The ORF1 of the gentamicin‐resistance operon (aac) of Pseudomonas aeruginosa encodes adenosine 5′‐phosphosulphate kinase

Summary The gentamicin‐resistance operon of Pseudomonas aeruginosa (aac) contains two cistrons for which only the second gene product has an identified function. The 813bp second cistron (ORF2) encodes a protein that confers gentamicin resistance by catalysis of the transfer of an acetyl group from acetyl Coenzyme A to gentamicin. The first open reading frame (ORF1) encodes a 23.9 kDa protein that we have found, by enzyme activity and immunological reactivity, to be adenosine‐5′‐phosphosulphate (APS) kinase. APS kinase catalyses the transfer of the gamma phosphoryl group of ATP to the 3′‐hydroxyl group of APS. The 70% sequence similarity between the Pseudomonas and Escherichia coli APS kinases suggests that the Pseudomonas enzyme may catalyse phosphoryl transfer to the 3′‐hydroxyl group of other nucleotides such as dephosphocoenzyme A, as does the purified E. coli APS kinase. In extracts of pseudomonad cells we have also detected a higher molecular mass (70 kDa) protein that cross‐reacts with an anti‐E. coli APS kinase antibody. This cross‐reactive protein is also present in Pseudomonas strains lacking the gentamicin‐resistance plasmid, and apparently reflects an APS kinase analogous to the nodQ‐encoded high‐molecular‐weight APS kinase present in Rhizobium meliloti. Production of the Pseudomonas aac APS kinase was repressed by cysteine when expressed in E. coli, as is E. coli APS kinase. However, cysteine did not repress production of the Pseudomonas enzyme when the aac ORF1 ‐encoded enzyme was expressed in a Pseudomonas strain, indicating differential regulation of gene expression in the two organisms.