Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

Abstract

Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been implicated as a host receptor of (certain) 936-type phages, is specified by a large gene cluster, which, among different lactococcal strains, contains highly conserved regions as well as regions of diversity. The regions of diversity within this cluster on the genomes of lactococcal strains MG1363, SK11, IL1403, KF147, CV56, and UC509.9 were used for the development of a multiplex PCR system to identify the pellicle genotype of lactococcal strains used in this study. The resulting comparative analysis revealed an apparent correlation between the pellicle genotype of a given host strain and the host range of tested 936-type phages. Such a correlation would allow prediction of the intrinsic 936-type phage sensitivity of a particular lactococcal strain and substantiates the notion that the lactococcal pellicle polysaccharide represents the receptor for (certain) 936-type phages while also partially explaining the molecular reasons behind the observed narrow host range of such phages. Lactococcal phages are classified into 10 groups based on morphology and DNA hybridization studies (1). Of these, three species are most frequently isolated from dairy environments, namely, the 936, c2, and P335 species (1). The 936 phages are strictly lytic and frequently cause problems for the dairy fermentation industry (2). For this reason, members of this phage species have attracted significant attention in recent years, and the genomes of several phages of this species are now available (3-8). The genome organization of these phages is well conserved and consists of three clusters: the early-, middle-, and late-expressed regions. Comparative genomic analysis of these phages has revealed that while most of the structural genes are highly conserved, there are particular regions of diversity within other regions of their genomes. Among these are the early-expressed genes, which are assumed to encode the replication functions of the phage, as well as the late-expressed region, particularly within the genes encoding the receptor binding protein (RBP) and the tail tape measure protein (TMP) (3,4,8). It has been suggested that the genomes of the 936-type phages can be subgrouped based on these variable regions, which appear to correspond to the subspecies grouping (i.e., Lactococcus lactis subsp. lactis or L. lactis subsp. cremoris) of the host(s) which they infect (3, 9). However, there are a number of exceptions to this in terms of phages that are capable of infecting both subspecies of L. lactis. Phages 645 and P475, for example, are capable of infecting certain members of both subspecies and possess a RBP which is different from those of the two major subgroups of the 936 phages (9). Recently, the RBP structures of phages p2 and bIL170 (or domai...

Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2

“…These saccharides are synthesized by glycosyltransferases and other enzymes whose genes are located in close proximity in the L. lactis genome, the so-called pellicle gene cluster. It has been shown that in various L. lactis strains, these cassettes differ by the type and order of their gene components, which should have an impact on the pellicle polysaccharide motif and, consequently, phage sensitivity (61). Here, we show that p2 RBPs have a much higher affinity for and longer residence time in their specific L. lactis MG1363 pellicle than TP901-1 RBPs.…”

“…Once near its host, the external accessible RBP sites of the baseplate in the nonactivated conformation may scan the hosts surface (Fig. 9C) in search of specific pellicle phosphohexasaccharide motifs, which differ between different L. lactis strains (61). If the number of specific binding events is large enough and Ca 2ϩ is available, the mechanical pull induced by this binding may destabilize the rest conformation of the baseplate and disrupt the interaction between the second Dit "arm" (14) and the RBP head domain.…”

Lactococcal 949 Group Phages Recognize a Carbohydrate Receptor on the Host Cell Surface

“…The majority of studies have focused on members of the 936 and P335 species, since these are among the most frequently isolated species in the dairy industry. Members of both of these species are believed to recognize a saccharide component of the cell wall polysaccharide (CWPS) that coats the surface of the cell (6,(10)(11)(12). Furthermore, phage 1358, which is the namesake of a rarely isolated lactococcal Siphoviridae phage species, is also predicted to recognize a CWPS component on the surface of its host, Lactococcus lactis SMQ-388 (13).…”

“…Lactococcal phages are currently classified into 10 species or groups, based on DNA hybridization studies and morphology (1). In recent years, lactococcal phages and their hosts have become an advanced model system for studying Gram-positive phage-host interactions due to the emergence of significant data regarding key molecular players involved in phage adsorption (the phage-encoded receptor binding protein [RBP] and the host-encoded receptor) to the host and the impact of phage infection on sensitive bacterial strains (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). The majority of studies have focused on members of the 936 and P335 species, since these are among the most frequently isolated species in the dairy industry.…”

Molecular Insights on the Recognition of a Lactococcus lactis Cell Wall Pellicle by the Phage 1358 Receptor Binding Protein

“…These surface polysaccharides were shown to be composed of repeating units of hexasaccharide phosphate that are distinct from other bacterial polysaccharides (11,28). A comparative analysis of the gene cluster of the pellicle revealed diversity among L. lactis strains but also an apparent correlation between the lactococcal pellicle genotype of a given strain and the host range of tested 936-type phages (29). This correlation was recently confirmed through the characterization of the pellicle of L. lactis 3107, the host of phage TP091-1 (28).…”