Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system

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.     

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

ABSTRACT: BACKGROUND: Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrhealsyndrome may result from the secretion of various virulence factors including hemolysin BLand nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulatedby the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr isa member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so farbeen studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genesis thus to obtain and characterize holoFnr. RESULTS: Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UVvisibleand EPR spectroscopic analyses together with the chemical estimation of the ironcontent indicated that Fnr binds one [4Fe-4 S]2+ cluster per monomer. Atmospheric O2 causesdisassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- andapoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has ahigher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnrinteract with ResD and PlcR to form a ternary complex. CONCLUSIONS: Overall, this work shows that incorporation of the [4Fe-4 S]2+ cluster is not required for DNAbinding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of aternary complex with ResD and PlcR. This points to some new unusual properties of Fnr thatmay have physiological relevance in the redox regulation of enterotoxin gene regulation.     

Characterization of aerobic and anaerobic vegetative growth of the food-borne pathogen Bacillus cereus F4430/73 strain

The Gram-positive bacterium Bacillus cereus is a facultative anaerobe that is still poorly characterized metabolically. In this study, the aerobic vegetative growth and anaerobic vegetative growth of the food-borne pathogen B. cereus F4430/73 strain were compared with those of the genome-sequenced ATCC14579 strain using glucose and glycerol as fermentative and nonfermentative carbon sources, respectively. Uncontrolled batch cultures on several defined media showed that B. cereus strains had high amino acid or pyruvate requirements for anaerobic fermentative growth. In addition, growth performance was considerably improved by maintaining the pH of the culture medium near neutrality. Spectra of fermentation by-products were typically (per mole of glucose) 0.2-0.4 acetate, 1.1-1.4 L-lactate, 0.3-0.4 formate, and 0.05-0.2 ethanol with only traces of succinate, pyruvate, and 2,3-butanediol. These spectra were drastically changed in the presence of 20 mmol nitrate x L(-1), which stimulated anaerobic growth. During anaerobic and aerobic respiration, the persistent production of acetate and other by-products indicated overflow metabolisms. This was especially true in glucose-grown cells for which respiratory complex III made only a minor contribution to growth. Surprisingly, oxygen uptake rates linked to the cytochrome c and quinol branches of the respiratory chain were maintained at high levels in anaerobic, respiring, or fermenting cells. Growth and metabolic features of B. cereus F4430/73 are discussed using biochemical and genomic data.     

Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression

In the present study, a food-borne pathogen strain of Bacillus cereus (F4430/73) was anaerobically grown in controlled-batch conditions under low initial oxidoreduction potential (ORP=–148 mV) using hydrogen gas as reducing agent. Its physiological characteristics, including growth, glucose fermentation capacity and enterotoxin production, were compared with anaerobic conditions generated by nitrogen gas (ORP=+ 45 mV). The results showed that low ORP affected growth mainly during the early stages. Maximal specific rates of growth and glucose consumption were reduced, and drastic changes in time profiles of fermentation product concentration were observed. Production of lactate was promoted at the expense of acetate. Nevertheless, low ORP did not affect final biomass yield. Under both ORP conditions, Non-haemolytic enterotoxin (Nhe) was produced early during the exponential growth phase as a first enterotoxin and Haemolysin BL (Hbl) later during the early stationary growth phase as a second enterotoxin. The major effect of low ORP was the strong stimulation of Hbl production and, to a lesser extent, Nhe production. This control was complex, involving different levels of regulation. We discussed the regulation of enterotoxin expression and the involvement of the pleiotropic regulator PlcR.     

蜡状芽胞杆菌群菌株中部分病原相关因子的检测

对26株蜡状芽胞杆菌群菌株进行了肠毒素基因及其它病原相关因子的检测。PCR结果表明,17株蜡状芽胞杆菌群菌株中含有病原调控因子plcR的同源序列。采用3组溶血肠毒素hbl基因和3组非溶血肠毒素nhe基因特异性引物,分别可从73%的菌株中至少扩增出一个与预期DNA片段大小一致的片段,其中,苏云金芽胞杆菌菌株中溶血素hbl基因和非溶血素nhe基因的阳性检出率为83%。蜡状芽胞杆菌DBt248完全没有溶血活性,而且在溶血素hbl和非溶血素nhe基因的3个亚基以及病原调控因子plcR的PCR检测中均为阴性,有望作为宿主菌用于苏云金芽胞杆菌晶体蛋白的表达和应用。     

Production and characterization of antibodies against each of the three subunits of the Bacillus cereus nonhemolytic enterotoxin complex

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.     

From soil to gut: Bacillus cereus and its food poisoning toxins

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed dodecadepsipeptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne. The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K. Hbl and Nhe are homologous three-component toxins, which appear to be related to the monooligomeric toxin cytolysin A found in Escherichia coli. This review will focus on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation.     

Effects of porcine bile on survival of Bacillus cereus vegetative cells and Haemolysin BL enterotoxin production in reconstituted human small intestine media

AIMS: To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM). METHODS AND RESULTS: The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3.0 g l(-1) were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 x 10(4) CFU ml(-1) into each GM at pH 5.0 for 30 min at 37 degrees C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3.0 g of PB per litre at pH 6.5 and incubated at 37 degrees C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0.6, 1.5 and 1.2 g l(-1), respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7-10 h incubation period were 0.3, 0.9, 0.9 and 3.0 g l(-1), respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0.6, 0.6, 0.3 and 0.9 g l(-1), respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile. CONCLUSIONS: The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation. SIGNIFICANCE AND IMPACT OF THE STUDY: The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.     

The Production of Bacillus cereus Enterotoxins Is Influenced by Carbohydrate and Growth Rate

Enterotoxin production is a key factor in Bacillus cereus food poisoning. Herein, the effect of the growth rate (μ) on B. cereus toxin production when grown on sucrose was studied and the Hemolytic BL enterotoxin (HBL) and nonhemolytic enterotoxin (Nhe) production by B. cereus was compared according to carbohydrate at μ = 0.2 h⁻¹. The anaerobic growth was carried out on continuous cultures in synthetic medium supplemented with glucose, fructose, sucrose, or an equimolar mixture of glucose and fructose. Concerning the HBL and Nhe enterotoxin production: (1) the highest enterotoxin production has occurred at μ = 0.2 h⁻¹ when growing on sucrose; (2) HBL production was repressed when glucose was consumed and the presence of fructose (alone or in mixture) cancelled glucose catabolite repression; (3) the consumption of sucrose increased Nhe production, which was not affected by the catabolite repression. Furthermore, analysis of the fermentative metabolism showed that whatever the μ or the carbon source, B. cereus used the mixed acid fermentation to ferment the different carbohydrates. The enterotoxin productions by this strain at μ = 0.2 h⁻¹ are highly influenced by the carbohydrates that do not involve any fermentative metabolism changes.     

Regulation of beta-1, 4-Glucosidase Expression by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 expressed beta-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed beta-glucosidase expression (<0.3 U/ml); however, this yeast did produce beta-glucosidase when the initial glucose concentration was 相似文献   

Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses

Haemolysin BL (HBL) and non-haemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL-L(2) and NheB component ranged from 0.02 to 5.6 microg mL(-1) and from 0.03 to 14.2 microg mL(-1), respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.     

Changes in the cytochrome composition of Rhodopseudomonas sphaeroides grown aerobically, photosynthetically and on dimethyl sulphoxide.

Several strains and mutants of Rhodopseudomonas sphaeroides can be grown anaerobically in the dark in the presence of dimethyl sulphoxide as an electron acceptor. During adaptation to this fermentative mode of growth, two major c-type cytochromes are synthesized, one with Mr 45 000 and the second with Mr 20 000 and a midpoint potential of +120 mV. These cytochromes are barely detectable in membranes prepared from cells grown in aerobic or photosynthetic conditions. An electrophoretic method is presented for the detection of the b-type and c-type cytochromes of pigmented or unpigmented membranes. The method resolves three b-type cytochromes and four c-type cytochromes in membranes from aerobically and photosynthetically grown cells.     

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis

Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning. Microbiological Societies.     

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.     

Mitochondrial function in cell wall glycoprotein synthesis in Saccharomyces cerevisiae NCYC 625 (Wild type) and [rho(0)] mutants

We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho(0)] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho(0)] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho(0)] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.