Inhibitory effect of prophage SPβ fragments on phage SP10 ribonucleotide reductase function and its multiplication in Bacillus subtilis

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Bacteria have evolved various kinds of defense mechanisms against phage infection and multiplication. Analysis of these mechanisms is important for medical and industrial application of phages as well as for their scientific study. Strains of Bacillus subtilis Marburg strain carrying both nonA and nonB mutations are susceptible to the Bacillus phage SP10. The nonB mutation has been shown to have a compromised intrinsic restriction system. The nonA mutation represents the cured state of prophage SPβ whose genome is 135 kb in length and contains 187 ORFs. For this study we investigated the molecular mechanism behind the inhibitory activity of the wild type nonA function against phage SP10 development. The progression of phage-developmental stages was examined in cells harboring wild type nonA, i.e. prophage SPβ. After phage adsorption and DNA injection into host cells, the synthesis of phage specific mRNA proceeded normally. However, phage DNA synthesis was severely inhibited by some effect of wild type nonA. We thus systematically deleted portions of the prophage SPβ region from the B. subtilis genome and the resultant mutant strains were examined as to whether they still retained sufficient wild type nonA functionality to inhibit SP10 phage development. The SPβ region encompassing the bnrdEF gene, which codes for a putative ribonucleotide reductase (RRase), turned out to be responsible for the wild type nonA function. The phage SP10 possesses its own xnrdE gene coding for a putative RRase that complements the temperature-sensitive mutation of the host RRase gene nrdE. This complementation was blocked by an artificially induced transcription from a non-coding strand of the bnrdEF region. It is thus likely that the transcript from the bnrdEF region of SPβ inhibits ribonucleotide reductase function of SP10, resulting in arrest of DNA synthesis during phage SP10 development.

Inhibitory effect of prophage SPβ fragments on phage SP10 ribonucleotide reductase function and its multiplication in Bacillus subtilis

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