Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm

The biofilm proteome of a dairy-associated Bacillus cereus strain (B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.     

Differential efficacy of a chlorine dioxide-containing sanitizer against single species and binary biofilms of a dairy-associated Bacillus cereus and a Pseudomonas fluorescens isolate

AIMS: Daily exposure to 100 p.p.m. chlorine dioxide of single species and binary biofilms of dairy-associated Bacillus cereus DL5 and Pseudomonas fluorescens M2, attached to stainless steel surfaces in a laboratory flow system, was studied. METHODS AND RESULTS: Surfaces were sampled daily before and after sanitizer treatment and cells and spores dislodged and enumerated by standard methods. Duplicate surfaces were prepared for confocal scanning laser microscopy (CSLM) and scanning electron microscopy. Higher counts of Ps. fluorescens M2 were obtained in single species biofilms, microcolonies stained green (viable) in CSLM images and were closely packed on attachment surfaces. By contrast, higher counts of B. cereus DL5 were obtained in binary biofilms, microcolonies stained green in CSLM images, but were more spread out. Lower spore counts were obtained for B. cereus DL5 in binary biofilms. The survival of Ps. fluorescens M2 cells after exposure to chlorine dioxide was apparently enhanced by the presence of B. cereus DL5 in binary biofilms. By contrast, B. cereus DL5 showed increased susceptibility to sanitizer treatment in the presence of Ps. fluorescens M2. CONCLUSIONS: Co-cultured bacteria in biofilms influence each other with respect to attachment capabilities and sanitizer resistance/susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Binary biofilms endemic in food-processing industries can survive sanitation regimes and may represent reservoirs of product contamination leading to subsequent spoilage and/or food safety risks.     

Adaptation of the food-borne pathogen Bacillus cereus to carvacrol

Carvacrol, a natural antimicrobial compound present in the essential oil fraction of oregano and thyme, is bactericidal towards Bacillus cereus. A decrease of the sensitivity of B. cereus towards carvacrol was observed after growth in the presence of non-lethal carvacrol concentrations. A decrease of the melting temperature (Tm) of membranes from 20.5 degrees C to 12.6 degrees C was the immediate effect of the addition of carvacrol. Cells adapted to 0.4 mM carvacrol showed a lower membrane fluidity than nonadapted cells. Adaptation of 0.4 mM carvacrol increased the Tm from 20.5 degrees C to 28.3 degrees C. The addition of carvacrol to cell suspensions of adapted B. cereus cells decreased Tm again to 19.5 degrees C, approximately the same value as for the non-adapted cells in the absence of carvacrol. During adaptation, changes in the fatty acid composition were observed. The relative amount of iso-C13:0, C14:0, and iso-C15:0 increased and cis-C16:1 and C18:0 decreased. The head-group composition also changed, two additional phospholipids were formed and one phospholipid was lacking in the adapted cells. It could be concluded that B. cereus adapts to carvacrol when present at non-lethal concentrations in the growth medium by lowering its membrane fluidity by changing the fatty acid and headgroup composition.     

Cytotoxicity of alkaline-tolerant dairy-associated Bacillus spp

The cytotoxicity of five Bacillus spp. isolated from alkaline cleaning solutions in South African dairies was evaluated against McCoy mouse cells using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)-based assay, confocal scanning laser microscopy and scanning electron microscopy. According to the MTT-based assay, two of the Bacillus isolates (Bacillus licheniformis 5 and B. pumilus 122) were cytotoxic to McCoy cells and the cytotoxic components were heat labile. Confocal scanning laser microscopy combined with fluorescent staining using propidium iodide and fluorescein diacetate indicated that cytotoxic effects occurred within 3 h, appeared to be membrane active and resulted in cell necrosis. Scanning electron microscopy showed that McCoy cells exposed to the cytotoxic components exhibited morphological damage.     

Thermostable pullulanase from a mesophilic Bacillus cereus isolate and its mutant UV7.4

Summary A mesophilicBacillus cereus strain was isolated from soil. Since it produced fairly good amounts of extracellular pullulanase, mutations with ultraviolet light and nitrosoguanidine were done to improve the productivity. One of the second generation mutants, UV7.4, produced larger amounts of pullulanase at 37°C than most (mesophilic or thermophilic) organisms reported. The pullulanase was highly active and stable for up to one hour at 70°C. If developed further, UV7.4 can be used commercially for the saccharification of starch.     

Laundry detergent compatibility of the alkaline protease from Bacillus cereus

The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.     

Engineering of the pH optimum of Bacillus cereus beta-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type

The optimum pH of Bacillus cereus beta-amylase (BCB, pH 6.7) differs from that of soybean beta-amylase (SBA, pH 5.4) due to the substitution of a few amino acid residues near the catalytic base residue (Glu 380 in SBA and Glu 367 in BCB). To explore the mechanism for controlling the optimum pH of beta-amylase, five mutants of BCB (Y164E, Y164F, Y164H, Y164Q, and Y164Q/T47M/Y164E/T328N) were constructed and characterized with respect to enzymatic properties and X-ray structural crystal analysis. The optimum pH of the four single mutants shifted to 4.2-4.8, approximately 2 pH units and approximately 1 pH unit lower than those of BCB and SBA, respectively, and their k(cat) values decreased to 41-3% of that of the wild-type enzyme. The X-ray crystal analysis of the enzyme-maltose complexes showed that Glu 367 of the wild type is surrounded by two water molecules (W1 and W2) that are not found in SBA. W1 is hydrogen-bonded to both side chains of Glu 367 and Tyr 164. The mutation of Tyr 164 to Glu and Phe resulted in the disruption of the hydrogen bond between Tyr 164 Oeta and W1 and the introduction of two additional water molecules near position 164. In contrast, the triple mutant of BCB with a slightly decreased pH optimum at pH 6.0 has no water molecules (W1 and W2) around Glu 367. These results suggested that a water-mediated hydrogen bond network (Glu 367...W1...Tyr 164...Thr 328) is the primary requisite for the increased pH optimum of wild-type BCB. This strategy is completely different from that of SBA, in which a hydrogen bond network (Glu 380...Thr 340...Glu 178) reduces the optimum pH in a hydrophobic environment.     

pH gradients through colonies of Bacillus cereus and the surrounding agar.

pH-sensitive microelectrodes, constructed with a tip diameter of about 4 microns, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 microns into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1.45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.     

Metabolomic Analysis of Cooperative Adaptation between Co-Cultured Bacillus cereus and Ketogulonicigenium vulgare

The cooperative adaptation of subcultivated Bacillus cereus and Ketogulonicigenium vulgare significantly increased the productivity of 2-keto-L-gulonic acid, the precursor of vitamin C. The mechanism of cooperative adaptation of the serial subcultivated B. cereus and K. vulgare was investigated in this study by culturing the two strains orthogonally on agar plates. It was found that the swarming distance of B. cereus along the trace of K. vulgare on the plate decreased after 150 days'' subcultivation. Metabolomic analysis on these co-cultured B. cereus and K. vulgare strains showed that their cooperative adaptation was accomplished by three key events: (i) the ability of nutrients (e.g., amino acids and purines) searching and intaking, and proteins biosynthesis is increased in the evolved B. cereus; (ii) the capability of protein degradation and amino acids transportation is enhanced in evolved K. vulgare; (iii) the evolved B. cereus was found to provide more nutrients (mostly amino acids and purines) to K. vulgare, thus strengthening the oxidation and energy generation of K. vulgare. Our results provided novel insights into the systems-level understanding of the cooperative adaptation between strains in synergistic consortium.     

Differential Involvement of the Five RNA Helicases in Adaptation of Bacillus cereus ATCC 14579 to Low Growth Temperatures

Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ΔcshA, ΔcshB, and ΔcshC strains were cold sensitive. Growth of the ΔcshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ΔcshA strain and partially for the ΔcshB strain but not for the ΔcshC strain, suggesting different functions at low temperature.Bacillus cereus is a human pathogenic sporulated bacterium which is associated with emetic and diarrheal types of food-borne illnesses (4). B. cereus is widespread in the environment and in a wide range of foods. The growth domains of B. cereus strains range from psychrotrophic to nearly thermophilic and correlate with several phylogenetic clusters (15), which presumably permit B. cereus to colonize many different habitats with different thermal regimes. Many foods are stored refrigerated before consumption, and in such cases, B. cereus has to adapt to low-temperature conditions.B. cereus growth at low temperature takes place with a lag phase which may correspond to an adaptation phase (12). Cold is a stress which dramatically affects membrane fluidity, protein synthesis, and also the topology of nucleic acids (22). When exposed to low temperature, bacteria have to face a transient inhibition of protein synthesis mainly due to the presence of secondary structures in mRNA that are stabilized by cold conditions (16, 19). To overcome the translation interruption, cold-shocked cells synthesize cold-induced RNA helicases, which remove secondary structures from RNA duplexes in the presence of ATP, such as CsdA of Escherichia coli (19) or CshA of Bacillus subtilis (1). csdA and srmB deletion mutants of E. coli showed a cold-sensitive phenotype, and these RNA helicases have been described as involved in the biogenesis of the ribosomal 50S subunit at 20°C (10, 11). RNA helicases could also be involved in the degradation of mRNA by unwinding double-stranded mRNA, thereby allowing the action of RNase (8).We have recently shown that the deregulation of the expression of one RNA helicase gene of B. cereus ATCC 14579 increased the lag phase of B. cereus at a low temperature (7). In this context, our aim was to investigate the role of the five putative RNA helicases present in the genome of B. cereus ATCC 14579 in its adaptation at low temperature, close to the growth limit.     

Synthesis of d-phenylalanine oligopeptides catalyzed by alkaline d-peptidase from Bacillus cereus DF4-B

Synthesis of d-phenylalanine oligopeptides from d-phenylalanine methylester has been demonstrated by use of alkaline d-peptidase (ADP) from Bacillus cereus. An expression plasmid pKADP was constructed by placing the PCR-amplified ADP gene (adp) under the tac promoter of pKK223-3. Oligomerization of d-phenylalanine methylester by use of the purified ADP from the transformant Escherichia coli was investigated under several conditions. d-Phenylalanine dimer, (d-Phe)₂, and trimer, (d-Phe)₃, were produced in 25.4% and 8.6% yield, respectively, when 50 mM of the substrate was incubated for 8 h with ADP (2.0 U/ml and 0.4 U/ml, respectively) in 100 mM triethylamine–HCl (pH 11.5). Addition of dimethylsulfoxide to the reaction mixture resulted in the production of tetramer, (d-Phe)₄ in 6.7% yield with the decrease of the (d-Phe)₂ and (d-Phe)₃ production. This is the first study on the synthesis of d-phenylalanine oligomers by use of a d-stereospecific endopeptidase.     

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237

Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H₃PO₄-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely hydrolyzed at all. Received: April 28, 1997 / Accepted: May 24, 1997     

Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent

The present investigation describes microbial production of an alkaline protease and its use in dehairing of buffalo hide. Bacillus cereus produced extracellular protease when grown on a medium containing starch, wheat bran and soya flour (SWS). The ammonium sulphate precipitated (ASP) enzyme was applied for dehairing of buffalo hide. Microscopic observation of longitudinal section of buffalo hide revealed that the epidermis was completely removed and hair was uprooted leaving empty follicles in the hide. The ASP enzyme was stable for one month at ambient temperature between 25–35 °C. Enzymatic dehairing may be a promising shift towards an environment-friendly leather processing method.     

Effects of sporulation pH on the heat resistance and the sporulation of Bacillus cereus

Spores of Bacillus cereus ATCC 7004, 4342 and 9818 were obtained in nutrient agar at several pH from 5·9 to 8·3. The optimum pH for sporulation was around 7, but good production of spores was obtained in the range 6·5–8·3. With all three strains, D -values clearly dropped with sporulation pH, decreasing by about 65% per pH unit. z -Values were not significantly modified ( P > 0·05) by this factor. Mean z -values of 7·13 °C ± 0·16 for strain 7004, 7·67 °C ± 0·04 for 4342 and 8·80 °C ± 0·64 for 9818 were obtained.     

A small (2.4 Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3 Mb) Bacillus cereus chromosome

We have determined the sizes of the chromosomes of six Bacillus cereus strains (range 2.4–4.3 Mb) and constructed a physical map of the smallest B. cereus chromosome (2.4 Mb). This map was compared to those of the chromosomes of four B. cereus strains and one B. thuringiensis strain previously determined to be 5.4-6.3 Mb. Of more than 50 probes, 30 were localized to the same half of the larger B. cereus and B. thuringiensis chromosomes. All 30 were also present on the small chromosome. Twenty of the probes present on the other half of the larger chromosomes were either present on extrachromosomal DNA, or absent from the B. cereus strain carrying the small chromosome. We propose that the genome of B. cereus and B. thuringiensis has one constant part and another less stable part which is more easily mobilized into other genetic elements. This part of the genome is localized to one region of the chromosome and may be subject to deletions or more frequent relocations between the chromosome and episomal elements of varying sizes up to the order of megabases.     

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8

Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71?kDa) and reducing conditions (35?kDa and 22?kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22?kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22?kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8?kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70?°C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li²⁺ and Co²⁺ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52.