NITRATE AND NITRITE-MEDIATED TRANSCRIPTION ANTITERMINATION CONTROL OFNASF (NITRATE ASSIMILATION) OPERON EXPRESSION IN KLEBSIELLA-PNEUMONIAE M5AL

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sou rces during aerobic growth. Nitrate is converted through nitrite to am monium by assimilatory nitrate and nitrite reductase, respectively. En zymes required for nitrate assimilation are encoded by the nasFEDCBA o peron of K. pneumoniae; nasF operon expression is subject to both gene ral nitrogen control and pathway-specific nitrate/nitrite induction, m ediated by the NtrC and NasR proteins, respectively. Sequence inspecti on revealed a presumptive sigma(N) (sigma(54))-dependent promoter as w ell as two presumptive upstream NtrC protein binding sites. Site-speci fic mutational and primer extension analyses confirmed the identity of the sigma(N)-dependent promoter. Deletions removing the apparent NtrC protein binding sites greatly reduced NtrC-dependent regulation, indi cating that these sites are involved in general nitrogen control. Howe ver, deletions removing most of the sequence upstream of the promoter had little effect on nitrate/nitrite regulation, suggesting that the n asF leader region is involved in nitrate/nitrite regulation. The 119 n ucleotide long transcribed leader region contains an apparent factor-i ndependent transcription terminator. Promoter replacement experiments demonstrated that the leader region is involved in nitrate/nitrite reg ulation of nasF operon expression. Deletions removing the transcriptio n terminator structure resulted in a nitrate-blind constitutive phenot ype, indicating that the transcription terminator structure serves a n egative function. Other deletions, removing proximal portions of the l eader region, resulted in an uninducible phenotype, indicating that th is region serves a positive function. These results indicate that nitr ate/nitrite regulation of nasF operon expression is determined by a tr anscription attenuation mechanism. We hypothesize that in the absence of nitrate or nitrite, the terminator structure abrogates transcriptio n readthrough into the nasF operon. In the presence of nitrate or nitr ite, the NasR protein mediates transcription antitermination, thereby allowing transcription to proceed into the nasF operon. (C) 1996 Acade mic Press Limited