Abstract
O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.
Highlights
Exopolysaccharides (EPS) and capsules are generally considered to be the outmost surface structure of a bacterial cell
The modulation of bacteriophage infectivity by OPS was first reported a long time ago[12]. The importance of this host structure is still underestimated,. This is due to the fact that the bulk of data derived from “classical” bacteriophage biology was obtained from studies of model coliphages such as T-series (T1 to T7), as well as phage lambda and a few other phages that were propagated on laboratory rough, i.e., depleted in O-antigen biosynthesis, E. coli strains
The overall time required for the whole procedure, starting from a bacterial colony, is about 4 hours, with the number of the samples treated in parallel is mainly limited by the number of available wells on the acrylamide gel
Summary
Exopolysaccharides (EPS) and capsules are generally considered to be the outmost surface structure of a bacterial cell. LPS molecules are comprised of lipid A, core oligosaccharide and O-antigen (or O-polysaccharide; OPS) These structures play a role in the interactions of bacteria with various surfaces and molecules, including other bacterial or eukaryotic cells[2], immunity factors[3,4,5], enzymes[6], and bacteriophages[7]. The importance of this host structure is still underestimated, In part, this is due to the fact that the bulk of data derived from “classical” bacteriophage biology was obtained from studies of model coliphages such as T-series (T1 to T7), as well as phage lambda and a few other phages that were propagated on laboratory rough, i.e., depleted in O-antigen biosynthesis, E. coli strains. This latter strategy is employed by some T5-like bacteriophages[17] and supposedly by many other siphoviruses[18]
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