Isolation and analysis of a fur mutant of Neisseria gonorrhoeae

Abstract

The pathogenic Neisseria spp. produce a number of iron-regulated gene products that are thought to be important in virulence. Iron-responsive regulation of these gene products has been attributed to the presence in Neisseria spp. of the Fur (ferric uptake regulation) protein. Evidence for the role of Fur in neisserial iron regulation has been indirect because of the inability to make fur null mutations. To circumvent this problem, we used manganese selection to isolate missense mutations of Neisseria gonorrhoeae fur. We show that a mutation in gonococcal fur resulted in reduced modulation of expression of four well-studied iron-repressed genes and affected the iron regulation of a broad range of other genes as judged by two-dimensional polyacrylamide gel electrophoresis (PAGE). All 15 of the iron-repressed spots observed by two-dimensional PAGE were at least partially derepressed in the fur mutant, and 17 of the 45 iron-induced spots were affected by the fur mutation. Thus, Fur plays a central role in regulation of iron-repressed gonococcal genes and appears to be involved in regulation of many iron-induced genes. The size and complexity of the iron regulons in N. gonorrhoeae are much greater than previously recognized.

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

“…3). This cluster included 26 Fur-regulated genes, subjected either to gel-shift analysis in this study, or to gel-shift and͞or footprint analyses in N. gonorrhoeae (17,18,26). Our cluster analysis indicated that a few genes demonstrating a positive shift in the gel-shift analysis grouped in clusters with genes that were negative in the gel-shift assay, indicating that not all of the genes found to bind to the Fur protein had a similar RNA expression profile.…”

“…The genes of this group are characterized by an expression profile of constant downregulation, which for some genes was as low as 10-fold and never Ͻ1.5-fold with respect to expression under iron-replete conditions. Considering that 32 of the 68 down-regulated transcriptional units were subjected to gel-shift analysis and that previous studies had demonstrated the Fur-binding activity of 5 additional down-regulated transcriptional units (17,18), this analysis appears to be a powerful tool to predict Fur-regulated genes (gel-shift data available for 46 genes, corresponding to 58% of all down-regulated genes). Considering that in the promoter region of some of these genes a canonical Fur-box sequence cannot be recognized, cluster analysis, rather than Fur-box prediction, appears to be the most reliable approach for predicting Fur-binding activity.…”

“…The gel-shift analysis described above allowed us to identify 32 DNA fragments capable of interacting with both Neisseria and E. coli Fur, 27 of which had a predictable Fur-box. We therefore hypothesized that sequence alignment of these fragments, together with the alignment of the promoter-proximal regions of previously characterized Neisseria Fur-dependent transcriptional units (17,18,26), should define a sufficiently reliable N. meningitidis Fur consensus sequence. To facilitate this alignment, we first scanned each fragment for the presence of a E. coli-like Fur sequence, and then launched the homology search on a limited portion of each of these fragments of Ϸ70 nt with the E. coli-like Fur-binding sequence.…”

“…However, studies on the Fur-dependent iron regulation of Neisseria genes have been hampered because of the inability to construct a fur null mutant. A missense mutant of Neisseria gonorrhoeae fur was shown to be altered in the iron regulation of a broad range of genes supporting the existence of a large set of genes that may be under Fur control (17). Our more recent studies in N. gonorrhoeae have established that the gonococcal Fur protein binds to the promoter regions of several genes involved in diverse metabolic pathways (18).…”

Defenses against Oxidative Stress inNeisseria gonorrhoeae: a System Tailored for a Challenging Environment

“…It has proved more difficult to identify genes under Fur control that are activated in response to high levels of iron. Attempts to generate a Fur null mutant have so far been unsuccessful, which may indicate that Fur plays a critical role in gonococcal survival (232). However, fur has since been successfully deleted in N. meningitidis (65), and attempts to disrupt Fur in N. gonorrhoeae may have failed due to a polar effect on an essential downstream gene.…”