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

Abstract

Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages. Dairy fermentations rely on the application of strains of Lactococcus lactis for the production of a wide variety of cheeses. However, these strains are under consistent pressure due to the presence of (bacterio)phages. 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 (2-11). 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. 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, 10-12). 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). The CWPS-encoding operons of currently sequenced lactococcal strains are classified into three genetic groups (types A, B, and C) (10), which have been correlated with phylogenetic subgroups of the 936 phage RBPs (10).Confirmation that CWPS indeed acts as the receptor for phages belonging to the P335 and 936 groups was obtained by "swapping" the CWPS types produced by different lactococcal strains. In this approach, the operons encoding the two C-type CWPS were compared, and an island of three genes encoding glycosyltransferases was identified as differential between the strains L. lactis NZ9000 (a C 1 -type strain that is resistant to infection by the P335 phages TP901-1 and LC3 and the 936 phage Viridus JM2) and...

Identification and Analysis of a Novel Group of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus

“…The morphology of the Siphoviridae family has been well documented (group B as defined by Bradley [9]), and siphophages infecting L. lactis and S. thermophilus exhibit the expected morphology, with icosahedral heads and noncontractile tails. Phages infecting S. thermophilus in general possess longer tails than their lactococcal counterparts (with an exception being the 949 group of lactococcal phages [40,70]), their long tails being consistent with their long TMP-encoding genes (71). Upon electron microscopic analysis, it was found that phages 9871-4 exhibit icosahedral heads and relatively short tails (Fig.…”

Phage Biodiversity in Artisanal Cheese Wheys Reflects the Complexity of the Fermentation Process

“…Each of the 50 samples was tested against a panel of 25 L. lactis strains to isolate as wide a diversity of lactococcal phages as possible. The bacterial strains selected for this analysis were of known genotypes based on cell wall polysaccharide (CWPS) operon sequences from a previous study, as these are known to encode the receptor material for a variety of lactococcal phages [11,29]. Phage screening was performed using the standard double agar plaque assay as previously described [27] and a number of single plaque isolates from phage-containing samples were propagated on the relevant lactococcal host and plaque-purified at least twice to ensure homogeneity of the phage population.…”

“…DNA for sequencing of the selected phage genomes was extracted from 20 mL of fresh phage lysate (>10 8 pfu·mL −1 ) as described previously [11]. For genome sequencing, five µg of extracted DNA was used as verified by nanodrop quantification.…”

“…Among these, several phage isolation studies have reported that members of the 936, c2 and P335 groups are the most frequently encountered in the dairy fermentation environment [5,6,7,8,9]. In contrast, members of the remaining seven lactococcal phage groups are much less frequently encountered [4,6,10,11]. The description of a lactococcal phage with a distinctively long tail in 1951 [12] marked the first observation of what is now called the 949 group of lactococcal phages.…”

“…More than 60 years later, the first genome sequence of a member of this group was published [13], revealing a genome of 114,768 kb with limited homology to other lactococcal phages. Subsequently, two additional members of this group have been genomically characterised, namely phiL47 and WRP3 [11,14], which were isolated from grass and a Sicilian cheese whey, respectively. The latent period of 949 (period during which phages replicate and assemble inside the host cell), was calculated to be approximately 70 min, which may in part explain the irregularity of its apparent occurrence [8].…”