1. The somatic antigen of Phase II Shigella sonnei can be isolated by extracting phenol-killed microorganisms with water. This substance inactivates all T phages to which this bacillus is susceptible. 2. The Phase II antigen is constituted from a protein and a phosphorylated lipocarbohydrate. The major portion of the protein component can be removed by digestion with pancreatin. The enzymatically degraded antigen thus obtained can be further dissociated into its protein and lipocarbohydrate components by treatment with 90 per cent phenol. Glucose, galactose, glucosamine, and an aldoheptose have been identified as the monosaccharide constituents of the lipocarbohydrate.
For some years now this laboratory has been concerned with the nature of the substances involved in the interaction between phage and bacterium. For these studies the dysentery bacillus Shigella sonnei was chosen as host and the T coil-dysentery phages as the viral parasite. Five of the seven viruses within this group attack Phase H Shigdl~ sonr~ and perpetuate themselves at the expense of the bacterial cell. These viruses begin the infectious process b y combining with a particular substance distributed on the bacterial surface--a complex of carbohydrate, protein, and phospholipid, which serves as the viral receptor (1, 2). In vitro however it is not necessary to have the complete bacterial antigen to bring about inactivation of the coli dysentery phage T4. The lipocarbohydrate component alone suffices. In the following account it will be shown that this substance, obtained from the bacterial antigen b y chemical degradation, will bring about a rapid inactivation of the T4 virus and its ultimate disintegration. The factors which govern this phenomenon will be described and the nature of the reaction between the phage and lipocarbohydrate will be discussed. Materials and Methods Strains of Microorganisms.~Cultures of Escherichia coli B and Phase H Shigella sonneiwere originally obtained from Dr. Mark H. Adams of New York University and from the United States Army Medical School. The organisms were subeultured daily on nutrient agar slants. The wild type strain of T4 phage used in this study was also supplied by Dr. Adams. The virus was maintained by transferring on E. toll B grown in nutrient broth.Viral Assays and Viscosimetric Measurements.--The action of the lipocarbohydrate on the T4 phage was studied by measuring changes in the infectivity fitre and the viscosity of phage-lipocarbohydrate mixtures. Viral assays were performed by the poured agar layer technique using Phase II Sk. sonnei as host (3). Viscosity was measured in Ostwaid viscosimeters maintained in a thermostat at 37 -4-0.1°C. Viscosimeters of 5 ml. capacity and having water values of 58 to 76 seconds were employed.Aqueous lipocarbohydrate solutions used in the tests were prepared by dissolving the material in distilled water so as to contain 5 mg./ml.Sterile solutions of the lipocarbohydrate were prepared as follows. A weighed sample of material was placed in a sterile test tube. The latter was flamed for most of its length, and 733on May 9, 2018 jem.rupress.org Downloaded from
Colicins differ from other antibiotics in that they are proteins endowed with an extremely narrow range of bactericidal activity (1,17). As a rule they are toxic only for those bacteria which belong to the same or to a closely related species as the colicinogenic microorganism. Moreover, bacteria which are sensitive to one type of colicin can be fully resistant to another (1). To account for this it was postulated by Fredericq as early as 1946 that bacteria which are sensitive to a certain colicin contain a specific receptor substance on their surface which serves as the site of attachment for the bacteriocin (2). It was also observed that microorganisms which are sensitive to colicins M, K, or E were always attacked by bacteriophage T1, T6, or BF23, respectively, and that mutants which had lost their sensitivity to one of the colicins were resistant to the corresponding phage. I t was suggested therefore that in each of these instances the receptor for the bacteriocins and virus was identical (3, 4). I n the ensuing years the receptor hypothesis was substantiated by the work of others. Thus, Bordet and Beumer have shown that extracts of colicin-sensitive bacteria inhibit bacteriocins in vitro (5). Mayr-Harting has demonstrated that colicin E2-sensitive microorganisms absorb the bacteriocin from solution and that their ability to do so is destroyed by heating or by chemical treatment (6). More recently, Guterman and Luria found that colicin B is inactivated by lipopolysaccharides of Escherichia coli strains which are sensitive to the bacteriocin (7).The location of the receptors in the bacterial cell has been studied by Nomura and his coworkers. They have shown that bacteria which were exposed to colicins can be rescued by treating them with trypsin and therefore they suggested that the bacteriocin is adsorbed to the surface of the cell (8). This observation was corroborated by the finding that approximately 90 % of radioactively labeled colicin E2 is absorbed to a cellular fraction containing the bacterial envelopes (9).
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The colicinogenic factor K has been transferred from E. coli K 235 to Proteus mirabilis. The DNA of the colicinogenic Proteus has been shown to contain a small amount of a satellite DNA which presumably harbors the Col K factor. In the presence of mitomycin C the colicinogenic Proteus secretes colicin K into the growth medium. The bacteriocin has been purified by chromatography and obtained as an immunologically homogeneous substance unconjugated with other antigens of the Proteus bacillus. Proteus colicin K is a protein of relatively low molecular weight. It contains all of the usual amino acids except cysteine and is free of lipids and polysaccharides. The bacteriocin can be separated by electrofocusing into two major components. The latter have the same biological properties but differ in their specific electrical charges.
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