Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis ofNeisseria meningitidisgroup 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 Gramnegative 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.

Expression of the Iron-ActivatednspAandsecYGenes inNeisseria meningitidisGroup B by Fur-Dependent and -Independent Mechanisms

“…1D). As expected, we did not observe differences in the transcript levels of the rmp gene (a gene which is not regulated in response to iron) (17) in N. meningitidis MC58 grown under iron-replete or -depleted conditions (data not shown). Our cotranscription results are in agreement with previous studies of E. coli indicating that transcription of the spc operon initiates from its own promoter and continues into the downstream operon containing additional ribosomal protein genes (3).…”

“…1B and Table 1) and RNA isolated from N. meningitidis MC58 cultures grown for 4 h under iron-depleted and iron-replete conditions. For these studies, N. meningitidis MC58 was grown in chemically defined medium (CDM) under iron-replete (100 M ferric nitrate) and -depleted (12.5 M desferal) conditions, as described previously (17). We observed growth restriction of the N. meningitidis MC58 cultures during growth under iron-depleted conditions (data not shown).…”

“…Recent studies indicate that in some organisms, Fur may also function as a positive regulator of gene transcription, together with iron (8,(9)(10)(11)(12)24), although the mechanism of iron activation by Fur is not well elucidated. Our recent studies of N. meningitidis group B, using a combination of microarray technology, computational analysis, and in vitro binding studies, revealed that a large number of genes are activated during growth in the presence of iron and that a number of these iron-activated genes have putative Fur-binding sequences to which Fur was demonstrated to bind (17). However, the biological significance of Fur binding to the operator regions of these genes has not been defined, as an N. meningitidis fur mutant had not been constructed at the time of that study.…”

“…These DNA-free RNA samples served as the template for subsequent RT-PCR with the primers listed in Table 1. All RT-PCRs were performed as previously described (17). Briefly, the reaction mixture contained 250 ng of DNA-free RNA, 25 l of 2ϫ reaction mixture, 100 ng of each primer, 1 l of RT-Taq mix, and diethyl pyrocarbonate-treated water to a final volume of 50 l. cDNA synthesis was performed at 50°C for 30 min, followed by predenaturing at 94°C for 2 min.…”

Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens

“…The most-well-studied bacterial iron-responsive regulator is the ferric uptake repressor (fur) (5,21,23,49). The Fur protein is the founding member of a family of proteins containing a wide array of metal-responsive transcriptional regulators and is present in many lineages of bacteria, including Escherichia coli and Bacillus subtilis (34).…”