Hemolysin II (HlyII) is a pore-forming toxin of the opportunistic pathogen Bacillus cereus. Despite our understanding of the mechanism of HlyII cytotoxicity in vitro, many of its characteristics, including potential target cells, conditions of its action and expression, are not known. Here we report that the expression of hlyII in Bacillus subtilis renders the bacteria hemolytic and is able to kill the crustacean Daphnia magna. The hemolytic activity of hlyII-encoded B. subtilis strains in culture media is positively correlated with virulence in D. magna. Fluorescence microscopy reveals postinfection changes in the mitochondrial potential of intestinal tissue, suggesting that the formation of ionic pores leads to cell death. In the presence of the transcriptional regulator HlyIIR, HlyII expression decreases 200-fold, and B. subtilis expressing both hlyII and hlyIIR remains hemolytic, but not pathogenic to the crustacean.

The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression.

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.

We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential (V(m)) and plasma membrane resistance (R(m)) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V(m) and R(m) of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca(2+)-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.

A novel approach to contaminant toxicity screening is proposed. The use of fluorescent microscopy with fluorescent dyes allows for assessing intoxication of Daphnia magna tissues, at various stages of exposure, to contaminants present in water. As shown, D. magna may not only be used as a test species in toxicity tests based on its lethality, but due to its translucency and application of fluorescent probes, separate steps of its intoxication and dying can be visualized. Using a variety of fluorescent probes, the present study also contributes to a better understanding of the toxicity mechanisms.