The nac gene of Klebsiella aerogenes encodes a bifunctional transcription factor that activates or represses the expression of several operons under conditions of nitrogen limitation. In experiments with purified components, transcription from the nac promoter was initiated by 54 RNA polymerase and was activated by the phosphorylated form of nitrogen regulator I (NRI) (NtrC). The activation of the nac promoter required a higher concentration of NRIϳP than did the activation of the Escherichia coli glnAp 2 promoter, and both the promoter and upstream enhancer element contributed to this difference. The nac promoter had a lower affinity for 54 RNA polymerase than did glnAp 2 , and uninitiated competitor-resistant transcription complexes formed at the nac promoter decayed to competitor-sensitive complexes at a greater rate than did similar complexes formed at the glnAp 2 promoter. The nac enhancer, consisting of a single high-affinity NRI-binding site and an adjacent site with low affinity for NRI, was less efficient in stimulating transcription than was the glnA enhancer, which consists of two adjacent high-affinity NRI-binding sites. When these binding sites were exchanged, transcription from the nac promoter was increased and transcription from the glnAp 2 promoter was decreased at low concentrations of NRIϳP. Another indication of the difference in the efficiency of these enhancers is that although activation of a nac promoter construct containing the glnA enhancer was relatively insensitive to subtle alterations in the position of these sites relative to the position of the promoter, activation of the natural nac promoter or a nac promoter construct containing only a single high-affinity NRIϳP binding site was strongly affected by subtle alterations in the position of the NRIϳP binding site(s), indicating a face-of-the-helix dependency for activation.Gram-negative enteric bacteria such as Escherichia coli, Salmonella typhimurium, and Klebsiella aerogenes regulate the expression of glnA, encoding glutamine synthetase, and several other genes and operons, collectively known as the Ntr regulon, in response to the availability of the preferred nitrogen source, ammonia (reviewed in references 3, 20-23, and 48). The Ntr genes and operons encode products that permit the use of alternative nitrogen sources when ammonia is absent. Within the Ntr regulon, the hut and put operons of K. aerogenes encode products that permit the utilization of histidine and proline as the sole carbon or nitrogen source (3, 6). The K. aerogenes hut and put operons are also part of the globally controlled response to carbon starvation (Cer regulon [23]), and transcription of hut and put can be activated either by carbon starvation (by the cyclic AMP [cAMP]-cAMP response protein [CRP] complex) or by nitrogen starvation (6, 27, 32, 37; reviewed in reference 23).The expression of glnA and the Ntr regulon in response to nitrogen limitation is activated by the phosphorylated form of the glnG (ntrC) product, nitrogen regulator I (NRI) (30). The re...

A 32-kDa polypeptide corresponding to NAC, the product of the Klebsiella aerogenes nac gene, was overexpressed from a plasmid carrying a tac-nac operon fusion and purified to near homogeneity by taking advantage of its unusual solubility properties. NAC was able to shift the electrophoretic migration of DNA fragments carrying the NAC-sensitive promoters hutUp, putPp1, and ureDp. The interaction between NAC and hutUp was localized to a 26-bp region centered approximately 64 bp upstream of the hutUp transcription initiation site. Moreover, NAC protected this region from DNase I digestion. Mobility shift and DNase I protection studies utilizing the putP and ureD promoter regions identified NAC-binding regions of sizes and locations similar to those found in hutUp. Comparison of the DNA sequences which were protected from DNase I digestion by NAC suggests a minimal NAC-binding consensus sequence: 5-ATA-N 9 -TAT-3. In vitro transcription assays demonstrated that NAC was capable of activating the transcription of hutUp by 70 -RNA polymerase holoenzyme when this promoter was presented as either a linear or supercoiled DNA molecule. Thus, NAC displays the in vitro DNA-binding and transcription activation properties which have been predicted for the product of the nac gene.The enteric bacterium Klebsiella aerogenes can use a large number of nitrogen-containing compounds as its sole source of nitrogen. In virtually every case, the expression of the operons encoding these catabolic activities is regulated by the quality and quantity of the nitrogen source present in the growth medium (26). When ammonium, the preferred nitrogen source, is abundant, operons allowing the utilization of growth rate-limiting nitrogen sources are weakly expressed and operons facilitating the assimilation of ammonium are derepressed. When cells are growth rate limited by the nitrogen source in the medium, the converse is true: operons that allow the utilization of alternative nitrogen sources are derepressed and those requiring elevated ammonium levels to effectively assimilate ammonium are repressed. In every case known, this regulation in response to the supply of nitrogen requires the action of a phosphorylated form of the transcription activator NTRC (26). Phosphorylation of NTRC allows the activation and repression of operons in response to nitrogen limitation, and the lack of phosphorylation or lack of NTRC prevents the activation and repression of such operons. The action of this nitrogen regulatory (NTR) system and the path of signal transduction through this network are the subject of intense investigation in a number of laboratories (1,13,36,37).Several NTRC-dependent operons of K. aerogenes also require the product of the nac gene for nitrogen regulation (5,25,42). The operons which facilitate the catabolism of histidine, proline, and urea are fully activated in response to nitrogen limitation only if the nac gene is intact. Similarly, the operons encoding glutamate dehydrogenase and glutamate synthase are repressed in response to nitr...

ToxR facilitates TcpP-mediated activation of the toxT promoter in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin secretion and toxin coregulated pilus expression. ToxR binds a region from ؊104 to ؊68 of the toxT promoter, from which ToxR recruits TcpP to the TcpP-binding site from ؊53 to ؊38. To precisely define the ToxR-binding site within the toxT promoter, promoter derivatives with single-base-pair transversions spanning the ToxR-footprinted region were tested for transcription activation and DNA binding. Nine transversions between ؊96 to ؊83 reduced toxT promoter activity 3-fold or greater, and all nine reduced the relative affinity of the toxT promoter for ToxR at least 2-fold, indicating that activation defects were due largely to reduced binding of ToxR to the toxT promoter. Nucleotides important for ToxR-dependent toxT activation revealed a consensus sequence of TNAAA-N 5 -TNAAA extending from ؊96 to ؊83, also present in other ToxR-regulated promoters. When these consensus nucleotides were mutated in the ompU, ompT, or ctxA promoters, ToxR-mediated regulation was disrupted. Thus, we have defined the core ToxR-binding site present in numerous ToxR-dependent promoters and we have precisely mapped the binding site for ToxR to a position three helical turns upstream of TcpP in the toxT promoter.

Transcription of the nitrogen-regulated nac promoter of Klebsiella aerogenes requires 54 RNA polymerase, is activated by the phosphorylated form of the transcription factor nitrogen regulator I (NRI) (NtrC), and is repressed by the product of the nac gene, Nac. Nac protects a large portion of the nac control region, extending from positions ؊130 to ؊70, from digestion by DNase I. This site(s) lies immediately upstream from the site at which 54 RNA polymerase binds, is downstream of a high-affinity binding site for the transcriptional activator NRIϳP, and partially overlaps a low-affinity NRIϳP-binding site. Binding of Nac to the DNA resulted in bending of the DNA but did not interfere with the binding of 54 RNA polymerase to the promoter or with the binding of NRIϳP to either the high-affinity site or low-affinity site. Furthermore, transcription assays with various wild-type and mutant templates suggested that Nac did not exclude NRIϳP from either the low-or high-affinity sites, nor did Nac interfere with the ability of the polymerase to form the open complex when the binding sites for NRIϳP were moved to different locations upstream from the promoter. Rather, Nac seemed to repress by an antiactivation mechanism in which the interaction of the NRIϳP, bound at its normal sites, with 54 RNA polymerase, bound to the promoter, was prevented.The nac gene of Klebsiella aerogenes encodes a transcription factor, Nac, that activates the transcription of the histidine utilization (hut) genes, proline utilization (put) genes, and other genes under conditions of nitrogen limitation (reviewed in reference 1). Nac negatively regulates the transcription of gdhA, encoding glutamate dehydrogenase, and negatively regulates its own expression in nitrogen-starved cells (reviewed in reference 1). The nac gene is transcribed by 54 RNA polymerase, and this transcription is activated by the phosphorylated form of the transcription factor nitrogen regulator I (NRI) (NtrC), which binds to a pair of sites that are functionally equivalent to an enhancer (7). The negative regulation of nac by Nac implies that 54 -dependent promoters may be subject to negative control.We studied the mechanism of repression of the nac promoter with purified components. Our results indicate that Nac acts directly to repress transcription of nac. The binding of Nac did not interfere with the binding of 54 RNA polymerase to the nac promoter, nor did Nac interfere with the binding of the transcriptional activator NRIϳP to its sites. Consequently, we propose that Nac repressed transcription from the nac promoter by an antiactivation mechanism in which the interaction of the enhancer-bound NRIϳP and 54 RNA polymerase at the promoter was prevented. MATERIALS AND METHODSTranscription assays, transcription templates, and DNase I footprinting. The methods for the transcription assays (initiated-complex assay) and DNase I footprinting were exactly as described in the accompanying manuscript (7). Supercoiled transcription templates containing nac or glnA control regions...