Bacillus cereus Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR

Abstract

BackgroundBacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrheal syndrome may result from the secretion of various virulence factors including hemolysin BL and nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulated by the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr is a member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so far been studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genes is thus to obtain and characterize holoFnr.ResultsFnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UV-visible and EPR spectroscopic analyses together with the chemical estimation of the iron content indicated that Fnr binds one [4Fe-4S]2+ cluster per monomer. Atmospheric O2 causes disassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- and apoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has a higher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary complex.ConclusionsOverall, this work shows that incorporation of the [4Fe-4S]2+ cluster is not required for DNA binding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of a ternary complex with ResD and PlcR. This points to some new unusual properties of Fnr that may have physiological relevance in the redox regulation of enterotoxin gene regulation.

Bacterial iron–sulfur cluster sensors in mammalian pathogens

“…Nhe and Hbl are pore-forming toxins each comprised of three protein components NheA, NheB, NheC and Hbl-B, Hbl-L 1 and Hbl-L 2 respectively 61 . FNR positively regulates expression of the nheABC and hbl operons and forms a ternary complex with ResD, the response regulator of the redox sensing two-component system ResDE and the virulence regulator PlcR 62, 63 . Under environments rich in oxygen, the oxygen-labile [4Fe-4S] cluster is lost resulting in accumulation of apo-FNR.…”

“…Under environments rich in oxygen, the oxygen-labile [4Fe-4S] cluster is lost resulting in accumulation of apo-FNR. Unlike E. coli and many other FNR containing organisms, apo-FNR of B. cereus is an active transcription factor with a binding affinity similar to [4Fe-4S]-FNR for certain promoters including the nheABC and hbl operons 62, 63 . However, under conditions of anaerobic growth, such as in the mammalian small intestine, the [4Fe-4S] cluster is stable and [4Fe-4S]-FNR binds with higher affinity to the fnr promoter region 62 .…”

“…Unlike E. coli and many other FNR containing organisms, apo-FNR of B. cereus is an active transcription factor with a binding affinity similar to [4Fe-4S]-FNR for certain promoters including the nheABC and hbl operons 62, 63 . However, under conditions of anaerobic growth, such as in the mammalian small intestine, the [4Fe-4S] cluster is stable and [4Fe-4S]-FNR binds with higher affinity to the fnr promoter region 62 . As such, there is an increase in FNR production, which subsequently leads to increased expression of the nheABC and hbl operons for maximal toxin production during B. cereus infection of the small intestine 62 .…”

Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

“…47,48 Although the B. cereus FNR has an O 2 -responsive [4Fe-4S] cluster, the cluster does not appear to be important for DNA-binding at the nhe promoter (there is evidence for monomeric apo-FNR binding) or for interaction with the redoxresponsive regulator ResD (see below). [48][49][50] Oxygen-sensing by the Shigella FNR protein has been shown to play a role in coordinating the function of a Type III secretion system (T3SS) that is important for virulence. In the anaerobic lumen of the gastrointestinal tract, FNR primes the bacterium for invasion by activating expression of the T3SS needles, while repressing the expression of spa32 and spa33, which regulate the function of the T3SS.…”

Cytochromec551and the CytochromecMaturation Pathway Affect Virulence Gene Expression in Bacillus cereus ATCC 14579

“…In B. cereus strain F4430/73, Nhe and Hbl expression increases during growth under anaerobic conditions (19,20) due to changes in the activity of the redox regulator Fnr and the two-component system ResDE in response to changes in oxidoreduction potential (ORP). ResDE and Fnr directly regulate expression of Hbl and Nhe and indirectly regulate all enterotoxin expression through changes to PlcR expression (20)(21)(22)). An additional two-component system, OhrRA, is also involved in regulating enterotoxin production in response to changes in ORP (23).…”