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Like eukaryotic and archaeal viruses, which coopt the host's cellular pathways for their replication, bacteriophages have evolved strategies to alter the metabolism of their bacterial host. SPO1 bacteriophage infection of Bacillus subtilis results in comprehensive remodeling of cellular processes, leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins that specifically shut down various processes in the bacterial host, including transcription, DNA synthesis, and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module, here we identified eight gene products that attenuated B. subtilis growth. Of the eight phage gene products that attenuated bacterial growth, a 25-kDa protein called Gp53 was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis. Our results further reveal that Gp53 is a phage-encoded adaptor-like protein that modulates the activity of the ClpCP protease to enable efficient SPO1 phage progeny development. In summary, our findings indicate that the bacterial ClpCP protease is the target of xenogeneic (dys)regulation by a SPO1 phage–derived factor and add Gp53 to the list of antibacterial products that target bacterial protein degradation and therefore may have utility for the development of novel antibacterial agents.

DNA mimicry by proteins is a strategy that employed by some proteins to occupy the binding sites of the DNA-binding proteins and deny further access to these sites by DNA. Such proteins have been found in bacteriophage, eukaryotic virus, prokaryotic, and eukaryotic cells to imitate non-coding functions of DNA. Here, we report another phage protein Gp44 from bacteriophage SPO1 of Bacillus subtilis, employing mimicry as part of unusual strategy to inhibit host RNA polymerase. Consisting of three simple domains, Gp44 contains a DNA binding motif, a flexible DNA mimic domain and a random-coiled domain. Gp44 is able to anchor to host genome and interact bacterial RNA polymerase via the β and β′ subunit, resulting in bacterial growth inhibition. Our findings represent a non-specific strategy that SPO1 phage uses to target different bacterial transcription machinery regardless of the structural variations of RNA polymerases. This feature may have potential applications like generation of genetic engineered phages with Gp44 gene incorporated used in phage therapy to target a range of bacterial hosts.

No abstract

SPO1 phage infection of Bacillus subtilis results in a comprehensive remodelling of processes leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins for the specific shut down of various host processes including transcription, DNA synthesis and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module we identified eight gene products which attenuated B. subtilis growth. Out of the eight gene products that attenuated bacterial growth, a 25 kDa protein, called Gp53, was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis. Results reveal that Gp53 functions like a phage encoded adaptor protein and thereby appears to alter the substrate specificity of the ClpCP protease to modulate the proteome of the infected cell to benefit efficient SPO1 phage progeny development. It seems that Gp53 represents a novel strategy used by phages to acquire their bacterial prey.Significance statementViruses of bacteria (phages) represent the most abundant living entities on the planet, and many aspects of our fundamental knowledge of phage–bacteria relationships remain elusive. Many phages encode specialised small proteins, which modulate essential physiological processes in bacteria in order to convert the bacterial cell into a ‘factory’ for phage progeny production – ultimately leading to the demise of the bacterial cell. We describe the identification of several antibacterial proteins produced by a prototypical phage that infects Bacillus subtilis and describe how one such protein subverts the protein control system of its host to benefit phage progeny development. The results have broad implications for our understanding of phage–bacteria relationships and the therapeutic application of phages and their gene products.