Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2

Abstract

A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-ype, phagesensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phagesensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further The specific adsorption of bacteriophages to host cell surface components has been well characterized in many bacteria. In Escherichia coli, for example, phages adsorb to oligosaccharides and proteins exposed on the outer surface of the host cell (40,54). Following adsorption, the mechanism of phage DNA translocation across the plasma membrane is less well understood, although host cell components required for this process have been identified (7,19), and a mechanism involving plasma membrane ion channels is likely in the case of phage T5 (5,20).Lactococci are gram-positive bacteria and have cell walls that are much thicker than those of gram-negative bacteria such as E. coli. Because of this, phages of lactococci and other gram-positive bacteria generally adsorb to the cell wall prior to interaction with the membrane (3, 28). However, it has been suggested that lactococcal phages may adsorb directly to the plasma membrane (32). This suggestion was based on an electron microscopic study of the surface of lactococci, which showed the plasma membrane protruding through holes in the cell wall (23), and the discovery of a phage-inactivating substance in the plasma membrane fraction of Lactococcus lactis subsp. lactis ML3 (32,33).Our laboratory has characterized phage receptors on the surface of two strains of L. lactis (44,46

Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1

“…Indeed, they interact only with host cell wall saccharides, putatively the phosphosaccharides present in the pellicle of Gram-positive bacteria, for specific recognition and attachment (8,10,33,(40)(41)(42). Furthermore, many other siphophages, such as SPP1 and the lactococcal phages belonging to the c2 species, bind reversibly to saccharidic receptors in a first step before interacting irreversibly with a membrane protein that initiates infection (5,(43)(44)(45)(46)(47)(48)(49). As the affinity between phage antireceptors and their saccharidic partners is generally moderate (in the low micromolar range), several RBPs are involved in binding to ensure strong interactions based on avidity (50,51).…”

Fluorescence Correlation Spectroscopy To Study Diffusion and Reaction of Bacteriophages inside Biofilms

“…exposed to the cell surface (54). In the case of c2-like prolate phage infection, a subsequent interaction occurs via PIP, a plasma membrane-associated protein required for the ejection of phage DNA (22,36,37). It was hypothesized that after the ejection of c2 phage DNA inside the host cell, phage development in L. lactis IL-1403 bacteria was blocked due to the action of an unidentified gene product encoded by one of the six prophages present in the IL-1403 genome (9; A. Aucouturier, personal communication).…”

Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5

“…DauC has significant similarity with the product of the bioC gene, which encodes an uncharacterized step in the biotin biosynthesis pathway (30). DauC also has lower but still significant sequence similarities with the gene products of the germination factor gerC2 from Lactococcus lactis (15), E. coli ubiG (40), and R. sphaeroides pmtA (3), particularly in regions of conserved domains (Table 3). The functions of bioC and gerC2…”