Mutant citrate synthases from Acinetobacter generated by transformation [proceedings

Abstract

Citrate synthase (EC 4.1.3.7) from aerobic Gram-negative bacteria is specifically and allosterically inhibited by NADH and de-inhibited by AMP (Weitzman & Jones, 1968; Weitzman & Danson, 1976). The enzyme is tetrameric with a mol.wt. of about 250000 and is a typically 'large' citrate synthase, as opposed to the 'small' dimeric citrate synthases (mol.wt. approx. l00000), which are elaborated by Gram-positive bacteria and eukaryotic organisms (Weitzman & Dunmore, 1969). To explore the structure-function relationships between the 'large' citrate synthases and their specific response to NADH, it would be valuable to have mutant forms of the enzyme with altered structure and catalytic or regulatory behaviour. The investigation of such variants in comparison with the natural enzyme might contribute to an understanding of the molecular requirements for the production of a tetrameric, rather than a dimeric, enzyme and for the constitution of receptor sites for regulatory effectors. In the precedingcommunication(Harford & Weitzrnan, 1978) wedescribed thestrategy for generating mutant forms of citrate synthase from Escherichia coli by reversion from an original citrate synthase-deficient mutant. In the present study we report the production of mutant forms of the enzyme from Acinetobacter lwofi (a Gram-negative aerobe) by transformation. Juni has demonstrated that auxotrophic strains of Acinetobacter are competent for genetic transformation to prototrophy (Juni & Janik, 1969; Juni, 1972), and we have found that mutants of Acinetobacter lacking citrate synthase, and thus requiring glutamate for growth, may be transformed to prototrophy by crude preparations of DNA not only from Acinetobacter but also from Pseudomonas aeruginosa (S. Harford, C. J. Beecroft & P. D. J. Weitzman, unpublished work). We anticipated that, among the latter class of transformants, there might be some that produced altered citrate synthase. A. lwofi, strain 4B, was isolated from water. Citrate synthase-deficient mutants of this strain were obtained by selecting for resistance to fluoroacetate, the rationale for this approach being that the absence of citrate synthase would prevent the formation of fluorocitrate and hence confer resistance to the otherwise powerfully toxic effect of fluoroacetate (Harford, 1977). The methods described by Juni (1972) were adopted for the preparation of transforming DNA from Pseudomonas aeruginosa and for the examination of transformation of the citrate synthase-deficient mutants. The latter were applied, with and without transforming DNA, to a nutrient-agar plate supplemented with lOm~-gIutamate; after 6h at 30°C, the growths were streaked on to a plate containing 10mwsuccinate as sole carbon source and incubated at 30°C. Only cells transformed to prototrophy appeared as colonies and several of these were isolated as pure cultures. That these were all Acinetobacter strains was confirmed by examination of the properties of other tricarboxylic acid-cycle enzymes (Jones & Weitzman, 1974) and also by starting with a streptomycin-resistant citrate synthasedeficient strain of Acinetobacter and observing streptomycin resistance in the transformants. For the examination of their citrate synthases, the transformants were grown in liquid culture in nutrient broth at 30°C, collected by centrifugation and sonicated to yield cell-free extracts. Experiments were carried out on these extracts without further fractionation. The transformant citrate synthases fell into two distinct groups that we have termed groups I and 11. The sensitivity of the enzymes to regulation by NADH and AMP was first tested. Group 1 showed inhibition by NADH and complete de-inhibition by AMP, whereas the group-I1 enzymes were totally insensitive to NADH. Examination of the molecular sizes of the citrate synthases by gel filtration on Sephadex G-200