Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network

TheNeisseria gonorrhoeae f erricu ptaker egulator (Fur) protein controls expression of iron homeostasis genes in response to intracellular iron levels. In this study, using transcriptome sequencing (RNA-seq) analysis of anN. gonorrhoeae fur strain, we defined the gonococcal Fur and iron regulons and characterized Fur-controlled expression of an ArsR-like DNA binding protein. We observed that 158 genes (8% of the genome) showed differential expression in response to iron in anN. gonorrhoeae wild-type orfur strain, while 54 genes exhibited differential expression in response to Fur. The Fur regulon was extended to additional regulators, including NrrF and 13 other small RNAs (sRNAs), and two transcriptional factors. One transcriptional factor, coding for an ArsR-like regulator (ArsR), exhibited increased expression under iron-replete conditions in the wild-type strain but showed decreased expression across iron conditions in thefur strain, an effect that was reversed in afur -complemented strain. Fur was shown to bind to the promoter region of thearsR gene downstream of a predicted σ70 promoter region. Electrophoretic mobility shift assay (EMSA) analysis confirmed binding of the ArsR protein to thenorB promoter region, and sequence analysis identified two additional putative targets, NGO1411 and NGO1646. A gonococcalarsR strain demonstrated decreased survival in human endocervical epithelial cells compared to that of the wild-type andarsR -complemented strains, suggesting that the ArsR regulon includes genes required for survival in host cells. Collectively, these results demonstrate that theN. gonorrhoeae Fur functions as a global regulatory protein to repress or activate expression of a large repertoire of genes, including additional transcriptional regulatory proteins.IMPORTANCE Gene regulation in bacteria in response to environmental stimuli, including iron, is of paramount importance to both bacterial replication and, in the case of pathogenic bacteria, successful infection. Bacterial DNA binding proteins are a common mechanism utilized by pathogens to control gene expression under various environmental conditions. Here, we show that the DNA binding protein Fur, expressed by the human pathogenNeisseria gonorrhoeae , controls the expression of a large repertoire of genes and extends this regulon by controlling expression of additional DNA binding proteins. One of these proteins, an ArsR-like regulator, was required forN. gonorrhoeae survival within host cells. These results show that the Fur regulon extends to additional regulatory proteins, which together contribute to gonococcal mechanisms of pathogenesis.