Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

蜡状芽胞杆菌转录激活子PIcR调控多基因的表达

蜡状芽胞杆菌是一种条件致病菌 ,它能引起食物中毒和其它形式的疾病。潜在的致病因子包括磷脂酶C、溶血素、肠毒素和呕吐毒素等。在很多致病菌中致病因子的表达都是协同调控的。从蜡状芽胞杆菌模式菌株ATCC1 4579经转座子诱变的文库 ,筛选到一株磷脂酶阴性的突变子 ,该突变子的蛋白酶活性明显减弱了。插入位点的序列分析表明 ,一个高度同源于苏云金芽胞杆菌转录激活子PlcR的基因被插入失活了。研究结果表明 ,除在苏云金芽胞杆菌中能激活磷脂酰肌醇磷脂酶C基因的转录外 ,转录激活子PlcR至少还能调控卵磷脂酶和一个或多个蛋白酶基因的表达 ,这是一个多效调节因子。     

Dephosphorylation and inactivation of phosphorylase kinase: subunit specificity of rabbit skeletal muscle protein phosphatases

The dephosphorylation of phosphorylase kinase by four rabbit skeletal muscle protein phosphatases was studied. The four enzymes used were preparations of protein phosphatases C-I, C-II, H-I, and H-II. Phosphatases C-I, C-II, and H-II were obtained as homogeneous preparations using procedures previously developed. Phosphatase H-I was purified 644-fold from rabbit skeletal muscle for the purposes of this study, and was the major phosphorylase phosphatase activity in the tissue extract. Phosphatases C-I and H-I were relatively specific for removal of the beta subunit phosphate of phosphorylase kinase, this occurring at rates approximately 100 times more rapidly than the removal of the alpha subunit phosphate. In contrast, phosphatases C-II and H-II readily dephosphorylated both the alpha and beta subunits, although the alpha subunit phosphate release occurred at rates about twice that of the beta subunit phosphate. These studies show that skeletal muscle contains two phosphatases capable of acting on phosphorylase kinase, and that these have different specificities as represented by phosphatases H-I and C-I on the one hand, and phosphatases C-II and H-II on the other hand. These studies also provided unequivocal evidence that dephosphorylation of the beta subunit of phosphorylase kinase is solely involved in the inactivation of the cAMP-dependent protein kinase-activated enzyme. When autophosphorylated phosphorylase kinase was used as the substrate, the four phosphatases displayed similar general specificities as they did toward the cAMP-dependent protein kinase-activated enzyme. With none of the phosphatases examined was there any evidence that alpha subunit phosphorylation affected the rate of beta subunit dephosphorylation.     

The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms.

Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications. Site-directed mutagenesis was employed to construct a structural variant of subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine. Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide. This mutant subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth of Bacillus cereus T spores. However, this same mutant subtilin was fully active with respect to its ability to induce lysis of vegetative B. cereus T cells. Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.     

BACILLUS CEREUS PATHOGENICITY: A REVIEW

Pathogenic Bacillus cereus can be routinely isolated and identified in the laboratory from foods and other sources. Typing of B. cereus strains implicated in food poisoning outbreaks is helpful for confirmation of the origin of the outbreak and for epidemiological studies. Data concerning vegetative growth and spores are given. Different types of toxin are produced by B. cereus in the course of its growth: a so-called diarrheal enterotoxin and an emetic heat-stable toxin; their biochemical characteristics and the systems used for their detection are reviewed. Different types of hemolysins and phospholipases C are also produced and may play a role in pathogenicity. Nongastrointestinal infections were also traced to this species.     

Epidemiology and pathogenesis of Bacillus cereus infections

Bacillus cereus is a causative agent in both gastrointestinal and in nongastrointestinal infections. Enterotoxins, emetic toxin (cereulide), hemolysins, and phoshpolipase C as well as many enzymes such as beta-lactamases, proteases and collagenases are known as potential virulence factors of B. cereus. A special surface structure of B. cereus cells, the S-layer, has a significant role in the adhesion to host cells, in phagocytosis and in increased radiation resistance. Interest in B. cereus has been growing lately because it seems that B. cereus-related diseases, in particular food poisonings, are growing in number.     

Comparison of hemolysins of Vibrio cholerae non-O1 and Vibrio hollisae with thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin (Vh-rTDH) produced by Vibrio hollisae and hemolysin (NAG-rTDH) produced by Vibrio cholerae non-O1 were characterized and compared with hemolysin (Vp-TDH) produced by Vibrio parahaemolyticus. These three hemolysins are each composed of two subunits and have similar, but not identical, molecular weights. The amino acid compositions of Vp-TDH and NAG-rTDH are similar, but are different from that of Vh-rTDH. The three hemolysins showed similar lethal toxicities to mice. The effects of temperature on hemolysis and the time dependencies of hemolysis by the three hemolysins were similar. The three were concluded to be immunologically related, but not identical, and to have common and also unique antigenic determinants.     

Bacillus subtilis possesses a second determinant with extensive sequence similarity to the Escherichia coli mreB morphogene.

A gene with substantial sequence similarity to the mreB morphogene of Bacillus subtilis has been identified at 302 degrees on the chromosomal map by A. Decatur, B. Kunkel, and R. Losick (Harvard University; personal communication). Our characterization has revealed that the protein product of this determinant (termed mbl for mreB-like) is 55 and 53% identical in sequence to the MreB proteins of B. subtilis and Escherichia coli, respectively. The protein is 86% identical to a protein identified as MreB from Bacillus cereus, suggesting that the B. cereus protein is actually Mbl. Insertional inactivation of mbl indicated that this gene is not essential for cell viability or sporulation. Cells bearing mutant mbl alleles display a decreased growth rate and an altered cellular morphology. The cells appear bloated and are frequently twisted. Intergenic suppressor mutations which restore the growth rate to an approximately normal level arise within the mutant population. A second site mutation, designated som-1, was mapped to the hisA-mbl region of the chromosome by transduction.     

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α₂β₂ tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1^−/−) leads to embryonic lethality in mouse, whereas P4ha1^+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2^−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1^+/−;P4ha2^−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the T_m of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1^+/−;P4ha2^−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2^−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.     

A novel double heme substitution produces a functional bo3 variant of the quinol oxidase aa3 of Bacillus cereus. Purification and paratial characterization

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3. The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg-1 protein. The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa3 oxidase. The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 microM, a Vmax of 21 micromol of O2 min-1mg-1, and a calculated turnover of 55 s-1. The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 microM, respectively. Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.     

Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase

The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.     

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

A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular level. Nucleotide sequence determination revealed the presence of two open reading frames. Both open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.     

A red pigment produced by aerobic sporeforming bacteria

Summary The production of an extracellular, water-insoluble, red pigment by strains of Bacillus cereus, B. circulans, B. licheniformis, B. macerans, and B. subtilis was found to be dependent on the presence of iron in the medium. B. cereus (strain TS) produced approximately 13 mg of pigment per gram of cells (dry weight), the pigment being formed during growth and not accumulating when growth ceased. Pigment-less mutants were usually present in pigmented cultures, their frequency increasing as the cultures aged. The absorption spectra (UV and visible regions) of pigments from representatives of the above-mentioned species were identical, with a maximum at approximately 385 and one at 490 m. These spectra were remarkably similar to that of pulcherrimin, a red, iron-containing pigment produced by the yeast Candida pulcherrima.The pigment from B. cereus TS was purified by treatment with alkaline methanol, followed by precipitation with acid in the presence of excess FeCl₃. Chemical analysis of the purified pigment indicated that it consisted of a ferric complex of substituted pyrazine rings with isobutyl groups bound to positions 2 and 5. The structure of the bacterial pigment was found to be essentially identical with that of pulcherrimin, the degree of chelation in the former being less extensive than in the latter. The name pulcherrimin a is proposed for the pigment produced by B. cereus TS.     

Involvement of calcium and dipicolinic acid in the resistance of Bacillus cereus BIS-59 spores to u.v. and gamma radiations

The role of dipicolinic acid (DPA) in determining the resistance of Bacillus cereus spores to u.v. and gamma radiation was investigated. B. cereus BIS-59 spores containing varying amounts of DPA were prepared by appropriate compositional adjustments in the secondary media. Compared with spores containing 6 per cent DPA (dry weight) those containing 0.8 per cent DPA were far more sensitive to u.v. radiation. Similar u.v. radiation sensitivity was also found in respect of a DPA-less mutant of B. cereus T 6A 1. Pre-treatment of DPA deficient spores (of wild type or mutant B. cereus) with DPA or the presence of DPA during irradiation resulted in increased resistance of these spores to u.v. radiation. In the range 0.2 to 1 per cent DPA content of spores of B. cereus BIS-59, a striking inverse relationship could be discerned between the DPA content and the number of spore photo-products (5-thymidyl, 5,6-dihydrothymine) formed in DNA and spore viability. The resistance of B. cereus spores to gamma radiation did not seem to be influenced by their DPA content.     

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.     

Possible Involvement of β-Lactamase in Sporulation in Bacillus cereus

Nonreverting beta-lactamase-negative strains were isolated from the beta-lactamase-constitutive strain, Bacillus cereus 569 H. These strains differed from both beta-lactamase-inducible and -constitutive strains not only in failure to produce beta-lactamase but also in failure to autolyze on aging, delayed sporulation, and failure to release free spores from sporangia when produced. The addition of B. cereus beta-lactamase of 15% purity to a final concentration of 10 IU/ml stimulates sporulation and particularly the release of free spores in culture from sporangia of strain 569 (inducible wild-type), 569/H (constitutive mutant of 569), and HPen(-), a nonreverting beta-lactamase strain isolated from 569/H in this laboratory. Cultures of HPen(-) did not release free spores without this treatment. Similar stimulation of sporulation and spore release by beta-lactamase from B. cereus were observed in another beta-lactamase-negative strain derived from 569/H as well as in certain sporogeny mutants of B. subtilis. The beta-lactamase preparation used in these experiments was free of peptidases, proteases, and autolysins capable of solubilizing wall from vegetative cells. These results, taken with our previous finding that a soluble peptidoglycan inducer becomes available in cultures of B. cereus only at sporulation and that normal derepression of beta-lactamase accompanies normal sporulation, suggest that beta-lactamase in B. cereus may be involved in peptidoglycan metabolism during sporulation and possibly the breakdown of sporangial wall with the concomitant release of mature spores.     

Monoclonal Antibody Developed against a Hemolysin of Bacillus thuringiensis

A total of five hybridoma cell lines that produced monoclonal antibodies (MAb) against a hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis were established and characterized. All of these monoclonal antibodies reacted similarly not only to Bt-hemolysin but also to a hemolysin (Bc-hemolysin) produced by B. cereus, suggesting that the two hemolysins are immunologically indistinguishable. The MAb developed in this study was also successfully applied for rapid and simple purification of both Bt- and Bc-hemolysins by immunoaffinity column chromatography. The partial N-terminal amino acid sequence of the purified hemolysins was determined to be Ile-Glu-Gln-Thr.     

Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli.

The ply genes encoding the endolysin proteins from Bacillus cereus phages Bastille, TP21, and 12826 were identified, cloned, and sequenced. The endolysins could be overproduced in Escherichia coli (up to 20% of total cellular protein), and the recombinant proteins were purified by a two-step chromatographical procedure. All three enzymes induced rapid and specific lysis of viable cells of several Bacillus species, with highest activity on B. cereus and B. thuringiensis. Ply12 and Ply21 were experimentally shown to be N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). No apparent holin genes were found adjacent to the ply genes. However, Ply21 may be endowed with a signal peptide which could play a role in timing of cell lysis by the cytoplasmic phage endolysin. The individual lytic enzymes (PlyBa, 41.1 kDa; Ply21, 29.5 kDa, Ply12, 27.7 kDa) show remarkable heterogeneity, i.e., their amino acid sequences reveal only little homology. The N-terminal part of Ply21 was found to be almost identical to the catalytic domains of a Bacillus sp. cell wall hydrolase (CwlSP) and an autolysin of B. subtilis (CwlA). The C terminus of PlyBa contains a 77-amino-acid sequence repeat which is also homologous to the binding domain of CwlSP. Ply12 shows homology to the major autolysins from B. subtilis and E. coli. Comparison with database sequences indicated a modular organization of the phage lysis proteins where the enzymatic activity is located in the N-terminal region and the C-termini are responsible for specific recognition and binding of Bacillus peptidoglycan. We speculate that the close relationship of the phage enzymes and cell wall autolysins is based upon horizontal gene transfer among different Bacillus phages and their hosts.