Characterization of staphylococcal endolysin LysSAP33 possessing untypical domain composition.

Endolysin, a peptidoglycan hydrolase derived from bacteriophage, has been suggested as an alternative antimicrobial agent. Many endolysins on staphylococcal phages have been identified and applied extensively against Staphylococcus spp. Among them, LysK-like endolysin, a well-studied staphylococcal endolysin, accounts for most of the identified endolysins. However, relatively little interest has been paid to LysKunlike endolysin and a few of them has been characterized. An endolysin LysSAP33 encoded on bacteriophage SAP33 shared low homology with LysK-like endolysin in sequence by 41% and domain composition (CHAP-unknown CBD). A green fluorescence assay using a fusion protein for LysSAP33_CBD indicated that the CBD domain (157-251 aa) was bound to the peptidoglycan of S. aureus. The deletion of LysSAP33_CBD at the C-terminal region resulted in a significant decrease in lytic activity and efficacy. Compared to LysK-like endolysin, LysSAP33 retained its lytic activity in a broader range of temperature, pH, and NaCl concentrations. In addition, it showed a higher activity against biofilms than LysK-like endolysin. This study could be a helpful tool to develop our understanding of staphylococcal endolysins not belonging to LysK-like endolysins and a potential biocontrol agent against biofilms.

Glucose Decoration on Wall Teichoic Acid Is Required for Phage Adsorption and InlB-Mediated Virulence in Listeria ivanovii.

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall teichoic acid [WTA]) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene liv1070, which encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention on the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting that the trade-off between phage resistance and virulence attenuation may be a general feature in the genus Listeria. IMPORTANCE Listeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants and occasionally in immunocompromised humans. Recent investigations revealed that in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently results in disconnection of one of the bacterium's major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria, and the host may be a feature common to the genus Listeria.

Bacteriophage-encoded enzymes destroying bacterial cell membranes and walls, and their potential use as antimicrobial agents.

Appearance of pathogenic bacteria resistant to most, if not all, known antibiotics is currently one of the most significant medical problems. Therefore, development of novel antibacterial therapies is crucial for efficient treatment of bacterial infections in the near future. One possible option is to employ enzymes, encoded by bacteriophages, which cause destruction of bacterial cell membranes and walls. Bacteriophages use such enzymes to destroy bacterial host cells at the final stage of their lytic development, in order to ensure effective liberation of progeny virions. Nevertheless, to use such bacteriophage-encoded proteins in medicine and/or biotechnology, it is crucial to understand details of their biological functions and biochemical properties. Therefore, in this review article, we will present and discuss our current knowledge on the processes of bacteriophage-mediated bacterial cell lysis, with special emphasis on enzymes involved in them. Regulation of timing of the lysis is also discussed. Finally, possibilities of the practical use of these enzymes as antibacterial agents will be underlined and perspectives of this aspect will be presented.

Membrane insertion mechanism and molecular assembly of the bacteriophage lysis toxin ΦX174-E.

The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.

Identification and characterization of pectin related gene NbGAE6 through virus-induced gene silencing in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is a transient based reverse genetic tool used to elucidate the function of novel gene in N. benthamiana. In current study, 14 UDP-D-glucuronate 4-epimerase (GAE) family members were identified and their gene structure, phylogeny and expression pattern were analyzed. VIGS system was optimized for the functional characterization of NbGAE6 homologous genes in N. benthamiana. Whilst the GAE family is well-known for the interconversion of UDP-D-GlcA and UDP-D-GalA during pectin synthesis. Our results revealed that the downregulation of these genes significantly reduced the amount of GalA in the homogalacturunan which is the major component of pectin found in primary cell wall. Biphenyl assay and high performance liquid chromatography analysis (HPLC) depicted that the level of 'GalA' monosaccharide reduced to 40-51% in VIGS plants as compared to the wild type plants. Moreover, qRT-PCR also confirmed the downregulation of the NbGAE6 mRNA in VIGS plants. In all, this is the first comprehensive study of the optimization of VIGS system for the provision of rapid silencing of GAE family members in N. benthamiana, eliminating the need of stable transformants.

Structural basis for the adsorption of a single-stranded RNA bacteriophage.

Single-stranded RNA bacteriophages (ssRNA phages) infect Gram-negative bacteria via a single maturation protein (Mat), which attaches to a retractile pilus of the host. Here we present structures of the ssRNA phage MS2 in complex with the Escherichia coli F-pilus, showing a network of hydrophobic and electrostatic interactions at the Mat-pilus interface. Moreover, binding of the pilus induces slight orientational variations of the Mat relative to the rest of the phage capsid, priming the Mat-connected genomic RNA (gRNA) for its release from the virions. The exposed tip of the attached Mat points opposite to the direction of the pilus retraction, which may facilitate the translocation of the gRNA from the capsid into the host cytosol. In addition, our structures determine the orientation of the assembled F-pilin subunits relative to the cell envelope, providing insights into the F-like type IV secretion systems.

Phage Lysis: Multiple Genes for Multiple Barriers.

The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.

Phage single-gene lysis: Finding the weak spot in the bacterial cell wall.

In general, the last step in the vegetative cycle of bacterial viruses, or bacteriophages, is lysis of the host. dsDNA phages require multiple lysis proteins, including at least one enzyme that degrades the cell wall (peptidoglycan (PG)). In contrast, the lytic ssDNA and ssRNA phages have a single lysis protein that achieves cell lysis without enzymatically degrading the PG. Here, we review four "single-gene lysis" or Sgl proteins. Three of the Sgls block bacterial cell wall synthesis by binding to and inhibiting several enzymes in the PG precursor pathway. The target of the fourth Sgl, L from bacteriophage MS2, is still unknown, but we review evidence indicating that it is likely a protein involved in maintaining cell wall integrity. Although only a few phage genomes are available to date, the ssRNA Leviviridae are a rich source of novel Sgls, which may facilitate further unraveling of bacterial cell wall biosynthesis and discovery of new antibacterial agents.

Cell Wall Glycans Mediate Recognition of the Dairy Bacterium Streptococcus thermophilus by Bacteriophages.

Receptors on the cell surfaces of bacterial hosts are essential during the infection cycle of bacteriophages. To date, the phage receptors of the industrial relevant dairy starter bacterium Streptococcus thermophilus remain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrial S. thermophilus strains sensitive to different phages (pac type, cos type, and the new type 987), were selected to generate spontaneous bacteriophage-insensitive mutants (BIMs). Of these, approximately 50% were deselected as clustered regularly interspaced short palindromic repeat (CRISPR) mutants, while the other pool was further characterized to identify receptor mutants. On the basis of genome sequencing data, phage resistance in putative receptor mutants was attributed to nucleotide changes in genes encoding glycan biosynthetic pathways. Superresolution structured illumination microscopy was used to visualize the interactions between S. thermophilus and its phages. The phages were either regularly distributed along the cells or located at division sites of the cells. The cell wall structures mediating the latter type of phage adherence were further analyzed via phenotypic and biochemical assays. Altogether, our data suggested that phage adsorption to S. thermophilus is mediated by glycans associated with the bacterial cell surface. Specifically, the pac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCEStreptococcus thermophilus is widely used in starter cultures for cheese and yoghurt production. During dairy fermentations, infections of bacteria with bacteriophages result in acidification failures and a lower quality of the final products. An understanding of the molecular factors involved in phage-host interactions, in particular, the phage receptors in dairy bacteria, is a crucial step for developing better strategies to prevent phage infections in dairy plants.

Enzymes and Mechanisms Employed by Tailed Bacteriophages to Breach the Bacterial Cell Barriers.

Monoderm bacteria possess a cell envelope made of a cytoplasmic membrane and a cell wall, whereas diderm bacteria have and extra lipid layer, the outer membrane, covering the cell wall. Both cell types can also produce extracellular protective coats composed of polymeric substances like, for example, polysaccharidic capsules. Many of these structures form a tight physical barrier impenetrable by phage virus particles. Tailed phages evolved strategies/functions to overcome the different layers of the bacterial cell envelope, first to deliver the genetic material to the host cell cytoplasm for virus multiplication, and then to release the virion offspring at the end of the reproductive cycle. There is however a major difference between these two crucial steps of the phage infection cycle: virus entry cannot compromise cell viability, whereas effective virion progeny release requires host cell lysis. Here we present an overview of the viral structures, key protein players and mechanisms underlying phage DNA entry to bacteria, and then escape of the newly-formed virus particles from infected hosts. Understanding the biological context and mode of action of the phage-derived enzymes that compromise the bacterial cell envelope may provide valuable information for their application as antimicrobials.

Plant Cell Wall Dynamics in Compatible and Incompatible Potato Response to Infection Caused by Potato Virus Y (PVYNTN).

The cell wall provides the structure of the plant, and also acts as a barier against biotic stress. The vein necrosis strain of Potato virus Y (PVYNTN) induces necrotic disease symptoms that affect both plant growth and yield. Virus infection triggers a number of inducible basal defense responses, including defense proteins, especially those involved in cell wall metabolism. This study investigates the comparison of cell wall host dynamics induced in a compatible (potato cv. Irys) and incompatible (potato cv. Sárpo Mira with hypersensitive reaction gene Ny-Smira) PVYNTN-host-plant interaction. Ultrastructural analyses revealed numerous cell wall changes induced by virus infection. Furthermore, the localization of essential defensive wall-associated proteins in susceptible and resistant potato host to PVYNTN infection were investigated. The data revealed a higher level of detection of pathogenesis-related protein 2 (PR-2) in a compatible compared to an incompatible (HR) interaction. Immunofluorescence analyses indicated that hydroxyproline-rich glycoproteins (HRGP) (extensin) synthesis was induced, whereas that of cellulose synthase catalytic subunits (CesA4) decreased as a result of PVYNTN infection. The highest level of extensin localization was found in HR potato plants. Proteins involved in cell wall metabolism play a crucial role in the interaction because they affect the spread of the virus. Analysis of CesA4, PR-2 and HRGP deposition within the apoplast and symplast confirmed the active trafficking of these proteins as a step-in potato cell wall remodeling in response to PVYNTN infection. Therefore, cell wall reorganization may be regarded as an element of "signWALLing"-involving apoplast and symplast activation as a specific response to viruses.

Structural insights into the binding and catalytic mechanisms of the Listeria monocytogenes bacteriophage glycosyl hydrolase PlyP40.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases that specifically degrade the bacterial cell wall at the end of the phage lytic cycle. They feature a distinct modular architecture, consisting of enzymatically active domains (EADs) and cell wall-binding domains (CBDs). Structural analysis of the complete enzymes or individual domains is required for better understanding the mechanisms of peptidoglycan degradation and provides guidelines for the rational design of chimeric enzymes. We here report the crystal structure of the EAD of PlyP40, a member of the GH-25 family of glycosyl hydrolases, and the first muramidase reported for Listeria phages. Site-directed mutagenesis confirmed key amino acids (Glu98 and Trp10) involved in catalysis and substrate stabilization. In addition, we found that PlyP40 contains two heterogeneous CBD modules with homology to SH3 and LysM domains. Truncation analysis revealed that both domains are required for full activity but contribute to cell wall recognition and lysis differently. Replacement of CBDP40 with a corresponding domain from a different Listeria phage endolysin yielded an enzyme with a significant shift in pH optimum. Finally, domain swapping between PlyP40 and the streptococcal endolysin Cpl-1 produced an intergeneric chimera with activity against Listeria cells, indicating that structural similarity of individual domains determines enzyme function.

Structural changes of tailless bacteriophage ΦX174 during penetration of bacterial cell walls.

Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ΦX174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ΦX174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ΦX174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS.

Host recognition by lactic acid bacterial phages.

Bacteriophage infection of lactic acid bacteria (LAB) is one of the most significant causes of inconsistencies in the manufacture of fermented foods, affecting production schedules and organoleptic properties of the final product. Consequently, LAB phages, and particularly those infecting Lactococcus lactis, have been the focus of intensive research efforts. During the past decade, multidisciplinary scientific approaches have uncovered molecular details on the exquisite process of how a lactococcal phage recognises and binds to its host. Such approaches have incorporated genomic/molecular analyses and their partnership with phage structural analysis and host cell wall biochemical studies are discussed in this review, which will also provide our views on future directions of this research field.

Structure of the host-recognition device of Staphylococcus aureus phage ϕ11.

Phages play key roles in the pathogenicity and adaptation of the human pathogen Staphylococcus aureus. However, little is known about the molecular recognition events that mediate phage adsorption to the surface of S. aureus. The lysogenic siphophage ϕ11 infects S. aureus SA113. It was shown previously that ϕ11 requires α- or ß-N-acetylglucosamine (GlcNAc) moieties on cell wall teichoic acid (WTA) for adsorption. Gp45 was identified as the receptor binding protein (RBP) involved in this process and GlcNAc residues on WTA were found to be the key component of the ϕ11 receptor. Here we report the crystal structure of the RBP of ϕ11, which assembles into a large, multidomain homotrimer. Each monomer contains a five-bladed propeller domain with a cavity that could accommodate a GlcNAc moiety. An electron microscopy reconstruction of the ϕ11 host adhesion component, the baseplate, reveals that six RBP trimers are assembled around the baseplate core. The Gp45 and baseplate structures provide insights into the overall organization and molecular recognition process of the phage ϕ11 tail. This assembly is conserved among most glycan-recognizing Siphoviridae, and the RBP orientation would allow host adhesion and infection without an activation step.

Mycobacteriophage SWU1 gp39 can potentiate multiple antibiotics against Mycobacterium via altering the cell wall permeability.

M. tuberculosis is intrinsically tolerant to many antibiotics largely due to the imperviousness of its unusual mycolic acid-containing cell wall to most antimicrobials. The emergence and increasingly widespread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) revitalized keen interest in phage-inspired therapy. SWU1gp39 is a novel gene from mycobacteriophage SWU1 with unknown function. SWU1gp39 expressed in M. smegmatis conferred the host cell increased susceptibility to multiple antibiotics, including isoniazid, erythromycin, norfloxacin, ampicillin, ciprofloxacin, ofloxacin, rifampicin and vancomycin, and multiple environment stresses such as H2O2, heat shock, low pH and SDS. By using EtBr/Nile red uptake assays, WT-pAL-gp39 strain showed higher cell wall permeability than control strain WT-pAL. Moreover, the WT-pAL-gp39 strain produced more reactive oxygen species and reduced NAD(+)/NADH ratio. RNA-Seq transcriptomes of the WT-pAL-gp39 and WT-pAL revealed that the transcription of 867 genes was differentially regulated, including genes associated with lipid metabolism. Taken together, our results implicated that SWU1gp39, a novel gene from mycobacteriophage, disrupted the lipid metabolism of host and increased cell wall permeability, ultimately potentiated the efficacy of multiple antibiotics and stresses against mycobacteria.

Mutual association of Broad bean wilt virus 2 VP37-derived tubules and plasmodesmata obtained from cytological observation.

The movement protein VP37 of broad bean wilt virus 2 (BBWV 2) forms tubules in the plasmodesmata (PD) for the transport of virions between cells. This paper reports a mutual association between the BBWV 2 VP37-tubule complex and PD at the cytological level as determined by transmission electron microscopy. The generation of VP37-tubules within different PD leads to a different occurrence frequency as well as different morphology lines of virus-like particles. In addition, the frequency of VP37-tubules was different between PD found at different cellular interfaces, as well as between single-lined PD and branched PD. VP37-tubule generation also induced structural alterations of PD as well as modifications to the cell wall (CW) in the vicinity of the PD. A structural comparison using three-dimensional (3D) electron tomography (ET), determined that desmotubule structures found in the center of normal PD were absent in PD containing VP37-tubules. Using gold labeling, modification of the CW by callose deposition and cellulose reduction was observable on PD containing VP37-tubule. These cytological observations provide evidence of a mutual association of MP-derived tubules and PD in a natural host, improving our fundamental understanding of interactions between viral MP and PD that result in intercellular movement of virus particles.

Molecular dissection of phage lysin PlySs2: integrity of the catalytic and cell wall binding domains is essential for its broad lytic activity.

The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain to be established. In the present study, the lytic activity of the catalytic domain (CD, PlySc) and binding specificity of the cell wall binding domain (CBD, PlySb) of PlySs2 were examined. Our results showed that PlySc alone maintains very limited lytic activity. Enhanced green fluorescent protein (EGFP)-fused PlySb displayed high binding affinity to the streptococcal strains tested, including S. suis, S. dysgalactiae, and S. agalactiae, but not staphylococci, supporting its utility as a good CBD donor for streptococcal-targeted lysin engineering. EGFP-fused intact PlySs2 similarly displayed high affinity for streptococci, but not staphylococci. Notably, four truncated PlySb fragments showed no binding capacity. These findings collectively indicate that integrity of the PlySc and PlySb domains is an essential determinant of the broad lytic activity of PlySs2.

Dynamic attachment of Chlorovirus PBCV-1 to Chlorella variabilis.

Chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their algal hosts at the site of attachment, using an intrinsic digesting enzyme(s). Chlorovirus PBCV-1 stored as a lysate survived longer than virus alone, suggesting virus attachment to cellular debris may be reversible. Ghost cells (algal cells extracted with methanol) were used as a model to study reversibility of PBCV-1 attachment because ghost cells are as susceptible to attachment and wall digestion as are live cells. Reversibility of attachment to ghost cells was examined by releasing attached virions with a cell wall degrading enzyme extract. The majority of the released virions retained infectivity even after re-incubating the released virions with ghost cells two times. Thus the chloroviruses appear to have a dynamic attachment strategy that may be beneficial in indigenous environments where cell wall debris can act as a refuge until appropriate host cells are available.

Molecular insights on the recognition of a Lactococcus lactis cell wall pellicle by the phage 1358 receptor binding protein.

UNLABELLED: The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a "shoulder" domain linking the RBP to the rest of the phage and a jelly roll fold "head/host recognition" domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ∼8 Šfrom each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages. IMPORTANCE: Siphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage 1358 receptor binding protein (RBP) in complex with monosaccharides. The positions and nature of monosaccharides bound to the RBP are in agreement with the pellicle structure and suggest a general binding mode of lactococcal phages to their pellicle saccharidic receptor.