Structure and regulation of the carAB operon in Pseudomonas aeruginosa and Pseudomonas stutzeri: no untranslated region exists

Abstract

The carAB operons from Pseudomonas aeruginosa PAO1 and Pseudomonas stutzeri JM300 were characterized by Southern and DNA sequence analyses. The results show that the previously reported sequence for carA (S. C. Wong and A. T. Abdelal, J. Bacteriol. 172:630-642, 1990) is derived from P. stutzeri and not P. aeruginosa, as originally reported. Therefore, the amino-terminal sequence of the purified carA product is identical to that derived from the nucleotide sequence in both organisms, P. stutzeri having four additional amino acids. The results also show that while carA and carB are contiguous in P. stutzeri, as is the case in other bacteria, they are surprisingly separated by an open reading frame (ORF) of 216 amino acids in P. aeruginosa. S1 nuclease mapping experiments with RNA extracted under a variety of growth conditions, as well as experiments using different lacZ fusions, indicate that the carA-ORF-carB operon of P. aeruginosa is transcribed from a single promoter. Moreover, these experiments demonstrate that expression of this single transcript is controlled by both arginine and pyrimidines and that variation in arginine levels specifically modulates transcriptional initiation, while pyrimidine regulation is exerted subsequent to transcriptional initiation. Modification of a rho-independent terminator-like structure, which is present upstream of carA in P. aeruginosa, removed all transcriptional sensitivity of a carA::lacZ fusion to pyrimidines. This result, when coupled with the finding that translation of an 18-amino-acid leader polypeptide (associated with this putative rho-independent terminator), is inversely proportional to pyrimidine concentration in the cell, strongly suggests that regulation of carA by pyrimidines is mediated through an attenuation-type mechanism in P. aeruginosa.