No abstract

Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments.

The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx O26 -eae-␤1 (4.8%; 19 samples), stx-wzx O103 -eae-(1.3%; five samples), stx-ihp1 O145 -eae-␥1 (0.8%; three samples), and stx-rfbE O157 -eae-␥1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eae␤1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eae␤1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3 end of a tRNA Ser gene. Phage mv4 DNA integration into the tRNA Ser gene preserved an intact tRNA Ser gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA Ser gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.Gram-positive Lactobacillus strains are extensively used as preservatives in food products and as lactic acid producers in dairy fermentations, but development of bacteriophages is the main cause of fermentation failures. Taxonomic studies on phages from Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis led to the determination of two genetic groups (21, 32). The phage representative of the most widespread group, bacteriophage mv4, has been well characterized (13). Phage mv4 is a temperate phage which infects and lysogenizes L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains. The 36-kb genome of the phage has been mapped physically, and its DNA is circularly permuted (20). Several genes have been characterized, such as those encoding structural proteins (52) or genes involved in cell lysis (6). The phage attachment site (attP) has previously been located on the mv4 genome, and several attachment sites on the chromosome of independently isolated lysogens have been identified (20).Most temperate bacteriophages integrate their DNA into the host chromosome by a site-specific recombination process following the Campbell model (10). This mechanism involves two specific attachment sites, one on the bacterial chromosome (attB) and the other one on the phage genome (attP). The recombination process is catalyzed by a phage-encoded integrase. There are many well-characterized examples of sitespecific recombination in gram-negative bacteriophages, and the best-studied system is that of bacteriophage (for a review, see reference 22). The integration system of phages of grampositive bacteria is less well documented, but data on sitespecific recombination are available for phages L54a, 11, and 13 of Staphylococcus aureus (14,25,55,56), for mycobacteriophage L5 (26), and for phages of...