STUDIES ON SALT ACTION : X. THE INFLUENCE OF ELECTROLYTES UPON THE VIABILITY AND ELECTROPHORETIC MIGRATION OF BACTERIUM COLI.

1. The strain of Bacterium coli used in these experiments multiplies in distilled water at pH 6.0 and pH 8.0 and in Ringer-Locke solution at pH 6.0. Under all the other conditions studied the numbers decrease with the passage of time. 2. The electrophoretic charge of the cells is highest in distilled water at pH 6.0 and pH 8.0. Under all other conditions studied the velocity of migration is decreased, but the decrease is immediate and is not affected by more prolonged exposure. 3. A strongly acid solution (pH 2.0) causes a rapid death of the cells and a sharp decrease in electrophoretic charge, sometimes leading to complete reversal. 4. A strongly alkaline solution (pH 11.0) is almost as toxic as a strongly acid one, although in distilled water the organisms survive fairly well at this reaction. Electrophoretic charge, on the other hand, is only slightly reduced in such an alkaline medium. 5. In distilled water, reactions near the neutral point are about equally favorable to both viability and electrophoretic charge, pH 8.0 showing slightly greater multiplication and a slightly higher charge than pH 11.0. In the presence of salts, however, pH 8.0 is much less favorable to viability and somewhat more favorable to electrophoretic charge than is pH 6.0. 6. Sodium chloride solutions, in the concentrations studied, all proved somewhat toxic and all tended to depress electrophoretic charge. Very marked toxicity was, however, exhibited only in a concentration of .725 M strength or over and at pH 8.0, while electrophoretic migration velocity was only slightly decreased at a concentration of .0145 M strength. 7. Calcium chloride was more toxic than NaCl, showing very marked effects in .145 M strength at pH 8.0 and in 1.45 M strength at pH 6.0. It greatly depressed electrophoretic charge even in .0145 M concentration. 8. Ringer-Locke solution proved markedly stimulating to the growth of the bacteria at pH 6.0 while at pH 8.0 it was somewhat toxic, though less so than the solutions of pure salts. It depressed migration velocity at all pH values, being more effective than NaCl in this respect, but less effective than CaCl₂. 9. It would appear from these experiments that a balanced salt solution (Ringer-Locke''s) may be distinctly favorable to bacterial viability in water at an optimum reaction while distinctly unfavorable in a slightly more alkaline solution. 10. Finally, while there is a certain parallelism between the influence of electrolytes upon viability and upon electrophoretic charge, the parallelism is not a close one and the two effects seem on the whole to follow entirely different laws.     

CHANGES IN THE STABILITY AND POTENTIAL OF CELL SUSPENSIONS : I. THE STABILITY AND POTENTIAL OF BACTERIUM COLI.

1. Stability and potential of Bacterium coli suspensions depend, not only on the strain of the organism and the medium in which it is suspended, but also on the previous treatment of the suspension, and the length of time it has been in the medium. 2. When treated at acid reactions, the negative charge on the bacteria is diminished; with some strains, a positive charge is acquired. Changes in stability accompany the changes in potential. 3. Washing acid-treated bacteria at neutral or slightly alkaline reactions does not restore the original potential; the zone of flocculation is moved toward the alkaline side. 4. These changes are due to two factors: the extraction of a soluble protein which combines with the surfaces of the cells, and a further irreversible change of the cell or its membrane.     

The Self-Assembly of a Cyclic Lipopeptides Mixture Secreted by a B. megaterium Strain and Its Implications on Activity against a Sensitive Bacillus Species

Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300–800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.     

A vector for promoter trapping in Bacillus cereus

We constructed a promoter-trap plasmid, pAD123, for Bacillus cereus. This plasmid contains a promoterless gene that encodes a mutant version of the green fluorescent protein, GFPmut3a, that is optimized for fluorescence-activated cell sorting [Cormack, B.P., Valdivia, R.H., Falkow, S., 1996. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.]. The plasmid replicates and confers drug resistance in both Escherichia coli and B. cereus. We constructed a library in pAD123, which consists of 29 000 clones containing chromosomal DNA from B. cereus strain UW85. A portion of the library (988 clones) was screened for GFP expression in B. cereus UW85 using a 96-well microtiter dish assay. GFP expression was detected by visual inspection with a fluorimager. We identified 21 clones as fluorescing in the initial screen, and further characterized these clones by restriction analysis, sequencing, and quantification of fluorescence intensity. Flow cytometry and cell sorting efficiently separated B. cereus cells expressing GFP from a 10 000-fold excess of non-expressing cells. Selected clones provided useful markers to follow B. cereus populations on plant surfaces. Our results indicate that GFP and pAD123 are useful tools for identifying regulatory sequences in Bacillus cereus, and that flow cytometry and cell sorting is a useful method for screening large libraries constructed in this vector.     

Specificity of translation of spore polypeptides in bacillus in vitro systems

Cell free extracts prepared from exponentially growing Escherichia coli and Bacillus cereus as well as from Bacillus cereus at the end of exponential growth were optimized for various factors required for amino acid incorporation when programmed with Bacillus ribonucleic acid. All three preparations synthesized glutamine synthetase antigen when ribonucleic acid from a Bacillus subtilis strain that overproduces glutamine synthetase was added. The post exponential Bacillus cereus extract, however, was most active for the synthesis of Bacillus cereus spore coat antigen when supplemented with the appropriate ribonucleic acid. There appears to be some specificity in the translation of at least this sporulation messenger RNA.Non-Standard Abbreviations PMSF phenyl methyl sulfonylfluoride - GS glutamine synthetase - UDS 8 M urea, 1% (W/V) sodium dodecyl sulfate, 50 mM dithioerythritol, 2 mM PMSF, 5 mM cyclohexylaminoethane sulfonic acid, pH 9.6     

Optimization on Antimicrobial Effects of Natural Compound Preservative Against B. cereus and E. coli by RSM

In this paper, the sensitivity of food spoilage organisms (Bacillus cereus; Escherichia coli) to natural antimicrobial peptides (surfactin; polylysine; nisin) from microorganism was observed, and the optimization of antimicrobial effect in meat evaluated by a RSM was studied. Results showed that these strains were sensitive to them. MICs of surfactin and polylysine and nisin were 31.25 and 312.5 and 312.5???g/mL respectively against B. cereus, and MIC were 15.625 and 156.25 and 2,500???g/mL respectively against E. coli. The optimization result indicated that B. cereus and E. coli could be sterilized by six log cycles when the temperature was 14.05?°C, the action time was 10.95?h, and the concentration (surfactin/polylysine/nisin weight ratio 0.1:1:2) was 379.53???g/mL.     

FACTORS LIMITING BACTERIAL GROWTH : VI. EQUATIONS DESCRIBING THE EARLY PERIODS OF INCREASE

Simple assumptions have led to equations by which the latent period in multiplication and the bacterial numbers expected at any time during the phase of rapid growth may be predicted. Experimental data obtained under rather diverse conditions have given satisfactory agreement with calculated values. Since the mathematical expressions contain no arbitrary constants, more than accidental significance must be attached to this agreement. The hypotheses set forth appear completely to describe the early development of Bacterium coli and Bacterium dysenteriae in broth, without postulating differences other than size among individual cells, or cells obtained under different conditions.     

Low molecular weight chitosans—Preparation with the aid of pronase,characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5–8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 °C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (< 50%). IR and ¹H-NMR data showed decrease in the degree of acetylation (14–19%) in LMWC compared to native chitosan (∼ 26%). Minimum inhibitory concentration of LMWC towards 10⁶ CFU ml^− 1 of B. cereus was 0.01% (w/v) compared to 0.03% for 10⁴ CFU ml^− 1 of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.     

Effects of Lactic Acid Bacteria and Organic Acids on Growth and Germination of Bacillus cereus

Growth and germination of vegetative cells and endospores of Bacillus cereus were affected by Streptococcus lactis, Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus in nonfat milk medium and by salts of organic acids in broth medium. Growth of the lactic acid bacteria was not affected by B. cereus. B. cereus increased rapidly to about 10⁸ CFU/ml when cells were added at the beginning of growth of lactic acid bacteria; it was inactivated slowly when added after 24 h and rapidly when added after 72 h of lactic acid bacterial growth. Streptococci were more inhibitory to the growth of B. cereus than lactobacilli were at 24 h. Spore germination was not affected after 24 h, but it was inhibited after 48 and 72 h of lactic acid bacterial growth. Acetate was more inhibitory to the growth of vegetative cells, while formate was more inhibitory to spore germination. Acetate, formate, and lactate (all at 0.1 M) completely inactivated multiplication of B. cereus at pH 6.1, 6.0, and 5.6, respectively. Spores of B. cereus were more resistant to these organic acids compared with the resistance of vegetative cells. Formate, lactate, and acetate (all at 0.1 M) caused 50% inhibition of spore germination at pH 4.4, 4.3, and 4.2, respectively.     

Inhibition of Bacillus cereus Strains by Antimicrobial Metabolites from Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21

Bacillus cereus is an endospore-forming, Gram-positive bacterium able to cause foodborne diseases. Lactic acid bacteria (LAB) are known for their ability to synthesize organic acids and bacteriocins, but the potential of these compounds against B. cereus has been scarcely documented in food models. The present study has examined the effect of the metabolites produced by Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21 on the viability of select B. cereus strains. Furthermore, the effect of E. faecium SM21 metabolites against B. cereus strains has also been investigated on a rice food model. L. johnsonii CRL1647 produced 128 mmol/L of lactic acid, 38 mmol/L of acetic acid and 0.3 mmol/L of phenyl-lactic acid. These organic acids reduced the number of vegetative cells and spores of the B. cereus strains tested. However, the antagonistic effect disappeared at pH 6.5. On the other hand, E. faecium SM21 produced only lactic and acetic acid (24.5 and 12.2 mmol/L, respectively) and was able to inhibit both vegetative cells and spores of the B. cereus strains, at a final fermentation pH of 5.0 and at pH 6.5. This would indicate the action of other metabolites, different from organic acids, present in the cell-free supernatant. On cooked rice grains, the E. faecium SM21 bacteriocin(s) were tested against two B. cereus strains. Both of them were significantly affected within the first 4 h of contact; whereas B. cereus BAC1 cells recovered after 24 h, the effect on B. cereus 1 remained up to the end of the assay. The LAB studied may thus be considered to define future strategies for biological control of B. cereus.     

Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

Adhesion to the intestinal epithelium could constitute an essential mechanism of Bacillus cereus pathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacterium B. cereus NVH 0500/00 was exposed in vitro to gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherent B. cereus after simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated. B. cereus could grow and simultaneously adhere to mucin during in vitro ileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration of B. cereus in the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml⁻¹. At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retain B. cereus and to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.     

A COMPARATIVE ELECTRON MICROSCOPICAL STUDY OF RNA FROM DIFFERENT SOURCES

Electron micrographs of ribosomal RNA from Escherichia coli, microsomal RNA from calf, rat, and chick liver, Bacillus cereus RNA and E. coli soluble RNA are presented. Filaments of about 10 A in diameter could be observed in preparations obtained from aqueous solutions of high molecular weight RNA. When ammonium acetate solutions were used a tendency for coiling and aggregation was observed. E. coli soluble RNA appears as small, sometimes elongated particles the smallest diameter being of about 10 A.     

Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Autoinducer-2 Could Affect Biofilm Formation by Food-Derived Bacillus cereus

Bacillus cereus is a foodborne pathogen and cause a frequent problem due to the biofilms forming in equipment of food production plants. Autoinducer-2 (AI-2) involved in interspecies communication, plays a role in the biofilm formation of B. cereus. In this study, biofilm formation by thirty-nine B. cereus strains isolated from foods produced in Korea was determined. To investigate the effect of AI-2 on biofilm formation by B. cereus SBC27, which had the highest biofilm-forming ability, biofilm densities formed after addition of the AI-2 from Staphylococcus aureus and Escherichia coli were analysed. As a result, it was found that the quorum sensing molecule AI-2 could induce biofilm formation by B. cereus within 24 h, but it may also inhibit biofilm formation when more AI-2 is added after 24 h. Thus, these results improve our understanding of biofilm formation by food-derived B. cereus and provide clues that could help to reduce the impact of biofilms, the biggest problem in food processing environments, which has an impact on public health as well as the economy.     

Activity assay for nisin-like acidic bacteriocins using an optimal pH-conditioned gel matrix

A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine-sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP-CGM). The OP-CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8 kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS-polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.     

The inhibitory effect of albomycin (Grisein) on growth ofBacillus cereus andEscherichia coli

It was confirmed that albomycin inhibits the growth ofBacillus cereus andEscherichaa coli depending on a concentration of the antibiotic and physiological state of the culture. Salts of trivalen iron in a test medium reduce the time of the effect of albomycin, however, they also increase the growth of the control culture ofBacillus cereus and increase a value of maximal out-growth. An exponential growth of the culture is restored following a short lag when albomcycin is removed from a test medium. The antibiotic effect of albomycin A₁ onBacillus cereus andEscherichia coli is removed by adding a 10-fold concentration of albomycin A₃. The effect of Neilands ferrichrome is similar. On comparison withEscherichia coli, albomycin does no inhibit the growth ofBacillus cereus when cultivated at 28°C. The addition of CrCl₃ solution partially restored the antibiotic activity of albomycin deprived of iron. It was found by microbiological tests that a retored growth of the whole culture was a result of albomycin inactivation. However, we did not find what change in the albomycin molecule is responsible for this inactivation.     

Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus

D‐alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3) cells, which were prepared through embedding and cross‐linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700 mM with an enantiomeric excess of 99.99% and e.e._p of 99.50%. The immobilized recombinant B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N‐acetyl‐D‐alanine methyl esters.     

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

Type selective inhibition of microbial fatty acid synthases by thiolactomycin

The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g. Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g. Saccharomyces cerevisiae enzyme). We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus. Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin. E. coli synthase, as expected, was strongly inhibited by thiolactomycin. Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E. coli synthase, it is largely insensitive to thiolactomycin.     

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.