Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B

Abstract

Iron is limiting in the human host, and bacterial pathogens respond to this environment by activating genes required for bacterial virulence. Transcriptional regulation in response to iron in Gram-negative bacteria is largely mediated by the ferric uptake regulator protein Fur, which in the presence of iron binds to a specific sequence in the promoter regions of genes under its control and acts as a repressor. Here we describe DNA microarray, computational and in vitro studies to define the Fur regulon in the human pathogen Neisseria meningitidis group B (strain MC58). After iron addition to an iron-depleted bacterial culture, 153 genes were up-regulated and 80 were down-regulated. Only 50% of the iron-regulated genes were found to contain Fur-binding consensus sequences in their promoter regions. Forty-two promoter regions were amplified and 32 of these were shown to bind Fur by gel-shift analysis. Among these genes, many of which had never been described before to be Fur-regulated, 10 were up-regulated on iron addition, demonstrating that Fur can also act as a transcriptional activator. Sequence alignment of the Fur-binding regions revealed that the N. meningitidis Fur-box encompasses the highly conserved (NATWAT)3 motif. Cluster analysis was effective in predicting Fur-regulated genes even if computer prediction failed to identify Fur-box-like sequences in their promoter regions. Microarray-generated gene expression profiling appears to be a very effective approach to define new regulons and regulatory pathways in pathogenic bacteria.

Fig. 1.

RT-PCR validation of microarray expression ratios, and EMSA of promoter regions of up- and down-regulated genes. (A Left and Center) RT-PCR and relative quantification on rmp mRNA, used as negative control, obtained from a 2-h culture grown under iron-replete (Fe) and -depleted (Df) conditions. PCR cycles at which transcripts were analyzed are indicated above each lane. M, molecular weight. (A Right) Fur binding to the rmp promoter region by EMSA analysis. Lanes: 1, no protein; 2, E. coli Fur; 3, Neisseria Fur. (B) RT-PCR analysis of representative down- and up-regulated genes obtained from bacterial cultures sampled at 2, 3, and 5 h of growth in iron-replete (Fe) and -depleted (Df) conditions. (C) EMSA analysis of the promoter-proximal regions of the iron-regulated genes represented in B. Lanes: 1, no protein; 2, E. coli Fur; 3, Neisseria Fur.

Fig. 2.

Multiple sequence alignment of upstream regions of EMSA-positive genes. (Upper) Graph reporting the representation of each nucleotide of the derived consensus sequence above the MC58 genomic nucleotide frequency. (Lower) Pairwise alignment of the derived consensus with the previously proposed Fur-boxes. In red is highlighted the most conserved region of the derived consensus, matching with the previously proposed Fur-binding consensus sequence (–18).

Fig. 3.

Unsupervised hierarchical clustering of N. meningitidis iron-regulated genes. Genes are grouped according to the similarity of their transcriptional profile, over the growth period. Gene expression ratios are represented by bars; green and red indicate down- and up-regulated genes, respectively. Vertical lines indicate the clusters containing the genes analyzed by EMSA, the names of which are reported nearby, together with the results of EMSA analysis and the computational search of Fur-boxes. The dark-green vertical bars indicate the down-regulated gene clusters, and the light-green vertical bars indicate the cluster of highly down-regulated, Fur-dependent genes. The names of genes organized as single transcriptional units are visualized in the same color.