The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a very versatile regulatory protein. NAC activates transcription of operons such as hut (histidine utilization) and ure (urea utilization), whose products generate ammonia. NAC also represses the transcription of genes such as gdhA, whose products use ammonia. NAC exerts a weak repression at gdhA by competing with the binding of a lysinesensitive activator. NAC also strongly represses transcription of gdhA (about 20-fold) by binding to two separated sites, suggesting a model involving DNA looping. We have identified negative control mutants that are unable to exert this strong repression of gdhA expression but still activate hut and ure expression normally. Some of these negative control mutants (e.g., NAC 86ter and NAC 132ter ) delete the C-terminal domain, thought to be required for tetramerization. Other negative control mutants (e.g., NAC L111K and NAC L125R ) alter single amino acids involved in tetramerization. In this work we used gel filtration to show that NAC 86ter and NAC L111K are dimers in solution, even at high concentration (NAC WT is a tetramer). Moreover, using a combination of DNase I footprints and gel mobility shifts assays, we showed that when NAC WT binds to two adjacent sites on a DNA fragment, NAC WT binds as a tetramer that bends the DNA fragment significantly. NAC L111K binds to such a fragment as two independent dimers without inducing the strong bend. Thus, NAC L111K is a dimer in solution or when bound to DNA. NAC L111K (typical of the negative control mutants) is wild type for every other property tested: (i) it activates transcription at hut and ure; (ii) it competes with the lysine-sensitive activator for binding at gdhA; (iii) it binds to the same sites at the hut, ure, nac, and gdhA promoters as NAC WT ; (iv) the relative affinity of NAC L111K for these sites follows the same order as NAC WT (ure > gdhA > nac > hut); (v) it induces the same slight bend as dimers of NAC WT ; and (vi) its DNase I footprints at these sites are indistinguishable from those of NAC WT (except for features ascribed to tetramer formation). The only two phenotypes we know for negative control mutants of NAC are their inability to tetramerize and their inability to cause the strong repression of gdhA. Thus, we propose that in order for NAC WT to exert the strong repression, it must form a tetramer that bridges the two sites at gdhA (similar to other DNA looping models) and that the negative control mutants of NAC, which fail to tetramerize, cannot form this loop and thus fail to exert the strong repression at gdhA.

Summary The obligate intracellular bacterium Chlamydia has an unusual developmental cycle in which there is conversion between two forms that are specialized for either intracellular replication or propagation of the infection to a new host cell. Expression of late chlamydial genes is upregulated during conversion from the replicating to the infectious form, but the mechanism for this temporal regulation is unknown. We found that EUO, which is expressed from an early gene, binds to two sites upstream of the late operon omcAB, but only the downstream site was necessary for transcriptional repression. Using gel shift and in vitro transcription assays we showed that EUO specifically bound and repressed promoters of Chlamydia trachomatis late genes, but not early or mid genes. These findings support a role for EUO as a temporal repressor that negatively regulates late chlamydial genes and prevents their premature expression. The basis of this specificity is the ability of EUO to selectively bind promoter regions of late genes, which would prevent their transcription by RNA polymerase. Thus, we propose that EUO is a master regulator that prevents the terminal differentiation of the replicating form of chlamydiae into the infectious form until sufficient rounds of replication have occurred.

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. ␤-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position ؊59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions ؊120 to ؉67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions ؊83 to ؉67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

SUMMARY The pathogenic bacterium Chlamydia replicates in a eukaryotic host cell via a developmental cycle marked by temporal waves of gene expression. We have previously shown that late genes transcribed by the major chlamydial RNA polymerase, σ66 RNA polymerase, are regulated by a transcriptional repressor EUO. We now report that EUO also represses promoters for a second subset of late genes that are transcribed by an alternative polymerase called σ28 RNA polymerase. EUO bound in the vicinity of six σ28-dependent promoters and inhibited transcription of each promoter. We used a mutational analysis to demonstrate that the EUO binding site functions as an operator that is necessary and sufficient for EUO-mediated repression of σ28-dependent transcription. We also verified specific binding of EUO to σ66-dependent and σ28-dependent promoters with a DNA immunoprecipitation assay. These findings support a model in which EUO represses expression of both σ66-dependent and σ28-dependent late genes. We thus propose that EUO is the master regulator of late gene expression in the chlamydial developmental cycle.

Peptides that play critical physiological roles are often encoded in precursors that contain several structurally-related gene products. Differential processing of a precursor by cell-specific processing enzymes can yield multiple messengers with diverse distributions and activities. We have reported the isolation of SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide from adult Drosophila melanogaster. The peptides are encoded in the FMRFamide gene and have a common C-terminal FMRFamide but different N-terminal extensions. In order to investigate the processing of the FMRFamide polypeptide protein precursor, we generated antisera to distinguish among the structurally-related neuropeptides. Utilizing a triple-label immunofluorescent protocol, we mapped the distribution of the peptides. Each peptide has a unique, non-overlapping cellular expression pattern in neural tissue suggesting that the precursor is differentially processed. In order to identify a biological activity of the peptides, we established an in vivo heart rate assay. SDNFMRFamide decreases heart rate but DPKQDFMRFamide and TPAEDFMRFamide do not, indicating that the N-terminal residues are critical for this activity. SDNFMRFamide immunoreactivity is present in the aorta, implying that SDNFMRFamide acts locally to affect heart rate; DPKQDFMRFamide and TPAEDFMRFamide antisera do not stain cardiac tissue. Our data support the conclusion that Drosophila contains cell-specific proteolytic enzymes to differentially process a polypeptide protein precursor resulting in unique expression patterns of structurally-related, yet functionally distinct neuropeptides.