Characteristics of a group IV cholera phage.

Cholera bacteriophages have been classified into four distinct groups by Mukerjee (I963a). Of these, the group IV phages can differentiate Vibrio eholerae and V. eltor organisms (Mukerjee, 1963b) and are of practical importance. The hitherto unreported physiological and physico-chemical properties of the group IV cholera phage, ~M49, have been investigated by us and the results are presented below. Cholera bacteriophage ~br49 was propagated on the host V. cholerae strain OGAWA I54 following in general the method of Hershey, Kalmanson & Bronfenbrenner (I943). The phage stock thus obtained usually contained about Io 11 p.f.u./ml, as assayed by double agar layer technique (Adams, I959). This phage yielded clear plaques of diameter between o'5 and 1.5 mm. after I6 hr incubation at 37 °. Longer incubation produced a surrounding halo. In all cases, bacteria were grown in nutrient broth (NB) containing, in I 1. of distilled water: bactopeptone (Difco), Iog.; NaCI, 5 g. ; beef extract (ACAS, Italy), lo g.; pH 7"4Kinetics of adsorption was studied at an m.o.i, of o.ooi or o.ot following in general the method described by Adams (I959). Adsorption was biphasic with rate constants of I'O26 × IO -9 ml./min, up to 8I ~oo adsorption and 0"950 x io -1° ml./min, thereafter. Similar biphasic adsorption kinetics was also reported for the phage 7V of Pseudomonas aeruginosa (Feary, Fisher & Fisher, I964), phage X of Escherichia coli (Schade & Adler, 1967) and the phage ~WI4 ofP. acidovorans (Kropinski & Warren, 197o). Adsorption of this phage q5149 depended significantly on the pH of the medium. In NB media of pH 6, 7"4, 8 and 9, the percentages of phage adsorbed in I5 min. at 37 ° were 72, 85, 64 and 6o ~ , respectively, with a m.o.i, of 0.001. No significant adsorption to the V. eltor strain MAK 757 could be obtained even after 2o min. incubation in NB of pH adjusted to any of the above values. V. eltor was known to be resistant to group IV cholera phage but the basis of this resistance was unknown. It has now been shown that the cell wall of V. eltor does not have the specific phage receptors. Cell wall and lipopolysaccharide (LPS) were isolated from the host cell, following in general the methods of Keeler et al. (I966) and Westphal, Lfideritz & Bister (i952), respectively. The purity of ceil-wall preparation was checked by electron microscopy, u.v. and visible extinction (Sur & Chatterjee, 197o) and that of LPS by u.v. extinction, electron microscopy and chemical analysis (Chatterjee, Adhikari & Raychaudhuri, I97I; Adhikari, 1971). Assays of phage adsorption to cell walls (Io/#g./ml.) and to LPS (Io/#g./ml.) were done by the methods of Chatterjee (1969) and Lindberg (I967), respectively. Adsorption of cholera phage ~I49 to isolated cell wall and LPS followed first-order reaction kinetics resulting in 70 ~ inactivation by cell wall within 3o min. and 50 ~o by LPS within 6o rain. These results agree satisfactorily with those reported for mycobacteriophage GS-7 (Imaeda & San Blas, I969). The presence of phage receptors in these structures is in conformity with the observations of Levine & Frisch (I934) for Enterobacteria and of Lindberg (I967) for Salmonella minnesota. After treatment with 0"5 ~ (w/v) sodium deoxycholate for I hr at 37 °, cell wall as well as LPS largely lost their phage-inactivating capacity. The 50 ~ phage-inactivating concentration (IC5o) of LPS increased from the normal value of 7/zg./ml. to about 3"6 mg./ml, after sodium deoxycholate treatment. The loss of phage-inactivating capacity might be due to the fact that sodium deoxycholate dissociates LPS of Gram-negative bacteria into very small units with subsequent loss of biological activity (Ribi et aL I966; Lindberg, I967).