Summary auto-generated
This study examined how calcium ions affect infection of Bacillus subtilis by bacteriophage SF6. When SF6 infects B. subtilis without added calcium, phage numbers rapidly declined due to loss of infectivity. Adding calcium, barium, or strontium ions reversed this decline, with optimal recovery occurring at 7.5 × 10⁻² M calcium—substantially higher than required by other phage-host systems. Using radioactively labeled phage DNA and protein, researchers determined that calcium ions are specifically required for DNA penetration into host cells. At low calcium concentrations, phage DNA and protein dissociate from cells after triggering injection, with DNA released into the medium. Phage adsorption occurs normally regardless of calcium concentration. When DNA successfully penetrates at high calcium, it remains cell-associated until lysis occurs. Intracellular phage development does not require high calcium concentrations. The authors propose that calcium stabilizes the interaction between the phage tail and bacterial cell surface, preventing shearing forces from disrupting DNA injection. They hypothesize that triggering of DNA release occurs, but the weak tail-receptor bond at low calcium allows the ejected DNA to escape into the medium rather than completing penetration.
Key findings
- Calcium ions (and strontium/barium at equivalent concentrations) are absolutely required for productive SF6 infection at concentration 7.5 × 10⁻² M, markedly higher than other phage-host systems
- Phage DNA penetration into the host cell is the calcium-dependent step; adsorption and intracellular phage development proceed normally without added calcium
- At low calcium concentrations, phages undergo triggering and DNA ejection but the DNA dissociates from cells and is released into the medium rather than penetrating the cytoplasm
- Electron microscopy and chromatography confirmed that inactivated phages eject their DNA into the medium, resulting in empty phage shells
- Calcium likely stabilizes the phage tail-receptor interaction, preventing shearing forces from disrupting the DNA injection process
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Abstract
Infection of Bacillus subtilis 168Wt by SF 6 resulted in a rapid reduction in the number of phages. This could be counteracted by the addition of calcium, barium or strontium ions. At the optimum concentration of 7.5 x 10-2 M, the number of p.f.u. remained constant until lysis began. Although cultures of another host, B. subtilis 31 try- his-, at the end of the logarithmic growth phase produced a substance which inactivated free phages, this was not the major cause of the reduction in the numbers of p.f.u. during infection experiments at low Ca2+ concentrations. The diminution of the number of p.f.u. was therefore attributed to the fact that at least one of the steps of the lytic cycle was calcium dependent. Adsorption of SF 6 was equally effective in media containing high or low concentrations of calcium ions. Infection experiments with phages whose DNA had been labelled radioactively revealed that, at high concentrations of calcium ions, the label remained associated with the host cells until lysis commenced. At low concentrations, however, a dissociation between phage DNA and the host was found, although adsorption took place at a normal rate. From these experiments we concluded that a high concentration of calcium ions was required for the penetration of phage DNA. Similar experiments with phages whose protein coat had been labelled showed the same results, indicating that desorption of the inactivated phages occurred. Both electron microscopy and column chromatography with hydroxyapatite showed that a considerable fraction of the inactivated phages had ejected their DNA into the medium. A hypothesis explaining these results is presented.
* Present address: Laboratory for Electron Microscopy, University of Leiden, Leiden, The Netherlands.