Studies of bacteriophages induced from Streptococcus cremoris strain R1 : is R1 a double Lysogen? : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, New Zealand

Abstract

Early studies on Streptococcus cremoris strain R₁ suggested that it was polylysogenic. Later, it was reported that its induced lysates contained bacteriophages (phages) of two types which were believed to differ in their morphology, buoyant densities, immune specificities and in their responses to heterologous antiphage sera. Further work on the strain did not reproduce the above observations, but did often give results which were consistent with it being a double lysogen. This project was an in-depth investigation of phages induced from R₁, in an attempt to establish the single or double lysogenic nature of the strain. Mid-log phase R₁ cells were harvested, washed with homologous antiphage serum and induced to lyse with ultraviolet light (UVL). The resulting phage lysates were analysed on caesium chloride (CsCl) density gradients. Though the OD₂₅₄ (optical density at 254 nm) scans of the gradients detected the presence of only one phage band, p.f.u. (plaque forming unit) profiles of the gradient fractions on indicator strains R₁C and 368 revealed, in addition to the main phage peak, several minor p.f.u. peaks (termed satellite and shoulder peaks) as possible manifestations of different phage types in the R₁ lysates. Further CsCl density gradient analyses of phage stocks and pooled phage fractions of these minor p.f.u. peaks showed that the latter phages were identical with those of the main phage peaks of mean buoyant density of 1.485 g/ml. Further characterization of the phages recovered from the CsCl gradients by neutralization tests with homologous antiphage serum confirmed the existence of only one serological phage type in the R₁ lysates. Final verification of the unity in phage type in R₁ lysates came from SDS-gel electrophoreses of the phages recovered from the different p.f.u. peaks and from lysates, which showed the largely identical gel patterns of their protein components. Host-specificity tests of the phages provided the last piece of evidence for the conclusion that R₁ is a single lysogen, harbouring only one prophage in its genome. Review of past electron-microscopic studies of R₁ lysates substantially support this conclusion. In fact, reconstruction of R₁ by lysogenization of a cured strain (R₁C) yielded a strain (R₁r) which closely resembled the original in lysogenic properties. From the data collected in the course of this work, it was inferred that 368 lysates possibly contained defective phages. An attempt was made to cure 368 of its supposedly defective prophage in the hope of providing a 'cleaner' strain for studying the host-induced variation observed in the R₁C-368 system. Though possible cured derivatives were obtained, they did not prove to be an improvement over the parental strain 368 with respect to their efficiency of plating for R₁ phages. Finally, phage mutant isolation and recombination experiments were attempted in the hope of gaining an insight into the lysogenic system operating in the R₁ cells. Using UVL and nitrous acid (HNO₂) mutagenesis on the temperate ϕr₁/R₁C induced from R₁, about 75 independently arising clear plaque-forming mutants were isolated for mapping experiments. Pairwise crosses between the UVL and HNO₂- induced mutants were performed by coinfecting R₁C cells. Though far from conclusive, the preliminary results obtained indicated a general low occurrence of turbid-plaqued (wild type) phage recombinants, and hence a low frequency of recombination.