IDENTIFICATION OF 2 DISTINCT BACILLUS-SUBTILIS CITRATE SYNTHASE GENES

Two distinct Bacillus subtilis genes (citA and citZ) were found to enc ode citrate synthase isozymes that catalyze the first step of the Kreb s cycle. The citA gene was cloned by genetic complementation of an Esc herichia coli citrate synthase mutant strain (W620) and was in a monoc istronic transcriptional unit, A divergently transcribed gene, citR, c ould encode a protein with strong similarity to the bacterial LysR fam ily of regulatory proteins. A null mutation in citA had little effect on citrate synthase enzyme activity or sporulation. The residual citra te synthase activity was purified from a citA null mutant strain, and the partial amino acid sequence for the purified protein (CitZ) was de termined. The citZ gene was cloned from B. subtilis chromosomal DNA by using a PCR-generated probe synthesized with oligonucleotide primers derived from the partial amino acid sequence of purified CitZ. The cit Z gene proved to be the first gene in a tricistronic cluster that also included citC (coding for isocitrate dehydrogenase) and citH (coding for malate dehydrogenase). A mutation in citZ caused a substantial los s of citrate synthase enzyme activity, glutamate auxotrophy, and a def ect in sporulation.