Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

Abstract

A chimeric promoter with the nitrogen assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of ␤-galactosidase activity upon nitrogen limitation. Primer extension experiments showed that the nitrogen assimilation control protein activates lacZp 1 in a position-dependent manner.The enteric bacterium Klebsiella aerogenes responds to a decrease in the concentration of ammonia, the preferred nitrogen source, by modifying transcription rates at many operons. Some of these nitrogen-regulated operons allow the cell to assimilate ammonia in low concentration or to use alternative nitrogen sources. This global response to nitrogen starvation is regulated by the Ntr system, a regulatory network built around a two-component system (see references 16 and 27 for reviews). Essentially, nitrogen starvation results in the activation of NtrC, a DNA-binding protein that enhances transcription from 54 -dependent promoters. Although the Ntr system is necessary for the modulation of all nitrogen-regulated operons, transcriptional activity of a subset of these operons is also dependent on the nitrogen assimilation control protein (NAC) (4,5,15). NAC is a member of the LysR family of DNA binding transcriptional factors (23,24). The transcription of the nac gene is 54 dependent and under positive control by the Ntr system (8, 15). The apparent paradox posed by a second, positively regulated transcription factor in the Ntr cascade is explained by the functions NAC adds to the Ntr repertoire: NAC regulates 70 -dependent promoters and can either activate or repress transcription (see reference 2 for a review). Therefore, we see NAC as the coupling agent between 54 -dependent Ntr regulation and the 70 -dependent operons. NAC activates the expression of operons involved in the catabolism of alternative nitrogen sources: histidine utilization (hut), proline utilization (put), and urease (ure) (15). The bestcharacterized NAC-activated system is that of the hutUH operon (25). The transcriptional activation of the hut operon by NAC has been reproduced in vitro with purified components (11). These in vitro transcription experiments have shown that NAC is sufficient for the activation of 70 -directed transcription. In addition, the binding site for NAC has been determined by gel mobility retardation and footprinting assays (11). NAC protects a 26-bp fragment of the hutU promoter centered 64 bp upstream of the hutU transcriptional initiation site. A sequence comparison of the NAC-protected regions from the hutU, putP, and ureD promoters revealed a minimal NAC binding consensus: 5Ј-ATA-N 9 -TAT-3Ј.Interestingly, some NAC-regulated operons have functions other than the generation of reduced nitrogen, such as carbon and energy provision, and are regulated independently by other control signals. For example, the hutUH operon, whose products provide glutamate and energy, is activated by c...

Expanded Role for the Nitrogen Assimilation Control Protein in the Response of Klebsiella pneumoniae to Nitrogen Stress

“…25 to 28 bp) from DNase I (6,13,17). This region is centered on a 15-bp core binding site whose key components have been summarized as ATA-N 9 -TAT (13,32). At other promoters NAC binds to the DNA as a tetramer (at nacp and codBp) 4814 FRISCH AND BENDER J. BACTERIOL.…”

“…Fragment DNA was amplified from the library using the pUC19-specific primers M13-20 and M13-RSP (27). For comparison and as a positive control, the wellcharacterized NAC binding site from hutUp was included on each gel (13,32,33). At the concentration of NAC used, approximately one half of the hutU promoter fragment was shifted in the EMSA.…”

“…Unlike the Ntr protein, NAC binds close to the start of transcription at the promoters it regulates (6,11,14,17,19,29,33). In contrast to the complex regulatory features required for a promoter to be controlled by the Ntr system, NAC regulation requires as little as a 15-bp binding site that can be inserted within the promoter (32). This suggests that it requires a far less drastic alteration of the promoter region to bring genes into the nitrogen regulon through the use of NAC rather than direct control by the Ntr system.…”

Isolation of a Negative Control Mutant of the Nitrogen Assimilation Control Protein, NAC, in Klebsiella aerogenes

“…NAC can activate transcription from a variety of positions upstream of RNA polymerase (RNAP) (17), perhaps via distinct contacts with the various subunits of RNAP, as has been shown with other activator proteins, including the LysR family members CatR, TrpI, and OxyR (6,10,21). The consensus sequence of the sites where NAC activates transcription is ATA-N 6 -TNGTAT, and the site can be divided into two functional half-sites-a promoter-distal site involved in the binding of NAC to the DNA and a promoterproximal site.…”

Genetic Analysis of the Nitrogen Assimilation Control Protein from Klebsiella pneumoniae

“…Nonradioactive EMSA of DNA fragments with (lanes 1 to 4) or without (lanes 5 to 8) an NBS. DNA for lanes 1 to 4 was PCR amplified from plasmid pCB648 (34), which carries the NBS from hut. DNA for lanes 5 to 8 was PCR amplified from plasmid pCB903 (35), in which the NBS in pCB648 was "scrambled" by reversing the sequence without reversing the 5Ј-to-3Ј orientation.…”