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.

FIG. 1.

Functional categories of iron-repressed and iron-induced genes. Fold changes in expression (expression under iron-depleted conditions/expression under iron-replete conditions) for FA1090 genes on the microarray were calculated at 1, 2, 3, and 4 h, and the genes were categorized according to biological function. Each bar represents the actual number of genes.

FIG. 2.

(A) Effectiveness of the FURTA for the identification of novel gonococcal Fur box regions. A total of 34,000 clones from an FA1090 FURTA library were transformed into E. coli DEC40 in groups of 1,000 and were screened for the FURTA-positive phenotype as described in Materials and Methods. Following each transformation round, novel FB regions identified by the FURTA were compared to novel FB regions identified by in silico predictions. (B) Increased β-galactosidase activity in E. coli DEC40 FURTA-positive clones. Clones containing FB sequences were grown to mid-log phase in CDM-100 (iron-replete medium) and assessed for β-galactosidase activity. The activity of each individual clone is expressed as a percentage of the activity of E. coli DEC39, a strain constitutively expressing lacZ. The IgA1 and tbpA clones contain internal FB sequences. Data are means ± SEM and are representative of three separate experiments. Analysis of variance with a Newman-Keuls posttest was used. A P value of ≤0.05 was considered significant. Asterisks indicate results significantly different from that for E. coli DEC40 containing the negative-control plasmid pUC18.

FIG. 3.

FB logos. (A) FB sequence logo for FURTA-positive, iron-responsive genes. (B) FB sequence logo for FURTA-negative, iron-responsive genes. The bit score, or overall height, represents sequence conservation at a given position, while the height of each residue within each stack represents the frequency of that residue.

FIG. 4.

Comparison of the relative binding affinity of gonococcal Fur for FBs located in the promoter region to that for FBs located within the coding region of TbpA. EMSAs were carried out by incubation of 25 nM concentrations of each respective DNA target containing a predicted FB sequence with increasing concentrations of Fur protein (for FetB, 0 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 600 nM, 800 nM, 1μM, and 1.2 μM Fur; for the TbpA and NS DNA fragments, 0 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 μM, 1.25 μM, 1.5 μM, 2 μM, and 3 μM Fur). DNA was visualized on a 4% nondenaturing polyacrylamide gel using SYBR green. Bound and unbound bands were measured to determine the percentage of Fur that was bound. The K_D was defined as the concentration of Fur required for a 50% shift of the target DNA. NS is a nonspecific DNA fragment from NGO1809.

FIG. 5.

Gene tree cluster of transcriptional changes in FURTA-positive and FURTA-negative genes with predicted FBs at 4 h. Red indicates an increase, and green indicates a decrease, in transcription levels under iron-depleted or iron-replete growth conditions.

FIG. 6.

Gonococcal Fur regulon, showing the proposed regulatory cascade of Fur and potential secondary transcriptional regulators. *, predicted FB and FURTA positive; **, predicted FB.