Molecular cloning and characterization of two genes for the biotin carboxylase and carboxyltransferase subunits of acetyl coenzyme A carboxylase in Myxococcus xanthus

We have cloned a DNA. fragment from a genomic library of Myxococcus; xanthu s using an oligonucleotide probe representing conserved regions of biotin c arboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fra gment contained two open reading frames (ORF1 and ORF2), designated the acc B and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene,vas strikingly similar to biotin carboxylase subun its of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylas e, The putative motifs for ATP binding, CO2 fixation, and biotin binding we re found in AccA. The accB gene was located upstream of the accA gene, and they formed a two-gene operon, The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunit s of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA dec arboxylase, Carboxybiotin-binding and acyl-CoA-binding domains, which are c onserved in several carboxyltransferase subunits of acyl-CoA. carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type st rain, Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth ph ase. Based on these results, we propose that this M. xanthus acetyl-CoA car boxylase consists of two subunits, which are encoded by the accB and accA g enes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.