The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low tempera-ture. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these vir-ulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid-to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the pro-duction of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes. T he Bacillus cereus sensu lato complex, also known as the B. cereus group, is a subdivision of the Bacillus genus that in-cludes B. cereus sensu stricto, B. thuringiensis, B. mycoides, B. pseu-domycoides, B. anthracis, B. weihenstephanensis, and the recently proposed new species B. cytotoxicus. These bacteria are routinely isolated from various foods undergoing spoilage (rice, meat, eggs, and dairy products) and are of considerable importance in food microbiology (23, 40, 51). Many strains of the B. cereus sensu lato group are actually opportunistic pathogens that are able to colo-nize hosts as diverse as insects and mammals (5, 14, 15, 25). They can cause serious gastrointestinal diseases in humans (9, 36, 37, 52), and some strains are also responsible for severe nongastroin-testinal infections, such as endophthalmitis and meningitis, par-ticularly in immunocompromised patients and preterm neonates (28, 36, 37, 38). The food-poisoning strains can cause two types of food-borne illness. The first is characterized by nausea and vomiting with abdominal cramps and has an incubation period of 1 to 6 h (36). This is the emetic form, caused by a small, preformed, cyclic pep-tide known as the emetic toxin (10, 26). The second type of illness consists principally of abdominal cramps and diarrhea after an incubation period of 8 to 16 h. This is the diarrheal form, which is thought to be mediated by the heat-labile diarrheagenic entero-toxin Nhe and/or the hemolytic enterotoxins Hbl and/or CytK (3, 4, 33, 35, 41). All three enterotoxins are cytotoxic and active against cell membranes, in which they make pores (12, 27). Nhe and Hbl each have three different protein subunits (33, 34) which act together, whereas the third enterotoxin, CytK, is a single-com-ponent protein (35). All the strains of the B. cereus sensu lato complex seem to carry genes encoding at least one of the known diarrheal toxins (2, 11, 24), and the expression of these genes is under the control of the PlcR transcriptional regulator, which is activated at the onset of the stationary phase of growth (1). More-over, the strains of the B. cereus group are also known to synthesize several types of phospholipase C and various hemolysins, collage-nases, and proteases (52), which may be implicated in B. cereus pathogenesis (13, 42, 44, 52, 57, 58). These extracellular mem-brane-active and tissue-degrading proteins constitute a significant proportion of the proteins secreted by B. cereus strains at the start of the stationary growth phase (20). They may, together with one or more of the more specific enterotoxins Hbl, Nhe, and CytK, contribute to the rapid course and severity of the infections caused by this bacterium. The bacterial species B. weihenstephanensis (31), which in-cludes most of the psychrotolerant strains of the B. cereus sensu lato group, or B. cereus phylogenetic group VI according to the new classification proposed by Guinebretière and collaborators (25), is widespread in nature and can contaminate many raw ma-terials for food production and cold-stored foods (23). Moreover, the spores of these psychrotolerant B. cereus strains are sufficiently heat resistant to survive pasteurization, cooking, and most heat treatments apart from canning, allowing the bacterium subse-quently to increase from initially low levels to very high levels at refrigeration temperatures (23, 29). This raises questions about the potential health risk posed by these bacteria when present in refrigerated food products, as both the emetic toxin and the diar-rheagenic enterotoxins of food-borne pathogenic B. cereus iden-tified to date are present in psychrotolerant strains (50, 51, 55). In a previous study, we found that psychrotolerant B. weihenstepha-nensis strains were less virulent in the insect model, G. mellonella,