Lactobacillus phage J-1 baseplate: towards the functional and structural characterization"

MARIA EUGENIA DIETERLE; SILVIA SPINELLI; MARIANA PIURI; CHRISTIAN CAMBILLAU

Simposio; V International Symposium on Lactic Acid Bacteria; 2016

Bacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult using Lactobacillus casei ?Shirota? as starter. Two years later, a related phage, PL-1, was subsequently recovered while using a strain resistant to J-1. After complete genome sequencing we showed that J-1 and PL-1 were almost identical. Based on the place in the genome and annotation, gp16 and gp17 are putative tail proteins and gp16 differs between both phages. Immunoelectron Microscopy (IEM) has shown that gp16 is located at the tip of the tail and antibodies against this protein neutralized phage infection. The structure of gp16 was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in other Siphoviridae. However, two regions of the C-terminus of gp16 could not be modeled using this template and showed sequence similarity to carbohydrate-binding modules (CBMs). The N-term of gp17 was modeled based on the structure of phage T4 gp27 and resembles tail associated lysozyme proteins (Tal). GFP-gp16 and GFP-gp17 fusions bind specifically to Lactobacillus casei/paracasei cells, and addition of L-rhamnose inhibits binding of GFP-gp16. These results indicate that both proteins are involved in host recognition.In order to understand how Lactobacillus phages recognize their hosts, we studied the baseplate of J-1 and its components at the molecular level. Using a combination of EM reconstruction and X-ray diffraction a baseplate atomic model was constructed. J-1 baseplate is formed by an hexameric ?evolved? Dit protein (gp16) containing two CBMs (CBM1 and CBM2) attached to a trimeric Tal (gp17) with no Receptor Binding Proteins (RBPs) homologs to those present in Lactococcal phages. The experimental EM map was fit with the X-ray structure of Dit /Tal phage p2. Two series of 6 EM density bulbs could be undoubtedly assigned to both CBM domains and the baseplate was measured, ~125-210 Å width and ~110 Å high. Interestingly, EM shows that Tal has a ~600 Å extension beyond the baseplate. The CBM2 structure present in Dit was solved by X-ray crystallography. The structure of this domain consists of 19 ß-strands and a few long extended stretches and loops. CBM2 is at least one of the domains responsible for sugar recognition as mCherry-CBM2 fusion protein has proven to specifically bind to L. casei cells. These findings suggest that J-1 baseplate structure contains different CBMs that may replace RBPs in host cell-wall binding, a feature that may extend to many other phages from diverse origins. The detailed study of the machinery involved in host recognition in Lactobacillus spp could facilitate the design of new strategies to avoid phage propagation in the dairy industry.