A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage

Abstract

A cloned cytolytic determinant from the genome of Bacillus cereus Bacillus cereus, a common soil saprophyte, has been recognized as an opportunistic pathogen of increasing importance (reviewed in reference 41). Although food-borne gastroenteritis is the most common malady attributed to B. cereus (41), the most devastating is B. cereus endophthalmitis (1, 4, 17). B. cereus elaborates a variety of extracellular membrane-active enzymes and cytolytic toxins. These membrane-active proteins include a phospholipase C (34), sphingomyelinase (22), phosphatidylinositol phospholipase C (23), cereolysin (7; a cytolysin of the streptolysin 0, thiol-activated class), and a second, heat stabile cytolysin about which little is known (8, 10, 37). Phospholipase C, sphingomyelinase, and cereolysin have been highly purified and used in studies of membrane structure (6,29,43) and in studies on the evolution of cytolysins produced by diverse genera of gram-positive bacteria (13,38). Phospholipase C is a Zn metalloenzyme of 23,000 daltons (34) which shows a high degree of stability in chaotropic agents and surfactants (27,28). The sphingomyelinase produced by B. cereus is a protein of between 41,000 and 23,300 daltons, depending on the method of analysis used (40), and requires divalent cations for activity (21).As a first step in determining the contribution that extracellular membrane-active proteins make to the ecology and virulence of B. cereus, a gene bank was established. Identification of a cloned cytolytic determinant from this B. cereuls GP-4 library has been reported (25). To gain insight into the relationship of the cloned cytolysin determinant to membrane-active proteins of B. cereus, nucleotide sequence and enzyme activity analyses were undertaken. The results reported here show that the cytolytic determinant cloned from B. cereus is composed of tandemly arranged genes for two distinct protein products, the activities of both being required to effect target cell lysis (hemolysis as tested). Moreover, the individual cytolysin components possess * Corresponding author. phospholipase C and sphingomyelinase activity, respectively. These data suggest that although the sphingomyelinase and phospholipase C of B. cereus have been studied in detail individually, their function in nature appears to be as a cytolytic unit representing the heat-stabile hemolysin previously observed. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in these experiments are listed in Table 1. Escherichia coli strains were routinely cultured in 2XYT medium (31) with aeration. Bacillus subtilis cultures were grown in HGP broth as previously described (25). Tetracycline (Sigma Chemical Co., St. Louis, Mo.) was incorporated into liquid and solid (1.2% agar) media at concentrations of 10 ,ug/ml for selection of resistant B. subtilis and E. coli strains. Ampicillin (Sigma) was used at 100 pRg/ml to select for recombinants cloned into the vectors pUC8,45). In addition, to screen for insertional ina...

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects

“…Two phosphatidylcholine esterases are known to be encoded by PlcRregulated genes (Agaisse et al, 1999). They are phospholipase C and sphingomyelinase, which together form cereolysin AB (Gilmore et al, 1989). We checked that the 407 Cry − ∆plcR strain did not form a halo on lecithin-agar plates, consistent with the genetic data (results not shown).…”

“…These results suggest that pathogenesis in mice and insects requires common PlcR controls pathogenicity extracellular factors that depend on the plcR regulon. Thus, PlcR-regulated genes, such as those encoding the enterotoxins Hbl (Beecher et al, 1995b) and Nhe (Granum et al, 1999 ;, the phospholipase PI-PLC (Kuppe et al, 1989 ;Lechner et al, 1989) and the cereolysin AB component (Gilmore et al, 1989), may be responsible for pathogenicity. These four genes were found in the B. thuringiensis 407 and B. cereus ATCC 14579 strains used in this study (Agaisse et al, 1999 ;.…”

“…B. thuringiensis 407 Cry − and B. cereus grown on sheepblood agar produced intense haloes of degradative (Callegan et al, 1999 ;Lindba$ ck et al, 1999). Thus, the specific activity of B. thuringiensis and B. cereus against human erythrocytes is probably due to the cereolysin AB component, which is known to cause the haemolysis of human erythrocytes (Gilmore et al, 1989).…”

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

“…The BLAST (Altschul et al, 1990) sequence database search detected four other bacterial SMase homologues from B. cereus strain GP-4 (PIR code, B32042; Gilmore et al, 1989), B. cereus strain SE-1 (S01950; Johansen et al, 1988), Staphylococcus aureus (S15766; Projan et al, 1989), and Leptospira interrogans (S22634; Segers et al, 1990). Their sequence identities with the query sequence (SO1 130; SMase from B. cereus strain IAM1208) were 95.1, 93.5, 58.5, and 46.5%, respectively.…”

Identification of contamination sources of Bacillus cereus in pasteurized milk

“…The phospholipase C and sphingomyelinase function synergisticaily to exhibit cytolysis. The enzyme complex (cereolysin AB) rnight be responsible for necrotic and lethal toxin activities of B. cereus (Gilmore et al, 1989). Cereolysin is also known as haemolytic 1 or mouse lethai factor 1 (Beecher and Macmillan, 1991;Granum, 1994 (Tumbull and Kramer, 1991;Ezzell and Wilhelmsen, 1993).…”