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

Abstract

Klebsiella pneumoniae is able to utilize many nitrogen sources, and the utilization of some of these nitrogen sources is dependent on the nitrogen assimilation control (NAC) protein. Seven NAC-regulated promoters have been characterized in K. pneumoniae, and nine NAC-regulated promoters have been found by microarray analysis in Escherichia coli. So far, all characterized NAC-regulated promoters have been directly related to nitrogen metabolism. We have used a genome-wide analysis of NAC binding under nitrogen limitation to identify the regions of the chromosome associated with NAC in K. pneumoniae. We found NAC associated with 99 unique regions of the chromosome under nitrogen limitation. In vitro, 84 of the 99 regions associate strongly enough with purified NAC to produce a shifted band by electrophoretic mobility shift assay. Primer extension analysis of the mRNA from genes associated with 17 of the fragments demonstrated that at least one gene associated with each fragment was NAC regulated under nitrogen limitation. The large size of the NAC regulon in K. pneumoniae indicates that NAC plays a larger role in the nitrogen stress response than it does in E. coli. Although a majority of the genes with identifiable functions that associated with NAC under nitrogen limitation are involved in nitrogen metabolism, smaller subsets are associated with carbon and energy acquisition (18 genes), and growth rate control (10 genes). This suggests an expanded role for NAC regulation during the nitrogen stress response, where NAC not only regulates genes involved in nitrogen metabolism but also regulates genes involved in balancing carbon and nitrogen pools and growth rate.

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…One explanation for this difference is that K. pneumoniae has Nac-regulated operons which are lacking in E. coli, like ure, encoding urease (211). Another explanation is that some Nac-regulated genes or operons, present in both organisms, have a point mutation in the Nac consensus binding site in the promoter of the gene/operon in E. coli (203,510), e.g., the dadAX operon in E. coli compared to the dadAB operon in K. pneumoniae, coding for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase, respectively (510).…”

“…However, unlike in E. coli, in K. pneumoniae, this regulon also contains genes involved in carbon and energy metabolism. Thus, not only does the Nac regulon of K. pneumoniae cover more metabolic functions than that of E. coli, it also has more genes in its regulon (211).…”

“…The minimal binding consensus sequence of Nac K , as determined by DNase I footprint and mobility shift assays, seems to be the 15-nucleotide consensus DNA binding site 5=-ATA-n 9 -TAT-3= (the first and last 3 nucleotides form a dyad symmetry) (206,211). When Nac K was cloned downstream of an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible promoter, the activity of hut and gdh, genes regulated by Nac K , depended only on IPTG, even when cells were grown with excess ammonium (212).…”

“…For 84 of the 99 regions, the association with purified Nac was confirmed by electrophoretic mobility shift assays in vitro. Regulation of 15 of the ChIP-identified fragments was tested further by primer extension analysis: 9 were activated, and 6 were repressed (211). As in E. coli, many detected genes of the Nac regulon in K. pneumoniae are involved in nitrogen metabolism.…”

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

“…Nac in turn activates and represses a set of genes whose products are mostly related to nitrogen metabolism. Nac-activated genes include those involved in the catabolism of histidine, proline, urea and alanine, among others [78][79][80]. Notably, Nac represses the genes coding for the two enzymes that synthesize glutamate, gdhA, encoding GDH, and gltAB, encoding GOGAT [81].…”