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. 2010 Oct;192(19):4812-20.
doi: 10.1128/JB.00931-09. Epub 2010 Mar 26.

Expanded role for the nitrogen assimilation control protein in the response of Klebsiella pneumoniae to nitrogen stress

Ryan L Frisch  1 , Robert A Bender
Affiliations

Expanded role for the nitrogen assimilation control protein in the response of Klebsiella pneumoniae to nitrogen stress

Ryan L Frisch et al. J Bacteriol. 2010 Oct.

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.

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Figures

FIG. 1.
FIG. 1.
Selectivity of NAC-DNA coimmunoprecipitation. (A) A schematic diagram of the regions targeted by the multiplex PCR of ChIP samples. The gray box indicates the promoter region containing the NAC binding site. Arrows indicate primers used to amplify target fragments within the promoter (P), 1 kb downstream (1kb DS), or 5 kb downstream (5kb DS) of the NAC binding site. (B) Results of multiplex PCR on immunoprecipitated DNA (IP) or total input DNA (T) from a chromatin immunoprecipitation experiment on nitrogen-limited Nac+ and Nac− strains. As a control, untreated genomic DNA from a Nac+ strain was also subject to multiplex PCR and run on the same gel (G). Multiplex PCR samples were separated on 5% TBE-PAGE gels and stained with ethidium bromide.
FIG. 2.
FIG. 2.
Electrophoretic mobility shift assay of ChIP clones. Fragments were selected to show the representative range of NAC binding seen for different fragments. The hutUH promoter fragment was used as a control on this and all gels. Reaction mixtures contained 0.24 pmol of DNA and either buffer 6 (−) or 0.48 pmol of NAC (+). Reaction mixtures were separated on 5% TBE-PAGE and stained with ethidium bromide. Fragment sizes were as follows: dppA, 179 bp; KPN_02730, 159 bp; hutU, 107 bp; oppA, 277 bp; KPN_01903, 232 bp; and KPN_04049, 130 bp. Bands corresponding to NAC-DNA complexes are labeled with a B.
FIG. 3.
FIG. 3.
Primer extension analysis of mRNA from Nac+ and Nac− strains grown under conditions of nitrogen limitation. Targets were chosen from those found in the ChIP screen (Table 1).
FIG. 4.
FIG. 4.
Template strand footprints of NAC bound to oppA or dppA promoter fragments. (A) End-labeled DNA fragments were mixed with buffer only or increasing concentrations of NAC prior to treatment with DNase I. A chemical A+G ladder of each fragment was also generated. Samples were separated on an 8% modified TBE urea gel, dried, and exposed to film. A solid line next to the gel shows protected regions. Arrowheads indicate hypersensitive sites. (B) The DNA sequence of footprinted regions of oppA and dppA fragments. Lines above the sequence indicate regions of the template strand protected from DNase I in the presence of NAC. Arrowheads indicate hypersensitive sites. Bold text indicates close matches to the NAC consensus site.
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