology,
985 Sanzo,
Faculty
Higashimura-cho,
EXPERIMENTAL
Fax(81-X49)36-2213. Ap, ampicillin;
B., Bacillus; bp, base
digf see section a; Idh, inositol dehydrogenase;
Idh; Ig, immunoglobulin;
iol, gene responsible
idh, for the
ability to grow on myo-inositol; IPTG, isopropyl-p-D-thiogalactopyranoor 1000 bp; LB, Luria-Bertani (medium); nt, nucle-
side;kb, kilobase
otide(s);
ORF, open reading
phoresis;
SD, Shine-Dalgarno
( ), denotes prophage denotes
a truncated
state;
AND
DISCUSSION
729-02 (Japan)
aa. amino acid(s);
d, deletion;
gene encoding
Department
University,
upon addition of inositol, and this induction is subjected to catabolite repression (Ramaley et al., 1979; Nihashi and Fujita, 1984). The enzyme was purified to homogeneity and characterized (Ramaley et al., 1979). The idh gene of B. subtilis is one of the iol genes which render cells able to grow on inositol. The M-6 mutation is located at 343” on the B. subtilis chromosome inside the digfdeletion (Fujita and Fujita, 1983). In this communication, we describe the cloning, sequencing and expression of the B. subtilis idh gene in E. coli.
frame;
PAGE,
sequence;
polyacrylamide-gel
SDS, sodium dodecyl
[ 1.denotes plasmid-carrier
gene at the indicated
side.
state;
electrosulfate;
’ (prime),
(a) Cloning of the Bacillus subtilis idh gene B. subtilis strain 61656 carries a large deletion (digf) covering loci including iol, gnt and fdp (Fujita and Fujita, 1983). Besides this strain, we isolated several iol mutants of our B. subtilis standard strain, 600 15 (101~ ), according to the method described previously (Fujita and Fujita, 1983). Among the iol mutant strains examined, strain YF 111 iol-4 1 as well as strain 6 1656 could not synthesize Idh at all (Table I). To clone the B. subtilis idh gene, we carried out prophage
122 TABLE
I of Idh in Bacilks subtilis and Eschericftia coli cells carrying
Synthesis
the
idh gene” Strain”
Inositol
in the medium
Idh activity” (nnlol/mi~
per mg protein)
B. subtilis 10
60015 iol+
363
61656 digf