Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon

Abstract

To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies-temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)-to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.The acquisition of iron is essential for bacterial pathogenesis. Because iron is insoluble in aqueous environments at neutral pH and has the potential to produce damaging free radicals, the mammalian host tightly sequesters iron (1, 51), lowering the level of free iron available to invading microorganisms to well below what is needed to survive (34). Since survival in the host and hence virulence is dependent on the acquisition of iron, pathogenic bacteria must be able to sense iron availability and globally regulate the transcription of iron acquisition genes. For many gram-positive and gram-negative organisms, including the sexually transmitted disease pathogen Neisseria gonorrhoeae, the crucial balance between acquiring enough iron to grow and avoiding the toxic effects of excess iron is controlled by the ferric uptake regulator (Fur) protein (12, 27, 49). Typically, Fur acts as a transcriptional repressor by binding as a dimer, along with ferrous iron as a corepressor, to regulatory Fur box (FB) sequences in the promoters of iron-regulated genes under iron-rich growth conditions. As intracellular iron stores are depleted, the Fur-Fe 2ϩ dimer dissociates from the promoter, allowing the entry of RNA polymerase and subsequent transcription (22). Fur acts as a global regulator controlling not only the expression of iron acquisition...

Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria gonorrhoeae

“…This gonococcal fur mutant strain does not exhibit a growth deficiency compared to the wild-type strain (Daou et al, submitted). To initially identify genes which were activated by Fur, we first screened N. gonorrhoeae F62 fur mutant and wild-type strains for the expression of a subset of genes which were previously reported in N. gonorrhoeae strain FA1090 to be activated in the presence of iron and to contain a predicted Fur box in the promoter region (21,38). Initial screening was performed in cultures grown under iron-replete and -depleted conditions using semiquantitative RT-PCR (data not shown).…”

“…Microarray analysis of N. gonorrhoeae FA1090 grown under iron-replete and -depleted conditions reported that 300 genes were repressed by growth under iron-replete conditions while 107 genes were induced (21,38). For those genes regulated by iron, putative Fur boxes were predicted based on the consensus Fur binding sequences from N. gonorrhoeae, P. aeruginosa, and E. coli (21,38). Using this analysis 92 gonococcal genes were shown to contain predicted Fur boxes, and of these 28 were shown to bind Fur in a Fur titration assay (FURTA), including 10 ironactivated genes (38).…”

“…For those genes regulated by iron, putative Fur boxes were predicted based on the consensus Fur binding sequences from N. gonorrhoeae, P. aeruginosa, and E. coli (21,38). Using this analysis 92 gonococcal genes were shown to contain predicted Fur boxes, and of these 28 were shown to bind Fur in a Fur titration assay (FURTA), including 10 ironactivated genes (38). However, the Fur-dependent repression or activation of these genes was not examined.…”

“…In contrast, the Fur regulon of N. gonorrhoeae is only beginning to be analyzed. Microarray analysis of N. gonorrhoeae FA1090 grown under iron-replete and -depleted conditions reported that 300 genes were repressed by growth under iron-replete conditions while 107 genes were induced (21,38). For those genes regulated by iron, putative Fur boxes were predicted based on the consensus Fur binding sequences from N. gonorrhoeae, P. aeruginosa, and E. coli (21,38).…”

The Iron Stimulon and Fur Regulon of Geobacter sulfurreducens and Their Role in Energy Metabolism

“…The binding of Fe(II) induces a conformational change that allows Fur to behave as a transcriptional repressor by blocking access of RNA polymerase to the DNA. This mechanism of Fur regulation has been identified across a diverse range of bacteria, including the model organism Escherichia coli and the human pathogens Helicobacter pylori and Neisseria gonorrhoeae as well as the Gram-positive bacterium Bacillus subtilis (7)(8)(9)(10). Although Fur was originally considered a regulator of genes involved in iron uptake, it is becoming more apparent that it functions as a global regulator involved in a variety of processes such as virulence, motility, and response to oxidative stress (11,12).…”