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Escherichia coil haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of [beta]-lactamase when the latter was fused to a Cterminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the [beta]-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion. DOI 10.1099/mic.0.038562-0

Cylindrospermopsin (CYN), a potent hepatoxin, occurs in freshwaters worldwide. Several cyanobacterial species produce the toxin, but the producing species vary between geographical regions. Aphanizomenon flos-aquae, a common algae species in temperate fresh and brackish waters, is one of the three well-documented CYN producers in European waters. So far, no genetic information on the CYN genes of this species has been available. Here, we describe the complete CYN gene cluster, including flanking regions from the German Aphanizomenon sp. strain 10E6 using a full genome sequencing approach by 454 pyrosequencing and bioinformatic identification of the gene cluster. In addition, we have sequenced a ~7 kb fragment covering the genes cyrC (partially), cyrA and cyrB (partially) of the same gene cluster in the CYN-producing Aphanizomenon sp. strains 10E9 and 22D11. Comparisons with the orthologous gene clusters of the Australian Cylindrospermopsis raciborskii strains AWT205 and CS505 and the partial gene cluster of the Israeli Aphanizomenon ovalisporum strain ILC-146 revealed a high gene sequence similarity, but also extensive rearrangements of gene order. The high sequence similarity (generally higher than that of 16S rRNA gene fragments from the same strains), atypical GCcontent and signs of transposase activities support the suggestion that the CYN genes have been horizontally transferred. DOI 10.1099/mic.0.036988-0

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coil (EPEC) type III secretion system effector NIeH has a broad-range anti-apoptotic activity. In this study we examined the effects of NIeH on cells challenged with TcdB. During infection with wild-type EPEC, NIeH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NIeH acts via a global anti-apoptotic pathway. DOI 10.1099/mic.0.037259-0

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB--encoded by a prophage in S. Typhimurium DT104--are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [[sup.32]P]NAD and ArtA, and the label was released by Hg[Cl.sub.2], which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized [ArtA.sup.6Arg-Ala] and [ArtA.sup.115Glu-Ala], in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA-and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX. DOI 10.1099/mic.0.028399-0

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMSl was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Gram-positive species.

The outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wail in mediating the fungicidal action of the cationic, [alpha]-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.

The current model for virulence gene regulation in Bacillus anthracis involves several trans-acting factors, the most important of which appears to be the anthrax toxin activator encoded by the atxA gene. AtxA is a positive regulator of the toxin genes pagA, cya and lef, and of a number of other plasmid- and chromosome-encoded genes. The AtxA protein (56 kDa) possesses a predicted winged-helix DNA-binding domain and phosphotransferase system-regulated domains, but the mechanism for positive regulation of AtxA target genes is not known. Sequence similarities in the promoter regions of AtxA-regulated genes are not apparent, and recombinant AtxA binds DNA with a high affinity in a non-specific manner. We hypothesized that the toxin genes possess common structural features or cis-acting elements that are required for positive regulation. We employed deletion analyses to determine the minimal sequences required for atxA-mediated toxin gene expression. In silico modelling and in vitro experiments using double-stranded DNA corresponding to the toxin gene promoter regions indicated significant curvature associated with these regions. These findings suggest that the structural topology of the DNA plays an important role in the control of anthrax toxin gene expression.

An unknown cytotoxin was identified in the culture supernatant of Clostridium perfringens type C. The cytotoxin, named TpeL, which was purified using mAb-based affinity chromatography, had a lethal activity of 62 minimum lethal dose (MLD) [mg.sup.-1] in mice and a cytotoxic activity of 6.2 x [10.sup.5] cytotoxic units (CU) [mg.sup.-1] in Vero cells. The nucleotide sequence of TpeL was determined. The entire ORF had a length of 4953 bases, and the same nucleotide sequence was not recorded in the GenBank/EMBL/DDBJ databases. The molecular mass calculated from the deduced amino acid sequence was 191 kDa, and a signal peptide region was not found within the ORF. The deduced amino acid sequence exhibited 30-39% homology to Clostridium difficile toxins A (TcdA) and B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi alpha-toxin (TcnA). The amino acid sequence of TpeL is shorter than these toxins, and the homologous region was located at the N-terminal site. Eighteen strains of C. perfringens types A, B and C were surveyed for the presence of the tpeL gene by PCR. The tpeL gene was detected in all type B (one strain) and C strains (five strains), but not in any type A strains (12 strains). TpeL was detected in culture filtrates of the five type C strains by dot-blot analysis, but not in the type B strain. It was concluded that TpeL is a novel toxin similar to the known large clostridial cytotoxins. Furthermore, the data indicated that TpeL is produced by many C. perfringens type C strains.

Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12. Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic [beta]-hairpin structure (the [beta]-tongue, residues 177-203) interacts with the lipid bilayer in target membranes. In seeming contradiction to this, the hlyE sequence from a pathogenic E. coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the [beta]-tongue. Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E. coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function. Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E. coli K-12. The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36-41 [Angstrom] diameter pores. However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components. TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE. Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.

A 1 x 4 kb positive regulatory element (ET[A.sup.exp]) that controls staphylococcal exfoliative toxin A (sETA) transcription was cloned from Staphylococcus aureus. ET[A.sup.exp] is located upstream of the cloned 5-8 kb eta gene (etaJ1) obtained from the chomosomal DNA of S. aureus ZM, the standard ETA-producing strain. The cETA prepared from an Escherichia coli transformant into which the recombinant plasmid petaJ1 (5 x 8 kb eta/pUC9) had been introduced was expressed at high levels in the culture supernatant and the ammonium-sulfate-precipitated culture supernatant fraction as shown by immunoblotting and the single radial immunodiffusion test. However, cETA produced by the recombinant plasmid petaJ3 containing the 1 x 7 kb eta sequence (etaJ3) with a 1 x 45 kb ET[A.sup.exp]-deficient eta fragment (1 x 7 kb eta/pUC9) obtained from the 5 x 8 kb eta sequence by subcloning was not detected in either the culture supernatant or the ammonium-sulfate-precipitated culture supernatant fraction (167-fold concentrate of the culture supernatant) by immunoblotting or the single radial immunodiffusion test. A large amount of cETA was produced by the 1 x 7 kb eta sequence when it was linked to ET[A.sup.exp] amplified by PCR (1 x 7 kb eta-ET[A.sup.exp]/pUC9), regardless of the orientation of ET[A.sup.exp] insertion. Northern blot hybridization showed lower levels of the transcripts of the 1 x 7 kb eta sequence than of the 5 x 8 kb eta sequence. The rsETA prepared from an S. aureus transformant into which the recombinant plasmid 3 x 4 kb eta-ET[A.sup.exp]/pYT3 (pYT3-etaJ6) had been introduced was expressed at high levels in the culture supernatant fraction as shown by the latex agglutination test. However, the agglutination titre in the culture supernatant fraction of rsETA produced by the recombinant plasmid (1 x 7 kb eta/pYT3) containing the 1 x 7 kb eta sequence carrying the 1 x 4 kb ET[A.sup.exp]-deficient eta fragment (pYT3-etaJ3) was 2500-4000 times lower than that of pYT3-etaJ6.

Shiga-toxin-2-encoding phages isolated from Escherichia coli O157:H7 have been studied relative to mosaic structure with the finding that frequent gene exchange between lambdoid phage genomes probably occurs. Restriction enzyme analysis, hybridization with probes for toxin genes and selected phage DNA such as the P gene, integrase gene and IS1203 were used for characterization. PCR studies and partial sequencing of selected DNA regions in the integrase to the stx(sub.2) region of the phages were used.

The genes and enzymes involved in producing the lacticin 3147 lantibiotic are described. This lantibiotic produced by Lactococcus lactis subsp. lactis DPC3147 produces ion-specific pores in the membrane of Gram-positive bacterial cells.

A 23-amino acid peptide called Sap-Ph is a pheromone that regulates bacteriocin production in Lactobacillus sakei Lb706. The peptide is produced by the orf4 gene, which is part of the sap gene cluster.

The immunity protein CbiA of Carnobacterium piscicola LV17A also protects against enterocin B as well as carnobacteriocin A and vice versa. Immunity proteins are proteins that protect bacteria from their own bacteriocins.

Interaction of three salmonellas with porcine and bovine terminal ileum in vivo is discussed. Comparative studies on invasiveness were done for Interaction of Salmonella choleraesuis, S. dublin and S. typhimurium. S. dublin could not be studied in some ways because of its tissue-destructive properties.

A mathematical model representing the dynamics of a colicinogenic and a colicin-sensitive population propagated under serial transfer culture conditions is presented. Also, a series of in vitro invasion experiments utilizing six representatives of the E colicin group were conducted, including the estimation of the growth rates and colicinogenic properties of the strains. Findings revealed that the properties of various colicinogenic strains cannot be employed to elucidate on the extensive variation in the relative abundance of different colicins in natural populations of bacteria.

Strain TA33a of Leuconostoc mesenteroides contains three leucocins designated as Leucocin A-TA33a, B-TA33a and C-TA33a. Leucocin B-TA33a has 31 amino acid residues and is homologous to mesenterocin 52B. Leucocin C-TA33a contains 36 amino acid residues and belongs to the class II bacteriocin superfamily. Leucocin A-TA33a is the same as leucocin A-UAL 187. These leucocins exhibit structural transitions adopting a beta structure when placed in an environment that mimics biological membranes based on circular dichroism spectra and gel electrophoresis.

A group of 10 site-directed mutants of Clostridium perfringens e-toxin was produced. All of the mutated proteins expressed in Escherichia coli were identified in immunoblots by a neutralizing mAb raised against wild-type native e-toxin. One mutation caused a loss of activity. This non-toxic protein was obtained by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated e-toxin caused a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant e-toxin.

Affinity chromatography of the haemolysin toxin from Streptococcus suis type 2 using a thiopropyl Sepharose 6B column reveals that the extracellular protein has a molecular mass of 65 kDa. The haemolysin's response to oxygen and oxidizing agents, suppression by low levels of cholesterol, stimulation by reducing agents and transmembrane pore formation enable its classification into a family of antigenically related cholesterol-binding cytolytic toxins. S. suis haemolysin haemolytic activity is prevented by anti-streptolysin antibodies.