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Isolation, and characterization of a bacteriophage active against pseudomonas acidovorans Kropinski, Andrew Maitland Boleslaw

Abstract

A phage was isolated from sewage which was active against the non-fluorescent Pseudomonad, P. acidovorans. It was given the designation ∅W-14. This is the first phage reported to be active against P. acidovorans. ∅W-14 was larger than previously reported Pseudomonas phages. It had an icosahedral head some 90 m in diameter and a contractile tail 140 mµ long and 20 mµ in diameter. The phage tended to form large aggregates. ∅W-14 formed haloed plaques on three strains of the host organism, but mutated at a high frequency (4 - 9 x 10ˉ⁴ ) to a clear plaque type when grown on P. .acidovorans #14. The haloed plaque form was considered to be the wild type and was given the designation ∅W-14a+. The clear plaque mutant appeared stable and was given the designation ∅W-14a. Adsorption of ∅W-14a+ was biphasic, with 35% of the phage population having a decreased adsorption capacity. The K value was 1.9 x 10ˉ⁹ ml/min. More normal kinetics were observed with ∅W-14a and with ∅W-14a+ in low-salt broth. The K value for ∅W-14a was 4.2 x IOˉ⁹. The burst size for ∅W-14a+ obtained from one-step growth experiments was 300, with a latent period of 67 minutes. ∅W-14a had a 50% higher burst size but the same latent period. The average burst size was markedly affected by the physiological age of the bacterial culture, and by the multiplicity of infection. Sensitivity of the phage to heat, pH, sonication and ultraviolet light was investigated. At least 70% of the lethal effects of UV irradiation could be reversed by photoreactivation. Thermal inactivation kinetics of ∅W-14a+ in broth were biphasic at 55 C and 60 C, the ΔH* being 75,700 calories/mole. ∅W-14a appeared to be more thermo-labile. Nucleic acid isolated from ∅W-14a+ was double-stranded DNA. There was a significant discrepancy between the moles % GC calculated from buoyant density determinations (1.666 g/cc³ -6% GC) and Tm determinations (98.4 C - 71.9% GC). The DNA was unusual also in that it yielded five bases upon hydrolysis. The fifth base was not one of those commonly found in DNA. It has not been identified. The base composition of the DNA as determined by chromatographic separation and quantitation of the bases was: Adenine, 21.8 moles %; Guanine, 28.2 moles %; Thymine, 11.1 moles %; Cytosine, 26.6 moles %; Unknown, 12.3 moles %; based upon the relationship: [ A + G ](over) [ T + C + Unknown ]=1

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