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A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.

A sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U [(mg protein).sup.-1]] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U [(mg protein).sup.-1] with methyl viologen and 833 U [(mg protein).sup.-1] with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 [micro]mol Fe and 2 [micro]mol Ni [(g protein).sup.-1]; in addition, 2-5 [micro]mol Mo [(g protein).sup.-1] was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.

Mid2p is a plasma membrane protein that functions in Saccharomyces cerevisiae as a sensor of cell wall stress, activating the PKC1-MPK1 cell integrity pathway via the small GTPase Rho1p during exposure to mating pheromone, calcofluor white, and heat. To examine Mid2p signalling, a global synthetic interaction analysis of a mid2 mutant was performed; this identified 11 interacting genes. These include WSC1 and ROM2, upstream elements in cell integrity pathway signalling, and FKS1 and SMI1, required for 1,3-[beta]-glucan synthesis. These synthetic interactions indicate that the Wsc1p sensor acts through Rom2p to activate the Fks1p glucan synthase in a Mid2p-independent way. To further explore Mid2p signalling a two-hybrid screen was done using the cytoplasmic tail of Mid2p; this identified ZE01 (YOL109w), encoding a 12 kDa peripheral membrane protein that localizes to the plasma membrane. Disruption of ZEO1 leads to resistance to calcofluor white and to a Mid2p-dependent constitutive phosphorylation of Mpk1p, supporting a role for Zeo1p in the cell integrity pathway. Consistent with this, zeo1-deficient cells suppress the growth defect of mutants in the Rho1p GDP-GTP exchange factor Rom2p, while exacerbating the growth defect of sac7[DELTA] mutants at 37[degrees]C. In contrast, mid2[DELTA] mutants have opposing effects to zeo1[DELTA] mutants, being synthetically lethal with rom2[DELTA], and suppressing an 18[degrees]C growth defect of sac7[DELTA], while overexpression of MID2 rescues a rom2[DELTA] 37[degrees]C growth defect. Thus, MID2 and ZEO1 appear to play reciprocal roles in the modulation of the yeast PKC1-MPK1 cell integrity pathway.

Many Gram-negative pathogens employ a specific secretion pathway, termed type III secretion, to deliver virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. Subsequent functions of many effector proteins delivered in this manner result in subversion of host-signalling pathways to facilitate bacterial entry, survival and dissemination to neighbouring cells and tissues. Whereas the secreted components of type III secretion system (TTSSs) from different pathogens are structurally and functionally diverse, the structural components and the secretion apparatus itself are largely conserved. TTSSs are large macromolecular assemblies built through interactions between protein components of hundreds of individual subunits. The goal of this project was to screen, using the standard yeast two-hybrid system, pair-wise interactions between components of the enteropathogenic Escherichia coliTTSS. To this end 37 of the 41 genes encoded by the LEE pathogenicity island were cloned into both yeast two-hybrid system vectors and all possible permutations of interacting protein pairs were screened for. This paper reports the identification of 22 novel interactions, including interactions between innermembrane structural TTSS proteins; between the type III secreted translocator protein EspD and structural TTSS proteins; between established and putative chaperones and their cognate secreted proteins; and between proteins of undefined function.

Cholic acid (CA) transport was investigated in nine intestinal Bifidobacterium strains. Upon energization with glucose, all of the bifidobacteria accumulated CA. The driving force behind CA accumulation was found to be the transmembrane proton gradient ([DELTA]pH, alkaline interior). The levels of accumulated CA generally coincided with the theoretical values, which were calculated by the Henderson-Hasselbalch equation using the measured internal pH values of the bifidobacteria, and a p[k.sub.a] value of 6.4 for CA. These results suggest that the mechanism of CA accumulation is based on the diffusion of a hydrophobic weak acid across the bacterial cell membrane, and its dissociation according to the [DELTA]pH value. A mixture of short-chain fatty acids (acetate, propionate and butyrate) at the appropriate colonic concentration (117 mM in total) reduced CA accumulation in Bifidobacterium breve JCM [1192.sup.T]. These short-chain fatty acids, which are weak acids, reduced the [DELTA]pH, thereby decreasing CA accumulation in a dose-dependent manner. The bifidobacteria did not alter or modify the CA molecule. The probiotic potential of CA accumulation in vivo is discussed in relation to human bile acid metabolism.

The effect of EDTA on Salmonella enterica serovar Typhimurium was studied in different growth phases with cells grown with or without [Ca.sup.2+] and [Mg.sup.2+] supplementation. EDTA affected the outer membrane much more strongly in the early exponential phase than in the mid- or late exponential phase, as indicated by uptake of l-N-phenylnaphthylamine (a nonpolar hydrophobic probe, [M.sub.r]219), and detergent (SDS) susceptibility. This effect was, however, not paralleled by LPS release (determined by measuring LPS-specific fatty acids or [sup.14C]-labelled LPS in cell-free supernatants, per a standardized cell density), which remained unchanged as a function of the growth curve. The conclusion from these results is that in the early exponential phase the effect of EDTA in S. enterica involves a component that is independent of LPS release.

Research has been conducted on the enteropathogenic Escherichia coli type III secretion system. Results indicate that this system can translocate and insert native Tir into the red blood cell membrane to bind intimin and to form an intimate bacterial attachment.

Research has been conducted on the listeriolysin O, the virulence factor secreted by the pathogenic Listeria monocytogenes acting as a pore-forming cytolysin. The truncated listeriolysin O proteins have been secreted into the L. monocytogenes culture supernatant to bind to cell membranes and the results of the experiment are reported.

A genetic difference between the majority of Salmonella serovars and Salmonella typhi can be seen in a chromosomal region surrounding the ompD porin gene. The majority of Salmonella serovars most frequently associated with the systemic infection of vertebrate hosts produce the major outer-membrane porin. It appears that the smvA gene is inactive in S typhi. Absence of ompD may suggest a role in host specificity.

Research has been conducted on Synechocystis sp. PCC 6803 cells with mechanosensitive ion channels MscL located in their plasma membrane. Results demonstrate that these cells can release Ca (super)2+ in depolarization response of the plasma membrane in the presence of K (super)+.

Research has been conducted on Salmonella enterica tetrathionate and thiosulfate reductases. Results demonstrate that the bacterium can utilize insoluble sulfur substrates via reactions at the cytoplasmic membrane.

It has been postulated that phenotypic variation in the relative expression of two chemically distinct types of lipopolysaccharide (LPS), a serotype-specific LPS (B-band) and a common antigen LPS (A-band) in Pseudomonas aeruginosa is an important mechanism enabling this opportunistic pathogen to alter its surface characteristics to mediate adhesion and to survive under extreme conditions. To further investigate this, the relative expression levels of the two distinct types of LPS in P. aeruginosa PA01 were investigated with cells grown in a chemostat at different dissolved oxygen tensions (p[O.sub.2]). The A-band LPS was constitutively expressed as p[O.sub.2] was increased from nearly zero to 350% of air saturation. In contrast, the B-band LPS showed a remarkable increase with increased p[O.sub.2]. Almost no B-band LPS was found in cells grown at a p[O.sub.2] of less than 3% of air saturation. Electron microscopic examination of cells revealed increased formation of membrane vesicles (MVs) on the surface of P. aeruginosa PAO1 under oxygen stress conditions. The toxicity of the supernatant of P. aeruginosa cultures to the growth of a hybridoma cell line significantly increased in samples taken from oxygen-stressed steady-state cultures. Furthermore, studies of adhesion in a continuous-flow biofilm culture revealed an increased adhesiveness for hydrophilic surfaces in P. aeruginosa PA01 grown at a higher p[O.sub.2]. The oxygen-dependent alterations of cell-surface components and properties observed in this work provide a possible explanation for the emergence of P. aeruginosa lacking the B-band LPS in chronically infected cystic fibrosis patients. The results are also useful for understanding the processes involved in the formation of MVs in P. aeruginosa.