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The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.Lactococcus lactis is the most important bacterium used for starter cultures by the dairy industry. An important problem in industrial milk fermentation is infection of the starter bacteria by bacteriophages, which leads to bacterial lysis. The consequences of phage infection are fermentation delay, alteration of product quality, and in severe cases loss of the product. All these outcomes result in considerable economic loss to dairies (5,22). Industrial phage ecology is dominated by three phage species: the predominant species 936 and species c2 and P335, which also have considerable importance (5,22).The first step in phage infection is adsorption of the phage to the host cell. Despite the fact that little information concerning the adsorption process of lactococcal phages is available, it seems to be a two-step process that starts with reversible binding to specific carbohydrates exposed on the surface of the cell wall (30, 37, 39), which is followed by, at least in species c2, irreversible binding to a protein in the cell membrane (14,30,38). Information on receptor-binding proteins (RBPs) in phages infecting gram-positive bacteria is sparse compared to the information available for phages infecting gram-negative bacteria. However, recently, Duplessis and Moineau identified the first RBP genes for the Streptococcus thermophilus phages DT1 and MD4 (11). These authors repo...

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that 645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and 645 that showed no homology in the C-terminal part.Lactococcus lactis is widely used in starter cultures for cheese production. Bacteriophage contamination during the fermentation process is a major problem, causing lysis of the starter bacteria and consequently slow or failed fermentation of the milk. Bacteriophage infection requires specific recognition between the phage receptor-binding protein (RBP) and the host cell receptor. A better understanding of this recognition mechanism should increase the possibility of preventing phage infection.Bacterial receptors have been well studied in gram-negative bacteria, especially Escherichia coli. Phages attacking E. coli recognize either lipopolysaccharides or specific proteins of the outer membrane. For example, phage lambda initially interacts reversibly and then interacts irreversibly with the outer membrane protein LamB (32, 35), which facilitates the diffusion of maltose into the cell (10). A slightly more complex mechanism is utilized by phage T5, which initially binds reversibly to polymannose O antigens in lipopolysaccharides at the E. coli surface (13, 14) and then binds irreversibly to the ferrichrome transporter FhuA (5, 19). Finally, phage T4 binds reversibly with its long tail fibers to B-type lipopolysaccharides or to the outer membrane porin OmpC (15,16,28), whereupon the additional short tail fibers bind irreversibly to the lipopolysaccharide core region (27).For gram-positive bacteria the information on phage receptors is sparser. However, phages attacking L. lactis seem to ...

Aims: To develop PCR assays able to distinguish between groups within lactococcal 936-species bacteriophages, as defined by their different receptor-binding protein (RBP) genes. Methods and Results: DNA sequences of RBP genes from 17 lactococcal bacteriophages of the 936-species were compared, and six phage groups were identified. For each phage group a specific primer pair targeting a variable region of the RBP genes was designed. In nine of 20 whey samples, from dairies with recorded phage problems, between one and six phage groups were identified by conventional PCR. The sensitivity and specificity of the method was improved by magnetic capture hybridization (MCH)-PCR using a capture probe targeting an 80-bp highly conserved region just upstream from the RBP gene in all the investigated phages. The MCH-PCR was performed on 100 ll whey samples and the detection limit of the assay was 10 2 -10 3 PFU ml )1 as opposed to the detection limit of 10 4 PFU ml )1 for conventional PCR performed on 1-ll whey samples. Conclusions:In this study, PCR assays have been developed to detect six different types of RBP genes in lactococcal 936-species bacteriophages.Significance and Impact of the Study: The PCR assays have practical applications at cheese plants for detection of 936-species phages with different RBP and thereby potentially with different host ranges. This knowledge will make it possible to improve starter culture rotation systems in the dairy industry.

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Transmission of different variants of PCV2 and viral dynamics in a research facility with pigs mingled from PMWS-affected herds and non-affected herds, Veterinary Microbiology (2008Microbiology ( ), doi:10.1016Microbiology ( /j.vetmic.2009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Aims:  To identify the main amino acids involved in the Flo11p‐mediated adhesion of Saccharomyces cerevisiae to the polystyrene surface PolySorp. Methods and Results:  Using a combination of phage display and competitive elution revealed that 12‐mer peptides of phages from competitive panning with S. cerevisiae FLO11 wild‐type (TBR1) cells had a higher consensus than those from competitive panning with S. cerevisiae flo11Δ mutant (TBR5) cells, suggesting that the wild‐type cells interact with the plastic surface in a stronger and more similar way than the mutant cells. Tryptophan and proline were more abundant in the peptides of phages from competitive elution with FLO11 cells than in those from competitive elution with flo11Δ cells. Furthermore, two phages with hydrophobic peptides containing 1 or 2 tryptophan, and 3 or 5 proline, residues inhibited the adhesion of FLO11 cells to PolySorp more than a phage with a hydrophobic peptide containing no tryptophan and only two proline residues. Conclusions:  Our results suggest a key role of tryptophan and proline in the hydrophobic interactions between Flo11p on the S. cerevisiae cell surface and the PolySorp surface. Significance and Impact of the Study:  Our study may contribute to the development of novel strategies to limit yeast infections in hospitals and other medical environments.