Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis, as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1

Abstract

e Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ؎ 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species. L actobacillus delbrueckii subsp. lactis is a member of the lactic acid bacteria (LAB) and is commonly used in the production of commercial fermented milk products, such as Emmental-like cheeses, where it is employed as a starter culture contributing to the acidification and the organoleptic properties of the final product (1, 2). Bacteriophage (or phage) infection of these starter strains represents a major hurdle to their technological activity, as this may cause lysis of the starter, which in turn may lead to reduced acidification activity and a poor(er)-quality product, with a consequent negative economic impact (3).The commercial importance of L. delbrueckii fermentations has catalyzed extensive research into the occurrence, diversity, and impact of its infecting phages. Currently, L. delbrueckii phages are organized into four distinct groups (designated groups a, b, c, and d), classified based on DNA homology by hybridization (4-6) and, more recently, by comparative genome analysis (7). L. delbrueckii phages belonging to groups a and c have enjoyed considerable scientific scrutiny, with phage LL-H representing a prototype Lactobacillus phage. LL-H was originally isolated in 1972 (8), and its full genome sequence and transcriptional map, which revealed two distinct phases of transcription, have been determined (9). Furthermore, research on phage-host interactions, culminating in the identification of gp71 as the receptor-binding protein of LL-H (10) and lipoteichoic acids as the recognized receptor, have significantly advanced our understanding of this phage and the way in which it recognizes its host (11, 12). Also, the prolate-head, temperate phage JCL1032, being a member of group c L. delbrueckii phages, has been subjected to considerable scientific characterization, including genome sequencing (13), analysis of genomic integration (14), and identification of lipoteichoic acid as its receptor (12).Due to their apparent increased frequency of isolation, research has recently focused on group b phages, facilitated by the sequencing of six phages, c5 and LL-Ku (13); Ld3, Ld17, and Ld25A (7); and ph...

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“…The prohead protease Pro was also identified in the LC-MS/MS analysis. The presence of the protease among the structural proteins was described previously for Lactobacillus delbrueckii -specific phages [ 51 , 52 ] but may be the result of lack of dissociation from the head after the assembly.…”

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“…The virus-containing supernatant was transferred to a new tube and stored at Ϫ80°C. After negative staining with 2% (wt/vol) uranyl acetate (Serva, Heidelberg, Germany), grid samples were analyzed with a transmission electron microscope (Tecnai10; FEI Thermo Fisher Scientific, Eindhoven, the Netherlands) equipped with a Megaview G2 charge-coupled-device (CCD) camera (Emsis, Münster, Germany) as described in detail previously (57).…”

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“…Cesium chloride purified phage samples were prepared as outlined above and subjected to electron microscopic analysis as described previously (Vegge et al, 2005 ; Casey et al, 2015 ).…”

“…Methanol-chloroform extraction of phage proteins, SDS-PAGE visualization and preparation of phage structural protein samples for mass spectrometry were performed as described previously (Casey et al, 2015 ). Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was performed as previously described (Ceyssens et al, 2008 ; Vanheel et al, 2012 ).…”