Isolation and preliminary characterization of bacteriophages of thermophilic Bacillus and Geobacillus species

Abstract

Thermophilic bacteriophages provide simple model systems for understanding biochemical and biological adaptation mechanisms at elevated temperatures. The essential objectives of this study were to characterise the physicochemical properties of select Geobacillus bacteriophages and to sequence their complete genomes. The later objective is believed to be an essential prerequisite to the engineering of a sitespecific integration vector for the stable cloning of exogenous genes into host bacteria. Bacteriophages were assayed at 55oC by the agar overlay technique using dry Karoo soils as source material. A pure strain of bacteriophage called GV1 (for Geobacillus stearothermophilus virus 1) was isolated with the strain Geobacillus stearothermophilus TAU3A1. Plaques were medium sized (2 to 4 mm diameters), with regular contour, clear, and without resistant cells. Host range specificity study showed that GV1 was lytic on thirteen thermophilic Bacillus-like strains tested, including strains of Geobacillus stearothermophilus, G. thermoglucosidasius, B. licheniformis, Anoxybacillus idirlerensis, and A. kuwalawohkensis. However, GV1 failed to infect a mesophilic strain of Bacillus megaterium. TEM analysis of semipurified particles revealed that the phage belongs to the family of Siphoviridae. Morphological characteristics included a long tail of approximately 100 nm and a hexagonal head of approximately 50 nm diameter. Viability and stability studies showed that the phage was best maintained at -80oC in PMN buffer supplemented with 20% glycerol. It was stable at a pH range of 5.5 to 7.5 and MgCl2 and CaCl2 concentration of 0.001 M. hermostability experiments, conducted over short periods of time, showed that GV1 was stable over the temperature range 50 to 75oC, with optimum at 55oC. The study of phage-host interactions showed that phage articles inhibited the initial growth of infected cultures in the first six hours post-infection, presumably while mature phages were released. This was followed by a steady recovery of the growth rate. Atempts to obtain pure particles and to extract and sequence phage DNA were unsuccessful due to the low titer nature of the phage.