Lichenysin is produced by most Bacillus licheniformis strains.

AIMS: The aim of this study was to elucidate the prevalence of lichenysin production in Bacillus licheniformis and to see whether this feature was restricted to certain genotypes. Secondly, we wanted to see whether cytotoxicity reflected the measured levels of lichenysin. METHODS AND RESULTS: Fifty-three genotyped strains of B. licheniformis, representing a wide variety of sources, were included. lchAA gene fragments were detected in all strains by polymerase chain reaction (PCR). All 53 strains produced lichenysins with four molecular masses as confirmed by LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis. The amounts of lichenysin varied more than two orders of magnitude between strains and were irrespective of genotype. Finally, there was a strong association between lichenysin concentrations and toxicity towards boar spermatozoa, erythrocytes and Vero cells. CONCLUSIONS: Lichenysin synthesis was universal among the 53 B. licheniformis strains examined. The quantities varied considerably between strains, but were not specifically associated with genotype. Cytotoxicity was evident at lichenysin concentrations above 10 µg ml(-1) , which is in accordance with previous studies. SIGNIFICANCE AND IMPACT OF STUDY: This study might be of interest to those working on B. licheniformis for commercial use as well as for authorities who make risk assessments of B. licheniformis when used as a food and feed additive.

Sphagnan--a pectin-like polymer isolated from Sphagnum moss can inhibit the growth of some typical food spoilage and food poisoning bacteria by lowering the pH.

AIMS: Investigate if the antibacterial effect of sphagnan, a pectin-like carbohydrate polymer extracted from Sphagnum moss, can be accounted for by its ability to lower the pH. METHODS AND RESULTS: Antibacterial activity of sphagnan was assessed and compared to that of three other acids. Sphagnan in its acid form was able to inhibit growth of various food poisoning and spoilage bacteria on low-buffering solid growth medium, whereas sphagnan in its sodium form at neutral pH had no antibacterial activity. At similar acidic pH, sphagnan had comparable antibacterial activity to that of hydrochloric acid and a control rhamnogalacturonan pectin in its acid form. CONCLUSIONS: Sphagnan in its acid form is a weak macromolecular acid that can inhibit bacterial growth by lowering the pH of environments with a low buffering capacity. SIGNIFICANCE AND IMPACT OF THE STUDY: It has previously been suggested that sphagnan is an antimicrobial polysaccharide in the leaves of Sphagnum moss with a broad range of potential practical applications. Our results now show that sphagnan in its acid form can indeed inhibit bacterial growth, but only of acid-sensitive species. These findings represent increased knowledge towards our understanding on how sphagnan or Sphagnum moss might be used in practical applications.

Antibacterial activity of sphagnum acid and other phenolic compounds found in Sphagnum papillosum against food-borne bacteria.

AIMS: To identify the phenolic compounds in the leaves of Sphagnum papillosum and examine their antibacterial activity at pH appropriate for the undissociated forms. METHODS AND RESULTS: Bacterial counts of overnight cultures showed that whilst growth of Staphylococcus aureus 50084 was impaired in the presence of milled leaves, the phenol-free fraction of holocellulose of S. papillosum had no bacteriostatic effect. Liquid chromatography-mass spectrometry analysis of an acetone-methanol extract of the leaves detected eight phenolic compounds. Antibacterial activity of the four dominating phenols specific to Sphagnum leaves, when assessed in vitro as minimal inhibitory concentrations (MICs), were generally >2.5 mg ml(-1). MIC values of the Sphagnum-specific compound 'sphagnum acid' [p-hydroxy-beta-(carboxymethyl)-cinnamic acid] were >5 mg ml(-1). No synergistic or antagonistic effects of the four dominating phenols were detected in plate assays. CONCLUSIONS: Sphagnum-derived phenolics exhibit antibacterial activity in vitro only at concentrations far in excess of those found in the leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: We have both identified the phenolic compounds in S. papillosum and assessed their antibacterial activity. Our data indicate that phenolic compounds in isolation are not potent antibacterial agents and we question their potency against food-borne pathogens.

Cytotoxicity in Bacillus mojavensis is abolished following loss of surfactin synthesis: implications for assessment of toxicity and food poisoning potential.

Bacillus subtilis and the closely related species Bacillus pumilus and Bacillus licheniformis have periodically been suggested to play a role in the aetiology of food poisoning despite the fact that the organisms do not possess the genes associated with enteropathogenicity in Bacillus cereus. We show here that Bacillus mojavensis, an organism closely related to B. subtilis, is able to produce toxic components which identify as a complex of three different surfactin analogues. These cyclic lipopeptides were soluble in methanol, heat stable after treatment in a boiling water bath for 10 min, resistant to enzymatic degradation by pepsin, trypsin, endoprotease V8 and proteinase K and formed pores in planar lipid bilayers. They were cytotoxic when tested in a series of commonly used laboratory cytotoxicity assays, namely, lactate dehydrogenase release, haemolysis, inhibition of both protein synthesis in Vero cells and motility in boar sperm. We show that such in vitro markers of enterotoxicity are due entirely to production of cyclic lipopeptides since deletion of sfp, a gene essential for surfactin synthesis which abolished the cytotoxicity to Vero cells, boar sperm motility and haemolytic activity. Thus, the relevance of cyclic lipopeptides as food poisoning toxins needs to be evaluated in assays other than the cell cytotoxicity assays in common use.

Inhibition of protein synthesis by a tryptic polypeptide of Clostridium perfringens type A enterotoxin.

The biological activity of Clostridium perfringens enterotoxin can be tested more precisely and with a much higher sensitivity by using the inhibition of protein synthesis by Vero cells, rather than the guinea pig skin test. Tryptic peptides of the enterotoxin produced in the presence of different concentrations of sodium dodecyl sulfate (0-1%) have been tested for biological activity (Vero cells) and inhibitory effect on cell-free protein synthesis (rabbit reticulocyte lysate). A fraction of tryptic peptides, about 16,000 daltons, was able to inhibit the cell-free protein synthesis, while the native enterotoxin had no such effect. The 16 kDa fraction had, however, lost the ability to disrupt the Vero cells (normal biological activity). It is probable that the enterotoxin has the double function (A and B chain), known from several other toxins, confined in its single polypeptide chain.

Trypsin activation of enterotoxin from Clostridium perfringens type A: fragmentation and some physicochemical properties.

Clostridium perfringens type A enterotoxin was activated about 3-fold by treatment with trypsin, without an observed change in molecular weight. On denaturation in 8 M urea, the trypsinated enterotoxin lost a small peptide of about 4000 daltons. The single cysteine residue of enterotoxin was in the small peptide together with seven out of nine residues of proline. Trypsin activation, without removal of the small peptide, increased the 'outside' number of amino groups from eight to eleven. The trypsin treatment of the enterotoxin did not change the antigenic properties of the protein. Glycine was the C-terminal residue of the native enterotoxin while the dansyl alpha-amino acid of the N-terminal could not be identified.

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins.

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Cloning and sequencing of the genes encoding acid-soluble spore proteins from Clostridium perfringens.

In Clostridium perfringens the acid-soluble spore proteins (ASSPs) of the alpha/beta type are encoded by two genes, sspC1 and sspC2. These genes, encoding ASSP C1 and C2 proteins, respectively, were cloned as parts of 3.5-kb genomic fragments. The genes were sequenced and polypeptides of 59 (C1) and 60 (C2) amino acids (aa) were deduced from the nucleotide sequence. The N-terminal Met is removed from the primary translation products in both cases, yielding mature polypeptides with Mrs of 6306 (C1) and 6553 (C2). The aa sequences of the two polypeptides were very similar and showed a closer relationship with C. bifermentans ASSPs than with ASSPs from Bacillus species.

The amino acid sequence of the enterotoxin from Clostridium perfringens type A.

The amino acid sequence of the enterotoxin from Clostridium perfringens type A was determined by analysis of peptides derived from the protein by digestion with trypsin chymotrypsin, thermolysin, pepsin, a lysine-specific protease. S. aureus V8 protease and a proline-specific protease, and fragments generated by cleavage with cyanogen bromide or by dilute acetic acid in 7 M guanidine HCl. The sequence which is complete except for the definite order of 3 small peptides between residues 88 and 103 consists of 309 amino acids and contains a correction to our preliminary announcement [(1984) FEMS Symp. 24, 329-330].

The chromatin-associated protein H-NS.

H-NS is a major component of chromatin in enteric bacteria. H-NS plays a structural role in organising the chromosome, and influences DNA rearrangements as well as the expression of many genes. The biochemical and functional characteristics of H-NS are distinct from those of 'typical' DNA-binding proteins and much remains to be learned about the mechanism(s) by which H-NS acts. In this article we review our current understanding of the role of H-NS, and describe possible models by which H-NS might influence DNA structure and gene expression.

Sphingomyelinase is part of the 'enterotoxin complex' produced by Bacillus cereus.

The three components of the 'enterotoxin complex' have been purified and the sequence of the first 14-15 amino acids of the proteins determined. Limited homology was found in the N-terminal sequence of the three proteins. The molecular mass of the proteins was determined to be 48, 40 and 34 kDa, respectively. Only the 40-kDa protein was toxic to Vero cells, whilst the 34-kDa protein was found to be hemolytic. The sequence of the first 14 N-terminal amino acids of this protein was identical to the sequence of the sphingomyelinase residues 28-41 (the N-terminal after loss of the signal sequence), except for a change from Gln to Glu in position 33 of the sphingomyelinase sequence.

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis.

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.

Bacillus cereus and its food poisoning toxins.

Bacillus cereus is becoming one of the more important causes of food poisoning in the industrialised world. It produces one emetic toxin and three different enterotoxins. The emetic toxin is a ring-shaped structure of three repeats of four amino and/or oxy acids: [D-O-Leu-D-Ala-L-O-Val-L-Val]3. This ring structure has a molecular mass of 1.2 kDa, and is chemically closely related to the potassium ionophore valinomycin. Two of the three enterotoxins have been shown to be involved in food poisoning. They both consist of three different proteins that act together. One of these enterotoxins is also a haemolysin. This haemolytic enterotoxin is transcribed from one operon. The third enterotoxin is a single component protein, but has not been shown to be involved in food poisoning.

Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak.

Three enterotoxic components have been isolated from a strain of Bacillus cereus which was involved in a large food poisoning outbreak in Norway in 1995. The components were purified by chromatography on three different columns. Three proteins of 39, 45 and 105 kDa, respectively, were found to be necessary for maximum cytotoxicity. The amino acid N-terminal sequences of the 39 and 45 kDa proteins were determined. The 45 kDa component was the same protein as the main antigen detected in the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra). The 39 kDa protein showed some similarity to the L1 protein of haemolysin BL from B. cereus. Furthermore, the three toxic components were all recognised by a polyclonal antiserum reported to detect enterotoxin from B. cereus. The proteins were different from the B- and L2-components of haemolysin BL, previously suggested to be a primary virulence factor, and had no detectable haemolytic activity.

The 105-kDa protein component of Bacillus cereus non-haemolytic enterotoxin (Nhe) is a metalloprotease with gelatinolytic and collagenolytic activity.

A sequence of 91 amino acids residues, probably starting from the N-terminal of the mature protein, was determined for the 105-kDa protein of the non-haemolytic enterotoxin of Bacillus cereus. The last part of this sequence was similar to parts of the N-terminal portions of two collagenases of Clostridium histolyticum and Clostridium perfringens. Zymography, with intact collagen fibril and gelatin as substrates, showed that the 105-kDa protein had collagenolytic and gelatinolytic activity. The 105-kDa protein also showed activity against a typical collagenase substrate, azocoll, and was inhibited by EDTA and 1,10-phenanthroline. We conclude that the 105-kDa protein is a collagenase.

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate.

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)2, similar to the transposition intermediates described for a number of IS elements.

CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia.

CytK is a cytotoxin isolated from a strain of Bacillus cereus cultured from cases of necrotic enteritis and the amino acid sequence of the protein suggests that it may belong to the family of beta-barrel pore-forming toxins. We show here in planar lipid bilayers the toxin is able to form pores which are weakly anion selective and exhibit an open channel probability close to one. The predicted minimum pore diameter is approximately 7 A. We also show that cytK is a potent cytotoxin against human intestinal Caco-2 epithelia. CytK, like other beta-barrel pore-forming toxins, spontaneously forms oligomers which are resistant to sodium dodecyl sulphate (SDS), but not to boiling. CytK represents a pore-forming toxin linked with human cases of necrotic enteritis.

The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus.

The non-haemolytic enterotoxin from Bacillus cereus has been sequenced. It is composed of three components, non-haemolytic enterotoxin A, B and C of 41.0, 39.8 and 36.5 kDa, respectively. Transcription of the operon seems to be positively regulated by plcR, a gene that also regulates phospholipase C expression. There is substantial similarity between the three proteins of non-haemolytic enterotoxin and between the non-haemolytic enterotoxin and haemolytic enterotoxin proteins.

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis.

Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning. Microbiological Societies.