Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis

Abstract

SPOl bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVBl. The injected DNA is partially degraded to small trichloroacetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPOl DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVBI. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPOl-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in heterogeneous in size. MATERIALS AND METHODS Bacteria and bacteriophage. B. subtilis CU403 divIVB1 thyA, thyB, metB (24) was used throughout this study as a source of minicells and bacteriophage host. SPOl bacteriophage was obtained from C. Yehle. SPOl phage stocks were produced by infecting host bacteria at an adsorbance at 660 nm of (A ...) 0.5 in TY medium (2) containing 5 x 10-3 M CaCl2 at 37 C with multiplicity of infection of 10. After cell lysis the lysate was cleared by centrifugation at 5,000 x g for 10 min. The supernatant was centrifuged at 27,000 x g for 3 h at 4 C and the phage pellet was allowed to resuspend overnight at 4 C in TBT buffer (2). The phage suspension was layered on top of a three-step CsCl gradient containing CsCl solutions in TBT of densities 1.

Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis

“…We have therefore investigated the effect of PEN on the incorporation of amino acids by minicells of B. subtilis (Table 3). It was found that Lor D-alanine was incorporated into hot trichloroacetic acid-precipitable material to a much larger extent than the other amino acids tested (L-histidine, D,L-lysine, L-leucine, L-proline) and, indeed, as previously reported (29,30) we were unable to detect incorporation of L- a SP82-infected (multiplicity of infection -5) or uninfected minicells (0.5 ml; A6" = 0.5) were incubated in minimal medium with or without methionine at 37°C for 60 min.…”

“…Protein synthesis induced in minicells is sensitive to CAM. It has been shown previously that protein synthesis occurs after phage infection of B. subtilis minicells (29). As a control for the effect of CAM and VAN on uninfected minicells, we checked the effect of CAM and VAN on phage-infected minicells.…”

“…An alternative method of introducing a deoxyribonucleic acid molecule into minicells has been to infect minicells ofE. coli (34,45) or Bacillus subtilis (29) with bacteriophage. The introduction of a phage genome results in the syntheses of ribonucleic acid and protein and permits an analysis of phage-coded products in the absence of host cell products.…”

“…SP82G was obtained from D. M. Green. Phage stocks were produced as previously described for bacteriophage SPOI (29).…”

Aberrant regulation of synthesis and degradation of viral proteins in coliphage lambda-infected UV-irradiated cells and in minicells

“…and SPP1 (22,50,58,63) express their entire genomes, T5, N4, SPOl, and SP82G (63, 68) do not proceed past the early stages of their development in infected minicells. Although repair of viral DNA (4) and synthesis of the replicative form of (X174 DNA (66) have been detected in infected minicells, replication of double-stranded phage DNAs has not been demonstrated (4,26,67,83,87).…”