Evidence for heterogeneity in populations of T5 bacteriophage. II. Some particles are unable to inject their second-step-transfer DNA

C Hulen, B Labedan, J Legault-Demare
1980 Journal of Virology  
A new class of bacteriophage was characterized in purified T5 stocks. Regardless of the host cell, these phages were irreversibly blocked at the first-step-transfer stage under conditions in which whole DNA injection normally takes place. However, they expressed their first-step-transfer functions. These observations confirmed the previously established heterogeneity of T5 bacteriophage populations and provided a new way to define a phage function necessary to release the blocking of T5 DNA
more » ... ction at the first-step-transfer stage. Populations of T5 bacteriophage are known to be made of different types of viral particles. It has been demonstrated that purified T5 stocks contain: (i) lytic phages, determined as PFU by the method of Adams (2); (ii) killing particles, which do not yield plaques, determined from the number of surviving bacteria after infection (10); and (iii) aberrant particles, which release all of their DNA outside the bacterium immediately after irreversible adsorption at any temperature (9). T5 phages are normally able to inject their DNA in two steps (13). Under conditions of a restricted energy supply (high bacterial concentration in buffer) which prevents DNA expression, only 8% in length of the chromosome is injected. Bacterium-phage complexes arrested at this step (first-step-transfer [FST]) may be kept unaltered for a certain time. Under conditions in which sufficient nutrients necessary for making FST-coded proteins are provided, the rest of the DNA (second-step transfer [SST]) is injected (12). However, under these latter conditions, it was consistently observed since the early work of Lanni (10) that only part of the DNA of adsorbed particles was actually transferred. The results presented here show that this nontransferable DNA originates from killing particles which do not inject their SST DNA after irreversible adsorption to host bacteria. The reasons why their SST DNA is not injected even if FST DNA is expressed are discussed. (This paper will be part of a doctoral thesis submitted by C.H. to the Faculty of Sciences of the University of Paris XI.) MATERIALS AND METHODS Phages and bacterial strains. Phage T5+ and its thermostable mutant T5stO (11) deleted in a region of the genome coding for tRNA's (4) were used. Escherichia coli F, a fast-adsorbing host for T5, E. coli B, E. coli K-12, and E. coli B41, an endonuclease Ideficient mutant (6), were from our collection. Preparation of DNA-labeled T5. Phage DNA was labeled with [2-'4C]uridine (0.4 ,uCi/ml) by the method of Labedan and Legault-D6mare (9) or with [methyl-14C]thymidine (0.5 1Ci/ml) in the presence of 2'-deoxyadenosine by the method of Hulen and Legault-Demare (7). Phages were purified by polyethylene glycol precipitation and banding on cesium chloride as previously described (9) . Washing of infected bacteria. E. coli F was grown in TCG medium (1) supplemented with 1 mM
doi:10.1128/jvi.36.3.633-638.1980 fatcat:glcps2hudnh4nnjryruledaotm