The gene encoding the citric acid cycle enzyme, citrate synthase, has been cloned from the thermoacidophilic archaebacterium, Thermoplasma acidophilum. We report the sequencing of this gene and its flanking regions, and the derived amino acid sequence of the enzyme is compared by multiple-sequence alignment analysis with those of citrate synthases from eubacterial and eukaryotic organisms. The similarity is < 30% between the archaebacterial and non-archaebacterial sequences, although the majority of residues implicated in the catalytic action of the enzyme have been conserved across all three kingdoms. The cloned archaebacterial gene has been expressed in Escherichia coli to produce catalytically active citrate synthase. This is the first reported sequence of citrate synthase from the archaebacteria.Citrate synthase, the first enzyme of the citric acid cycle, has been studied from organisms that represent all three primary kingdoms : the eukaryotes, the eubacteria and the archaebacteria [l -31. Two oligomeric forms of citrate synthase have been identified: a dimeric form found in eukaryotes and Gram-positive eubacteria, and a hexameric form found in Gram-negative eubacteria. Both types are made up of identical subunits, with M , values of approximately 50 000, the hexameric form appearing to behave functionally as a trimer of the basic dimer [4]. However, the two forms of the enzyme differ in their regulatory sensitivities: the dimer is inhibited isosterically by ATP whereas the hexameric form is inhibited allosterically by NADH and, in some cases, additionally by 2-oxoglutarate [l -31.The primary amino acid sequences of citrate synthase from the eukaryotes pig heart [5] and kidney [6], Arabidopsis thaliana [7] and Saccharomyces cerevisiae [8], and from the eubacteria Escherichia coli [9, lo], Rickettsia prowazekii [l 11, Acinetobacter anitratum [12] and Pseudomonas aeruginosa [ 131 have been determined, either by amino acid sequencing of the protein or from the DNA sequence encoding the gene. In addition, a high-resolution X-ray crystallographic structure is available for the pig heart enzyme [14, 151. This has allowed identification of 12 residues involved in the active site; multiple sequence comparisons [16] have indicated that the majority of these 12 active site residues are conserved between all citrate synthases.To extend our studies on the diversity of citrate synthases and the correlation of oligomeric structure and function, we have initiated investigations on the enzyme from the archaebacteria. These organisms are thought to constitute a phylogenetically distinct evolutionary group, in addition to Correspondence to M. J. Danson, Department of Biochemistry, Enzyme. Citrate synthase (EC 4.1.3.7). Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X5.5282.

The citrate synthase gene from the thermophilic archaebacterium. Thermoplasma acidophilum was expressed in Escherichia coli, yielding an active product of the expected molecular weight. Manipulation of the citrate synthase gene in a series of pUC19 constructs showed that the presumed Thermoplasma ribosome binding site is recognized by the E. coli ribosome. A rapid purification of the expression of the expression product to homogeneity was achieved, based on the thermostability of Thermoplasma citrate synthase.

Motility of the alkalophilic

Bacillus sp. strain C-125 has been chosen as a model alkalophilic bacterium to understand how adaptation to growth at high pH is achieved. To aid genetic analysis, we have started characterization of its genome. By using the two infrequently-cutting restriction endonucleases, A scI and Sse83871, in conjunction with pulsed-field electrophoretic techniques, the size of the genome was found to be 3.7 Mb. Southern blot analysis of single, double and partial digests of Bacillus sp. strain C-125 DNA, using AscI-linking clones, gene probes and purified Bacillus sp. strain C-125 restriction fragments, allowed a putative chromosome map to be constructed.

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