Isolation of a Virulent Lactobacillus brevis Phage and Its Application in the Control of Beer Spoilage

Randazzo

Neve

et al. 2015

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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...

Section: Methodsmentioning

confidence: 99%

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

Randazzo

Neve

et al. 2015

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“…Purification of phages Q33 and BM13 by CsCl gradient was performed as previously described (25). Adsorption of CsClpurified phages to freshly prepared carbon film floated from a freshly coated mica sheet and negative staining with 2% (wt/vol) uranyl acetate were done as described previously (26). The film was picked up with a 400-mesh copper grid (Agar Scientific, Essex, United Kingdom), and specimens were examined with a Tecnai 10 transmission electron microscope (TEM) (FEI, Eindhoven, The Netherlands) operated at an acceler- 11 PFU/ml) were concentrated with a NanoSep 300K column (Pall Corporation) and treated as previously described (27,28).…”

Section: Methodsmentioning

confidence: 99%

Identification of a New P335 Subgroup through Molecular Analysis of Lactococcal Phages Q33 and BM13

Martel

Tremblay

et al. 2013

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Lactococcal dairy starter strains are under constant threat from phages in dairy fermentation facilities, especially by members of the so-called 936, P335, and c2 species. Among these three phage groups, members of the P335 species are the most genetically diverse. Here, we present the complete genome sequences of two P335-type phages, Q33 and BM13, isolated in North America and representing a novel lineage within this phage group. The Q33 and BM13 genomes exhibit homology, not only to P335-type, but also to elements of the 936-type phage sequences. The two phage genomes also have close relatedness to phages infecting Enterococcus and Clostridium, a heretofore unknown feature among lactococcal P335 phages. The Q33 and BM13 genomes are organized in functionally related clusters with genes encoding functions such as DNA replication and packaging, morphogenesis, and host cell lysis. Electron micrographic analysis of the two phages highlights the presence of a baseplate more reminiscent of the baseplate of 936 phages than that of the majority of members of the P335 group, with the exception of r1t and LC3.

“…Adsorption of CsCl-purified phages to freshly prepared carbon film floated from a freshly coated mica sheet and negative staining with 2% (wt/vol) uranyl acetate were done as described previously (24). The film was picked up with a 400-mesh copper grid (Agar Scientific, Essex, United Kingdom), and specimens were examined with a Tecnai 10 transmission electron microscope (FEI, Eindhoven, The Netherlands) operated at an acceleration voltage of 80 kV.…”

Section: Electron Microscopy (Em)mentioning

confidence: 99%

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

Kot

Murphy

et al. 2013

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103

139

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...