Derepression of beta-lactamase (penicillinase in Bacillus cereus by peptidoglycans

In Bacillus cereus 569 a cellular inducer of beta-lactamase was isolated which has the same constituents and basic structure as the soluble peptidoglycan found in sporulation, extracts from spores, and germination extracts, and which was previously called "spore-peptide." The material has been extensively purified and characterized. Two acid-soluble, high-molecular-weight peptidoglycan fractions containing muramic acid, glucosamine, diaminopimelic acid, d-aspartate, and d- and l-alanine, -lysine, -glycine, and -glutamate, distinguishable on the basis of size and different amino acid to amino sugar ratios, have been found to be responsible for the observed induction. Both fractions are capable of inducing high levels of beta-lactamase in concentrations lower than those of benzyl penicillin required for optimal induction. Several experiments also suggest that it is the accumulation of such soluble peptidoglycan in penicillin-treated cells which leads to induction of beta-lactamase and not the penicillin itself. The "spore-peptide" inducer becomes available during sporulation, and endogenous derepression of beta-lactamase activity occurs simultaneously. Such derepression also occurs in a strain of B. cereus very sensitive to penicillin and in which both uninduced as well as "spore-peptide"-induced beta-lactamase is a small fraction of that produced by the typical penicillinase producer. These results suggest that beta-lactamase in B. cereus functions in cell wall metabolism during sporulation.     

Production of a variant of beta-lactamase II with selectively decreased cephalosporinase activity by a mutant of Bacillus cereus 569/H/9.

1. Mutants of Bacillus cereus 569/H/9 have been screened in a search for strains that synthesize variants of beta-lactamase II. 2. One of these mutants (strain 569/H/9/1) produces a beta-lactamase II-like enzyme that shows a selective decrease in cephalosporinase activity. 3. beta-Lactamase II from strain 569/H/9/1 has been purified to apparent homogeneity and its kinetic properties have been examined. This enzyme resembles the parent beta-lactamase II in its relative activity with benzylpenicillin as substrate when Zn(II) is replaced by other metal ions, but differs detectably from the parent enzyme in its isoelectric point.     

Induced release of Bacillus spores from sporangia by sodium sulphate

Incubation of sporulating cultures of Bacillus anthracis, B. cereus, B. subtilis and B. thuringiensis in 1°0 mol/l sodium sulphate markedly increased the release of free spores from sporangia. It is postulated that the release of spores is due to activation of latent autolysins which hydrolyse sporangial cell walls. Sodium sulphate-induced lysis of sporangia represents a novel and highly effective method for the recovery of spores from cultures of Bacillus species.     

Induced release of Bacillus spores from sporangia by sodium sulphate

Incubation of sporulating cultures of Bacillus anthracis, B. cereus, B. subtilis and B. thuringiensis in 1.0 mol/l sodium sulphate markedly increased the release of free spores from sporangia. It is postulated that the release of spores is due to activation of latent autolysins which hydrolyse sporangial cell walls. Sodium sulphate-induced lysis of sporangia represents a novel and highly effective method for the recovery of spores from cultures of Bacillus species.     

Induction of Accelerated Sporangial Lysis by Basic Peptide Antibiotics. A Novel Method of Preparation of Free Spores of Bacilli

An accelerated release of free spores from sporangia of Bacillus cereus NCIB-8122 and Bacillus subtilis SMYW was induced by the addition of the basic peptide antibiotics, polymyxin B or colistin (100 μg/ml), to sporangia formed in liquid Bactopeptone medium. Destruction of sporangial cell walls of B. cereus prelabelled with ³H-4-diaminopimelic acid commenced shortly after the addition of either antibiotic, the label being gradually released into the medium. Normal free spores were released following the addition of antibiotics to sporangia containing refractile spores (stages IV-V of sporogenesis). Earlier additions induced the lysis of both compartments of the sporangium, accompanied by the release of already-synthesized dipicolinic acid and alreadyaccumulated ⁴⁵calcium. The heat resistance and germination ability of spores released in the presence of the antibiotics were the same as those of control spores released by long-term spontaneous lysis of sporangia. Similar effects of the antibiotics were observed with B. subtilis SMYW. Results obtained were used firstly for fast preparation of relatively clean free spores and secondly for the characterization of the developmental stage of sporogenesis at which the spore becomes independent of the maternal cell. It reaches this property at the end of stage IV and during stage V.     

Production, purification and spectral properties of metal-dependent beta-lactamases of Bacillus cereus

New methods for the production of consistently high levels of metal-dependent beta-lactamases (beta-lactamhydrolase, EC 3.5.2.6) from strains 569/H/9 and 5/B/6 of Bacillus cereus are described which have significant advantages over those reported previously. For example, these techniques do not require a fermentor with pH-stat capabilities. We also describe rapid very-high-yield purification schemes for the metal-dependent beta-lactamases from these strains, employing high-performance ultrafiltration (HPUF) and mass ion exchange techniques. Furthermore, we have developed improved methods for the removal of the active site Zn(II) and reconstitution of the beta-lactamase enzymatic activity with Co(II), which result in higher recovery of the original activity than previously reported. In order to characterize the purified beta-lactamases II of B. cereus 569/H/9 and 5/B/6 we have examined the molecular weights, and steady state kinetic parameters of Zn(II) enzymes, and the electronic and EPR spectra of the Co(II)-reconstituted enzymes. EPR spectra of CO(II)-reconstituted beta-lactamase from B. cereus 5/B/6 have not been previously reported.     

Processing of Bacillus cereus 569/H beta-lactamase I in Escherichia coli and Bacillus subtilis

The gene for Bacillus cereus 569/H beta-lactamase I, penPC, has recently been cloned and sequenced (Mézes, P. S. F., Yang, Y. Q., Hussain, M., and Lampen, J. O. (1983) FEBS Lett. 161, 195-200). A typical prokaryotic signal peptide but with no lipoprotein modification site, as present in the Bacillus licheniformis 749/C beta-lactamase, was indicated by the DNA sequence for this secretory protein. We have here purified the beta-lactamase I products found in Escherichia coli and Bacillus subtilis carrying penPC and have determined the first 20 NH2-terminal amino acids of each of the forms. Processing of the beta-lactamase I in E. coli occurs at a single site which is characteristic for cleavage by a signal peptidase. B. subtilis secreted two distinct products to the culture medium which were both smaller than the single product formed in E. coli. Sequencing of [35S]Met-labeled pre-beta-lactamase I from phenylethyl alcohol-treated cells of B. cereus 569/H indicated that UUG is being utilized as the initiation codon for penPC. The same result was obtained for the pre-beta-lactamase I from similarly treated cells of the closely related B. cereus 5/B strain.     

Enumeration and Identification of Bacillus cereus in Foods: I. 24-Hour Presumptive Test Medium

An egg yolk-polymyxin medium (KG) for rapid enumeration of Bacillus cereus is described. The test is presumptive in that differentiation of B. cereus (and closely related organisms) from other species is based on the formation of turbidity in the agar surrounding the colonies of the cereus group organisms. The medium is formulated to encourage sporulation and release of free spores for serological confirmatory tests within the 24-hr incubation period. The production of turbidity in egg yolk and free-spore production by 25 strains of B. cereus on KG agar were measured. The recovery of food poisoning strains of B. cereus inoculated into nonsterile food slurries was assessed. A comparison of KG agar and mannitol-egg yolk-polymyxin-agar indicated that the two media were comparable in their abilities to recover low levels of B. cereus from naturally contaminated foods. Since KG agar enhances spore formation by B. cereus, thus permitting early serological testing, its use in screening food products is advocated.     

Beta-lactamase III of Bacillus cereus 569: membrane lipoprotein and secreted protein

A third beta-lactamase in Bacillus cereus 569 has been identified and characterized. It corresponds to gamma-penicillinase reported by Pollock [Pollock, M. R. (1956) J. Gen. Microbiol. 15, 154-169] but whose existence has been questioned since then. It will be called beta-lactamase III. It resembles the class A beta-lactamases but is immunologically distinct from the major class A secreted beta-lactamase I of B. cereus. As with several other Gram-positive beta-lactamases it occurs in two forms, membrane bound as a glyceride-cysteine lipoprotein and as a hydrophilic secreted protein formed by cleavage on the carboxyl side of the modified cysteine that is the membrane attachment site. It is produced in all B. cereus 569 strains tested but is absent in B. cereus 5/b. Antibody to beta-lactamase III interacts to varying degrees with all the known class A beta-lactamases, most strongly with that of B. licheniformis 749/C.     

Cloning, nucleotide sequence, and expression of the Bacillus cereus 5/B/6 beta-lactamase II structural gene.

Two forms of heat-stable, zinc-containing beta-lactamase II have been described for strains of Bacillus cereus and have been shown to differ in substrate specificity (R. B. Davies, E. P. Abraham, J. Fleming, and M. R. Pollock, Biochem. J. 145: 409-411, 1975). We report here the nucleotide sequence, inferred amino acid sequence, and expression of beta-lactamase II from B. cereus 5/B/6 and compare our results with those for its homolog characterized in B. cereus 569/H (M. Hussain, C. Anthony, M. J. Madonna, and J. O. Lampen, J. Bacteriol. 164: 223-229, 1985) to document amino acid differences contributing to the specific properties of these enzymes.     

Cloning and sequencing of the metallothioprotein beta-lactamase II gene of Bacillus cereus 569/H in Escherichia coli

The structural gene for beta-lactamase II (EC 3.5.2.6), a metallothioenzyme, from Bacillus cereus 569/H (constitutive for high production of the enzyme) was cloned in Escherichia coli, and the nucleotide sequence was determined. This is the first class B beta-lactamase whose primary structure has been reported. The amino acid sequence of the exoenzyme form, deduced from the DNA, indicates that beta-lactamase II, like other secreted proteins, is synthesized as a precursor with a 30-amino acid N-terminal signal peptide. The pre-beta-lactamase II (Mr, 28,060) is processed in E. coli and in B. cereus to a single mature protein (Mr, 24,932) which is totally secreted by B. cereus but in E. coli remains intracellular, probably in the periplasm. The expression of the gene in E. coli RR1 on the multicopy plasmid pRWHO12 was comparable to that in B. cereus, where it is presumably present as a single copy. The three histidine residues that are involved (along with the sole cysteine of the mature protein) in Zn(II) binding and hence in enzymatic activity against beta-lactams were identified. These findings will help to define the secondary structure, mechanism of action, and evolutionary lineage of B. cereus beta-lactamase II and other class B beta-lactamases.     

Transduction in Bacillus cereus by Each of Two Bacteriophages

The ability of phage CP-51 to mediate transduction both homologously and heterologously in some of its hosts was investigated. CP-51 was shown to transduce Bacillus cereus strains 6464, 9139, and T in addition to 569 which was reported earlier from this laboratory. Furthermore, CP-51 grown on B. thuringiensis was shown to transduce some mutants of B. cereus. During this investigation, a second transducing phage for B. cereus 569 was isolated from lysates of phage CP-51 grown on B. cereus 6464. This phage, designated CP-53, is carried by wild-type strain 6464 possibly as prophage. All auxotrophic mutants of B. cereus 569 tested, those requiring tryptophan, histidine, methionine, and leucine, were transduced to prototrophy by CP-53. Electron micrographs of the two phages revealed that CP-51 has a tail core surrounded by a contractile sheath and CP-53 has a long flexible tail without a contractile sheath. CP-53 is stable in the cold, whereas CP-51 is rapidly inactivated at 4 C.     

Induction of beta-lactamase influences the course of development in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.     

The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.     

Cloning and sequencing of the beta-lactamase I gene of Bacillus cereus 5/B and its expression in Bacillus subtilis.

The beta-lactamases of Bacillus cereus have attracted interest because they are secreted efficiently, because multiple enzymes are frequently present, and because their regulation has unusual features. beta-Lactamase I of strain 5/B is produced constitutively at a high level, and the exoenzyme appears to be several thousand daltons larger than the corresponding product of strain 569/H. We have cloned the gene for 5/B beta-lactamase I in Escherichia coli and B. subtilis and have sequenced the structural portion and the regulatory regions. The 5/B enzyme is produced at a low level in E. coli RR1(pRWY200) and remains cellbound. In B. subtilis it is formed in large amounts, and over 90% of it is released into the medium. There is a large degree of homology between the promoter and leader peptide regions of the 5/B and 569/H genes; both utilize UUG as the translation initiation codon (P. S. F. Mézes, R. W. Blacher, and J. O. Lampen, (J. Biol. Chem. 260:1218-1223, 1985). Although there are significant differences in the peptide segment where processing would be expected to occur, the NH2 terminus of the major 5/B product from B. subtilis BD170(pRWY215) is His-44, which is the same as the NH2 terminus of the major 569/H product from B. subtilis BD170(pRWM5).     

Role of sphingomyelinase in infectious diseases caused by Bacillus cereus

Bacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H(2)O(2) and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27.5 degrees C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10(4) spores/g whereas with 800 g loaves survival occurred with doughs containing 5.0 X 10(3) spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0.2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.