A positive, genetic selection against the activity of the nitrogen regulatory (NTR) system was used to isolate insertion mutations affecting nitrogen regulation in Kiebsiella aerogenes. Two classes of mutation were obtained: those affecting the NTR system itself and leading to the loss of almost all nitrogen regulation, and those affecting the nac locus and leading to a loss of nitrogen regulation of a family of nitrogen-regulated enzymes. The set of these nac-dependent enzymes included histidase, glutamate dehydrogenase, glutamate synthase, proline oxidase, and urease. The enzymes shown to be nac independent included glutamine synthetase, asparaginase, tryptophan permease, nitrate reductase, the product of the nifLA operon, and perhaps nitrite reductase. The expression of the nac gene was itself highly nitrogen regulated, and this regulation was mediated by the NTR system. The loss of nitrogen regulation was found in each of the four insertion mutants studied, showing that loss of nitrogen regulation resulted from the absence of nac function rather than from an altered form of the nac gene product. Thus we propose two classes of nitrogen-regulated operons: in class I, the NTR system directly activates expression of the operon; in class II, the NTR system activates nac expression and the product(s) of the nac locus activates expression of the operon.The enteric bacterium Klebsiella aerogenes is capable of using a large number of compounds as its sole source of nitrogen. The formation of the enzymes needed to metabolize these compounds is in most, if not all, cases regulated by the availability of the preferred nitrogen source, ammonium (22). The formation of the enzymes needed for the conversion of poor nitrogen sources to either ammonia or glutamate is increased when ammonia is absent. Conversely, the formation of the two enzymes ultimately responsible for assimilating ammonia into glutamate (glutamate dehydrogenase and glutamate synthase) is repressed when ammonia is absent. As early as 1973, Prival et al. (33) recognized a role for the glnA locus in the genetic control of nitrogen-regulation in K. aerogenes. After a false start suggesting that the product of the glnA gene (glutamine synthetase) was itself the activator of transcription for nitrogen-regulated genes (23), it eventually became clear that the glutamine synthetase encoded by the glnA locus was physiologically important for nitrogen regulation because it synthesized the internal signal of nitrogen excess (glutamine or a product thereof) from ammonia, but that glnA was not directly involved in the genetic control of nitrogen regulation. The work from several groups (for reviews, see references 19,22 can cause the conversion of promoter-bound RNA polymerase containing the novel sigma subunit sigma-54, encoded by rpoN (instead of the normal sigma-70), from a closed complex to an open complex-at least in the case of the glnA promoter (32). Thus NTRC can activate transcription directly.This transcriptional activation was directly demonstrated in vitro ...

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We initiated these studies to help clarify the roles of heme, ␦-aminolevulinic acid (ALA), hemA, and hemM in Escherichia coli heme synthesis. Using recombinant human hemoglobin (rHb1.1) as a tool for increasing E. coli's heme requirements, we demonstrated that heme is a feedback inhibitor of heme synthesis. Cooverexpression of rHb1.1 and the hemA-encoded glutamyl-tRNA (GTR) reductase increased intracellular levels of ALA and heme and increased the rate of rHb1.1 formation. These results support the conclusion that heme synthesis is limited by ALA (

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The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC.DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the ot and , subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.