Nitrate and Nitrite-mediated Transcription Antitermination Control of nasF(Nitrate Assimilation) Operon Expression in Klebsiella pneumoniaeM5al

Abstract

Klebsiella pneumoniaecan use nitrate and nitrite as sole nitrogen sources during aerobic growth. Nitrate is converted through nitrite to ammonium by assimilatory nitrate and nitrite reductase, respectively. Enzymes required for nitrate assimilation are encoded by the nasFEDCBAoperon of K. pneumoniae nasFoperon expression is subject to both general nitrogen control and pathway-specific nitrate/nitrite induction, mediated by the NtrC and NasR proteins, respectively. Sequence inspection revealed a presumptive σ N(σ 54)-dependent pro- moter as well as two presumptive upstream NtrC protein binding sites. Site-specific mutational and primer extension analyses confirmed the identity of the σ N-dependent promoter. Deletions removing the apparent NtrC protein binding sites greatly reduced NtrC-dependent regulation, indicating that these sites are involved in general nitrogen control. However, deletions removing most of the sequence upstream of the promoter had little effect on nitrate/nitrite regulation, suggesting that the nasFleader region is involved in nitrate/nitrite regulation. The 119 nucleotide long transcribed leader region contains an apparent factor-inde pendent transcription terminator. Promoter replacement experiments demonstrated that the leader region is involved in nitrate/nitrite regulation of nasFoperon expression. Deletions removing the transcription terminator structure resulted in a nitrate-blind constitutive phenotype, indicating that the transcription terminator structure serves a negative function. Other deletions, removing proximal portions of the leader region, resulted in an uninducible phenotype, indicating that this region serves a positive function. These results indicate that nitrate/nitrite regulation of nasFoperon expression is determined by a transcription attenuation mechanism. We hypothesize that in the absence of nitrate or nitrite, the terminator structure abrogates transcription readthrough into the nasFoperon. In the presence of nitrate or nitrite, the NasR protein mediates transcription antitermination, thereby allowing transcription to proceed into the nasFoperon.