Control of Enterotoxin Gene Expression in Bacillus cereus F4430/73 Involves the Redox-Sensitive ResDE Signal Transduction System

Abstract

In contrast to Bacillus subtilis, the role of the two-component regulatory system ResDE has not yet been investigated in the facultative anaerobe Bacillus cereus. We examined the role of ResDE in the food-borne pathogen B. cereus F4430/73 by constructing resDE and resE mutants. Growth performances, glucose metabolism, and expression of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) were analyzed in the three strains under distinct oxygenation and extracellular oxidoreduction potential (ORP) conditions. We show that growth and glucose metabolism were only moderately perturbed in both resDE and resE mutants under aerobiosis, microaerobiosis, and anaerobiosis generated under N 2 atmosphere (initial ORP ‫؍‬ ؉45 mV). The major effects of resDE and resE mutations were observed under low-ORP anaerobic conditions generated under hydrogen atmosphere (iORP ‫؍‬ ؊148 mV). These conditions normally favor enterotoxin production in the wild type. The resE mutation was more deleterious to the cells than the resDE mutation, causing growth limitation and strong deregulation of key catabolic genes. More importantly, the resE mutation abolished the production of enterotoxins under all of the conditions examined. The resDE mutation only decreased enterotoxin expression under anaerobiosis, with a more pronounced effect under low-ORP conditions. Thus, the ResDE system was found to exert major control on both fermentative growth and enterotoxin expression, and it is concluded that the ResDE system of B. cereus should be considered an anaerobic redox regulator. The data presented also provide evidence that the ResDE-dependent regulation of enterotoxins might function at least partially independently of the pleiotropic virulence gene regulator PlcR.The facultative anaerobic, spore-forming Bacillus cereus has gained notoriety as an opportunistic human pathogen capable of causing a wide variety of diseases ranging from periodontitis, endophthalmitis, and meningitis in immunocompromised patients to the more frequent gastrointestinal infections (24,44). B. cereus is associated with two types of gastrointestinal infection, both causing diarrhea and vomiting after the ingestion of a contaminated food. Recent evidence suggests that B. cereus cells are primarily ingested as spores and that the spores are able to adhere to the mucosal epithelium of the small intestine, where they can then germinate (52). The resulting vegetative cells produce three enterotoxins responsible for diarrheal symptoms (2,8,18). These enterotoxins are hemolysin BL (Hbl) (4), nonhemolytic enterotoxin (Nhe) (27), and the single cytotoxin protein Cyt K (11, 28). Hbl is composed of three proteins L2, L1, and B encoded by genes clustered into an operon with the transcriptional order hblC, hblD, and hblA (42). All three components need to be excreted to induce biological effects, which include enterotoxic activity and hemolytic and dermonecrotic activities (5, 6). Nhe is also a threecomponent enterotoxin. It was first isolated from an Hbl-negative strain of B. cereus i...

The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures

“…Recent research has revealed how a 2CS contributes to the high adaptability of B. cereus strains that enables these bacteria to persist in processed foods (25). Some 2CSs were shown to play diverse roles in B. cereus adaptation (26)(27)(28)(29)(30), but most of the 2CSs found among B. cereus sensu lato strains have an unknown function (31), and none has yet been shown to be involved in low-temperature adaptation.…”

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

“…In this study, using B. cereus strain ATCC 14579, we demonstrated increased plcR, nhe, hbl, and cytK transcription under aerobic conditions in response to the loss of cytochrome c maturation or CccB. As control of enterotoxin production occurs at both the transcriptional and posttranscriptional levels (20,47), increased hemolysis in the ⌬resBC and ⌬cccB functionally demonstrates increased enterotoxin production. These findings may reflect the previously noted differences in response to redox conditions between strains of B. cereus (49).…”

“…The ResDE twocomponent system, known to regulate some enterotoxin gene ex-pression, is encoded by genes downstream of resBC. In B. cereus and B. subtilis, resDE is transcribed both from a promoter upstream of resA, producing a resABCDE transcript, and from a promoter upstream of resD, producing a resDE transcript (20,52). A polar effect on expression of resDE is unlikely due to the markerless nature of the resBC deletion and the fact that addition of resBC in trans to the ⌬resBC mutant can complement the overexpression phenotype.…”

“…Enterotoxin production is sensitive to the oxidation-reduction potential through complex interactions between multiple regulators. 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)).…”

“…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).…”