The Htm1/EDEM protein has been proposed to act as a "degradation lectin" for endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins. In this study, we provide genetic and biochemical evidence that Yos9 protein in Saccharomyces cerevisiae is essential for efficient degradation of mutant glycoproteins. Yos9 is a member of the OS-9 protein family, which is conserved among eukaryotes and shows similarities with mannose-6-phosphate receptors (MPRs). We found that amino acids conserved among OS-9 family members and MPRs were essential for Yos9 protein function. Immunoprecipitation showed that Yos9 specifically associated with misfolded carboxypeptidase Y (CPY*), an ERAD substrate, but only when it carried Man8GlcNAc2 or Man5GlcNAc2 N-glycans. Our experiments further suggested that Yos9 acts in the same pathway as Htm1/EDEM. Yos9 protein is important for glycoprotein degradation and may act via its MRH domain as a degradation lectin-like protein in the glycoprotein degradation pathway.

Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G؉C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (ϳ6 kb) and K (ϳ20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G؉C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.Listeria monocytogenes is a gram-positive, opportunistic pathogen that can cause a wide spectrum of diseases, such as meningitis, septicemia, abortion, and gastroenteritis, associated with mortality rates as high as 25 to 30% (17, 62). Listeria bacteria are ubiquitously found in nature and can enter the food chain at many points (26, 66). They are able to proliferate over a wide range of external conditions, including low temperatures and high salt concentrations, thus posing severe problems to the food industry (2,22,15,48).Almost all of the Listeria bacteriophages described to date are temperate (38); only very little is known about virulent phages infecting this host. Listeria phages are valuable sources of biological information and useful tools for the study, differentiation, manipulation, and control of these bacteria (6,33,41). Phage A511 and some of its components have been particularly useful in phage typing schemes (35, 61), as reporter phage (39), with respect to their endolysins (16), and also for the control of Listeria contamination of foods (7; S. Guenther, D. Huwyler, S. Richard, and M. J. Loessner, submitted for publication). In contrast to temperate Listeria phages A118 and PSA (36, 71), A511 is a virulent (i.e., s...

The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3 overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3 end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize ؉1 and ؊1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual ؉1 frameshift at the 3 ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution.The opportunistic pathogen Listeria monocytogenes is ubiquitous in nature and can become endemic in food processing environments, causing contamination of dairy products, meats, vegetables, and processed ready-to-eat food (14). L. monocytogenes is the causative agent of epidemic and sporadic listeriosis. The risk of infection is markedly increased among immunocompromised patients, newborns, pregnant women, and the elderly and is associated with a mortality rate of about 20 to 30% (37).Although all strains of L. monocytogenes are considered potentially pathogenic, epidemiological evidence has shown that certain serovars are more frequently associated with both sporadic cases and larger food-borne outbreaks. However, genetic variation within the virulence genes of wild-type strains appears to be limited and could not be directly linked to differences in pathogenicity (30) or environmental distribution.It is becoming increasingly clear that bacteriophages have an important role in bacterial biology, diversity, and evolution, as indicated by the advances in genome sequencing which revealed a high incidence of phage-related sequences in bacterial genomes. Many phages have been described for the genus Listeria, and lysogeny appears to be widespread (28). Availability of the genome sequences of different L. monocytogenes and L. innocua strains also revealed the...

SummaryRecognition of the bacterial host and attachment to its surface are two critical steps in phage infection. Here we report the identification of Gp108 as the host receptor-binding protein of the broad host-range, virulent Listeria phage A511. The ligands for Gp108 were found to be N-acetylglucosamine and rhamnose substituents of the wall teichoic acids of the bacterial cell wall. Transmission electron microscopy and immunogold-labelling allowed us to create a model of the A511 baseplate in which Gp108 forms emanating short tail fibres. Data obtained for related phages, such as Staphylococcus phages ISP and Twort, demonstrate the evolutionary conservation of baseplate components and receptor-binding proteins within the Spounavirinae subfamily, and contractile tail machineries in general. Our data reveal key elements in the infection process of large phages infecting Grampositive bacteria and generate insights into the complex adsorption process of phage A511 to its bacterial host.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail.