Bacillus subtilis and B. mojavensis strains connected to food poisoning produce the heat stable toxin amylosin.

AIM: To screen and characterize toxic, heat-stable substances produced by food borne strains from Bacillus subtilis group. METHODS AND RESULTS: Using the boar sperm motility inhibition assay, six isolates from two outbreaks, out of the 94 isolates from 26 foods, were found to produce ethanol-soluble heat-stable substances that were toxic to sperm cells by depleting the mitochondrial membrane potentials. The toxic isolates were identified as Bacillus subtilis and B mojavensis. Colon carcinoma cells (Caco-2) were used to model the contact with the human digestive tract. The extract of B. subtilis F 2564/96 depolarized the mitochondria in intact Caco-2 cells similarly as in sperm cells. The substance responsible for these effects was purified using HPLC and identified by electron spray ionization ion trap mass spectrometry analysis as amylosin. The temperature requirement for amylosin production was 21-37 degrees C for B. subtilis and 11-21 degrees C for B. mojavensis. Both species produced amylosin in air as well as in 7-8% CO(2) with 8-9% O(2). CONCLUSIONS: Food borne illness related strains of B. subtilis and B. mojavensis, produced the heat-stable toxin amylosin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that suggests a role for the heat-stable, ion-channel forming toxin amylosin, as a virulence factor in food borne Bacillus.

Extracellular production of cloned alpha-amylase by Escherichia coli.

Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.

Characterization and polyelectrolyte precipitation of beta-galactosidase containing genetic fusions of charged polypeptides.

Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation. Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli. These fusion proteins were all shown to possess specific activity equal to that of the native enzyme. Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics. All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme. An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail. No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase.

Toxic Bacillus pumilus from indoor air, recycled paper pulp, Norway spruce, food poisoning outbreaks and clinical samples.

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.

Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails.

Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques. Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively. Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively). For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484). All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10. The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium. The enzymes were subsequently purified by affinity chromatography. The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme. The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins. Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully. The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation of Crocein Orange G by lignin peroxidase isoenzymes. Kinetics and effect of H2O2.

The ligninolytic enzyme system of Phanerochaete chrysosporium is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium of P. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from the N-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H2O2 concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 microM COG was obtained when the H2O2 concentration was 150 microM. This was also the lowest H2O2 concentration for maximal decolorization of 100 microM COG, regardless of the amount of lignin peroxidase used in the reaction.

Comparison of periplasmic and membrane associated beta-lactamase.

Beta-lactamase encoded by a plasmid pBR 322 was produced during the active growth phases of Escherichia coli IA 199. The maximal specific activity was about 15 times higher in shock fluid than in the cells disrupted by sonic disintegration. beta-Lactamase activity found in the membrane preparations increased gradually parallel to the cell growth. The amount of beta-lactamase in the membrane fraction, however, was only 0.2-0.4% of that found in shock fluid. beta-Lactamase was purified to homogeneity from shock fluid by a one-step procedure in a DEAE-Sepharose column. Most of the beta-lactamase activity present in the membrane fraction was released by salt extraction. beta-Lactamase solubilized treatment after salt extractions had the same molecular weight and immunological properties as beta-lactamase purified from the periplasmic space. Membrane associated beta-lactamase did not contain any covalently linked phospholipid.

Expression in Pichia pastoris and purification of Aspergillus awamori glucoamylase catalytic domain.

In this paper we report the expression in Pichia pastoris, purification, and characterization of the Aspergillus awamori glucoamylase catalytical domain (GAc). Pichia pastoris produced GAc to the level of 0.4 g per liter medium. This production level is about the same level as that gained for recombinant GA from Aspergillus and about 100-fold more than previously achieved by Saccharomyces cerevisiae. The GAc expressed in Pichia pastoris was purified by two independent chromatographic methods employing ion exchange or affinity chromatography to apparent homogeneity. The purified protein has a molecular weight of about 75,000 and specific activity of 78 units per milligram protein. The propeptide present in the glucoamylase N terminus was found to be removed correctly by P. pastoris. Glucoamylase produced by P. pastoris is N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed in P. pastoris. O-glycosides (studied to our knowledge for the first time in P. pastoris in this report) contribute about half of the total carbohydrate content in GAc. Purified GAc appears as multiple hands on isoelectric focusing with p1 values around 3.5, a value that is little higher than that for GAc produced in S. cerevisiae. GAc could be used as a versatile tool in studying protein expression in P. pastoris: as an affinity handle for other secreted proteins produced in P. pastoris, as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing in P. pastoris.

Human placental alkaline phosphatase: expression in Pichia pastoris, purification and characterization of the enzyme.

The soluble form of human placental alkaline phosphatase (PLAP) was expressed in the methylotrophic yeast Pichia pastoris and the expression product was purified and characterized. Yeast-derived PLAP (yPLAP) was secreted into the medium to the level of 2 mg/liter. yPLAP displayed kinetic properties similar to those reported earlier for the membrane-bound PLAP. Purified yPLAP had specific activity of 774 U/mg and appeared in two subunit sizes, ca. 62 and 65 kDa. This difference was due to heterogenous N-glycosylation. Purified yPLAP appeared as multiple forms in isoelectric focusing in pI range of 4.2 to 5.2. The expression system is discussed in comparison to previously reported expression systems.

Fusion tails for the recovery and purification of recombinant proteins.

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.

Purification of major lignin peroxidase isoenzymes from Phanerochaete chrysosporium by chromatofocusing.

The basidiomycete Phanerochaete chrysosporium produces several isoforms of lignin peroxidase, which catalyzes the oxidative depolymerization of lignin To date, ion-exchange chromatography and preparative isoelectric focusing (IEF) have been commonly used for isolation of lignin peroxidase isoenzymes. In this work we have purified major lignin peroxidases to high purity by a one-step chromatographic method, chromatofocusing. The purified isoenzymes were identified by analytical IEF using isoenzymes purified by preparative IEF as standards. The specific activities and spectral properties of the isoenzymes were comparable with the previously published data. The predominant isoenzyme under the growth conditions used was LiP 4.65. Almost 50% of the lignin peroxidase activity applied into the column was recovered in the LiP 4.65 fraction. The total recovery of the lignin peroxidase activity was over 80%.

Microscopic study of migration of microbes in food-packaging paper and board.

The microbiological barrier properties of food-packaging paperboards, coated with polyethylene, mineral pigment or a biodegradable polymer and of high-density paper were examined with confocal laser scanning microscopy. The results show that the spatial distribution of microscopically observable bacterial cells was uneven inside the paperboard. The concentration in the interface between the polyethylene coating and the cellulose fibers was 100-200 times higher than inside the cellulose matrix. The bacteria in the interface and the mineral coating layer grew in response to access to food and moisture, whereas no growth was observed inside the fiber web, not even after extended exposure for up to 90 days. The paper and paperboards studied contained soluble nutrients (C:N:P 54:9:1 to 309:3:1) and no measurable antimicrobial activity. The factor limiting growth and migration of bacteria inside the fiber web was most likely limited access to free water, even under conditions of extensive wetting. The studied paperboards functioned as efficient barriers against translocation of microbes. The microbes residing between the paperboard and its polymer coating facing food, was the only potential site from which microbes could leak into food. This emphasizes the need for high hygienic quality of surface-sizing chemicals. Mineral-coating pigments were a source of microbes and their application behind the PE coating facing food is contraindicated.

C-terminal truncations of a thermostable Bacillus stearothermophilus alpha-amylase.

A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues. The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The Km values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding. The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile. The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling. The liquefying amylases seem to require approximately 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium.

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.

Effects of signal peptide mutations on processing of Bacillus stearothermophilus alpha-amylase in Escherichia coli.

Bacillus stearothermophilus alpha-amylase has a signal peptide typical for proteins exported by Gram-positive bacteria. There is only one signal peptidase processing site when the protein is exported from the original host, but when it is exported by Escherichia coli, two alternative sites are utilized. Site-directed mutagenesis was used to study the processing in E. coli. Processing sites for 13 B. stearothermophilus alpha-amylases carrying mutations in their signal peptide were determined. Processing of the signal peptide was remarkably tolerant to mutations, because switching between the alternative sites was possible. The length and the sequence of the region between the hydrophobic core and the cleavage site was crucial for determining the choice of the processing site. Some mutations more distal to the cleavage site also affected the site preference.

Paenibacillus borealis sp. nov., a nitrogen-fixing species isolated from spruce forest humus in Finland.

Seven spore-forming, nitrogen-fixing bacterial isolates from spruce forest humus in Finland were studied using the polyphasic approach. PCR amplification of 16S rRNA gene fragment with specific primers showed that the isolates were members of Paenibacillus. Levels of 16S rDNA similarity between the isolates were 97.3-100.0% and those between the isolates and other Paenibacillus species were 90.3-96.5%. The highest similarities were observed with Paenibacillus azotofixans and Paenibacillus durus. Ribotyping with EcoRI and PvuII restriction showed a high diversity in the Paenibacillus species and distinguished the isolates from these closely related species. The main whole-cell fatty acids were anteiso-C15:0 (33-48%), straight-chain C14:0 (7-21%) and C16:0 (9-20%), and iso-C15:0 (6-15%). Electron microscopy revealed a unique striped morphology of the spore surfaces. Based on phylogenetic inference and phenotypic and chemotaxonomic characteristics, these isolates are proposed as a new species, Paenibacillus borealis sp. nov., the type strain of which is KK19T (= DSM 13188T = CCUG 43137T).

Recovery of a charged-fusion protein from cell extracts by polyelectrolyte precipitation.

Beta-galactosidase served as a model system to explore the feasibility of enhancing the selectivity of a low-cost, easily scaled separation method-precipitation. Enhanced selectivity was sought by fusing the enzyme with polypeptide tails including 5 and 11 aspartates. The unfused protein could not be selectively removed from the Escherichia coli cell extract by precipitation with polyethylenimine (PEI), but the longest fusion could be selectively removed. The presence of nucleic acids limited the purification attainable. Pretreatment with nuclease followed by diafiltration resulted in an extract from which the same fusion could be precipitated with greater than fivefold enrichment, while the untailed enzyme remained unenriched by the same precipitation step. Selectivity is attributed to the binding strength of the polyanionic tails to the polycationic PEI.

Psychosomatic symptoms in preadolescent children.

As part of a Finnish national epidemiological study on child psychiatric disorders, psychosomatic symptoms were studied in a sample (n = 1,100) of 8-year-old children on the basis of self-report questionnaires by the children, their parents and teachers. Psychosomatic symptoms were common, although constant symptoms were rare. There were no sex differences in the occurrence of symptoms, but interesting differences were observed in associations between symptoms and other factors. Psychosomatic symptoms were strongly associated with depression scores and school performance.