Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1

Abstract

The genes coding for two structurally different isocitrate dehydrogenase isozymes (IDH-I and IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, were cloned and sequenced. Open reading frames of the genes (iedI and iedII) are 1,248 and 2,229 bp in length, respectively. The amino acid sequences predicted from the open reading frames of iedI and icdII corresponded to the N-terminal amino acid sequences of the purified IDH-I and IDH-II, respectively. No homology was found between the deduced amino acid sequences of the isozymes; however, the IDH-I, a dimeric enzyme, had a high amino acid sequence identity (74.3%) (18,25). One, IDH-I, is a homodimer of thermostability comparable to that of the enzymes from mesophiles, and the other, IDH-II, is a monomer with extreme lability above 20°C. In addition, immunochemical properties and N-terminal amino acid sequences of the isozymes have been shown to be different from each other (18) but showed similarities to those of the respective types of other bacterial IDHs (12).The genes encoding dimeric IDHs of E. coli (35) and T thermophilus (21) have been cloned and sequenced, respectively. There is significant homology between these predicted amino acid sequences, and the amino acid residues which are suggested to interact with isocitrate (17) are also conserved in the two enzymes. Recently, homology between the deduced amino acid sequences of Saccharomyces cerevisiae (9) heart (32) mitochondrial IDHs were reported to be very high, but the eucaryotic enzymes exhibit low amino acid sequence similarity to the E. coli IDH. However, Soundar and Colman (32) reported that the amino acid residues which are implicated in metal-isocitrate binding in the E. coli enzyme (17) are located in the same general region of the eucaryotic enzymes. These findings have implications for the molecular evolution of IDH. But the relation between dimeric and monomeric IDHs is still unclear, since no sequence of a monomeric IDH has been determined.Since Vibrio sp. strain ABE-1 possesses structurally different IDH isozymes (18,25), investigation of the genes encoding the isozymes is necessary to elucidate the physiological and evolutionary basis of the coexistence of the isozymes at the molecular level. In this paper, we report the cloning and sequencing of the genes coding for the IDH isozymes. It was found that the two genes are closely adjacent, and the predicted amino acid sequence of the IDH-I is very similar to that of the E. coli enzyme but quite different from that of IDH-II. Some regulatory aspects of the expression of the cloned genes are also presented. MATERIALS AND METHODS Materials

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

“…Thirdly, a further indication that cold-active promoters might be involved in adaptation to low temperature has been found in a psychrophilic Vibrio sp. : this organism expresses two structurally and genetically different isozymes of isocitrate dehydrogenase, and one of these, IDH-II, is extremely thermolabile and is encoded by a gene that contains a CCAAT motif in its promoter region (Ishii et al, 1993). In the present study, we found that pnp is cold inducible in Y. enterocolitica, and we showed that a 0.33 kbp region upstream of pnp is sufficient to retain its cold induction.…”

Cloning, Sequencing, and Expression of a Gene Encoding the Monomeric Isocitrate Dehydrogenase of the Nitrogen-fixing Bacterium,Azotobacter vinelandii

“…This sequence has 82z and 76z identity with those of C. maris IDH-II 22) and V. parahaemolyticus IDH, 2) respectively. The optimum temperature for activity of the puriˆed IDH was found to be 409 C, indicating that it is a typical mosophilic en- zyme.…”

“…Conversely, no such a similarity was found in the amino acid sequence deduced from the ORF2, nor was found homologs in the nucleotide and protein databases. In addition, alignment of the amino acid sequences of the bacterial monomeric type IDHs showed that ORF1 has a high amino acid sequence identity (66.2z and 62z) to the enzymes of C. maris 22) and Corynebacterium glutamicum, 6) respectively (Fig. 4).…”

The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris a aThe GenBank accession number for the sequence reported in this paper is AB066287.

“…Two downstream primers were designed from the sequences of highly conserved regions of ICL genes cloned from various organisms (Rehman & McFadden, 1996) ; 5h-SCCATRTGNCCRCAYTTYTT-3h (20-mer) corresponding to KKCGHMA and 5h-CNARRTGN-CCRCAYTTYTT-3h (19-mer) corresponding to KKCGHLG. C. maris genomic DNA was isolated as described by Ishii et al (1993). Amplification was carried out for 30 cycles in a DNA thermal cycler 4800 (Perkin-Elmer) in 100 µl reaction mixture containing 1n3 µg genomic DNA, 200 pmol each forward and reverse primer and 2n5 U KOD DNA polymerase (Toyobo) in a buffer system prepared by the manufacturer.…”