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