STRUCTURE AND REGULATION OF THE CARAB OPERON IN PSEUDOMONAS-AERUGINOSA AND PSEUDOMONAS-STUTZERI - NO UNTRANSLATED REGION EXISTS

The carAB operons from Pseudomonas aeruginosa PAO1 and Pseudomonas stu tzeri 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 f rom P. stutzeri and not P. aeruginosa, as originally reported. Therefo re, the amino-terminal sequence of the purified carA product is identi cal to that derived from the nucleotide sequence in both organisms, P. stutzeri having four additional amino acids. The results also show th at while carA and carB are contiguous in P. stutzeri, as is the case i n other bacteria, they are surprisingly separated by an open reading f rame (ORF) of 216 amino acids in P. aeruginosa. S1 nuclease mapping ex periments with RNA extracted under a variety of growth conditions, as well as experiments using different lacZ fusions, indicate that the ca rA-ORF-carB operon of P. aeruginosa is transcribed from a single promo ter. Moreover, these experiments demonstrate that expression of this s ingle transcript is controlled by both arginine and pyrimidines and th at variation in arginine levels specifically modulates transcriptional initiation, while pyrimidine regulation is exerted subsequent to tran scriptional initiation. Modification of a rho-independent terminator-l ike structure, which is present upstream of carA in P. aeruginosa, rem oved all transcriptional sensitivity of a carA::lacZ fusion to pyrimid ines. This result, when coupled with the finding that translation of a n 18-amino-acid leader polypeptide (associated with this putative rho- independent terminator), is inversely proportional to pyrimidine conce ntration in the cell, strongly suggests that regulation of carA by pyr imidines is mediated through an attenuation-type mechanism in P. aerug inosa.