A host-shutoff early gene of Bacillus subtilis bacteriophage SPO1
Stewart, Charles R.
Doctor of Philosophy
Shutoff of host biosynthesis is one of the earliest and most dramatic events occurring in viral infection and requires the expression of viral early genes. To understand the mechanisms of bacteriophage SPO1 induced host-shutoff, two SPO1 early genes, e3 and e22, were cloned and sequenced, and the roles of e3 in host-shutoff and in phage growth were studied. Both e3 and e22 are novel genes, and are actively expressed during the first few minutes of infection before being promptly shut off. Expression of a plasmid-borne e3 gene, in either B. subtilis or E. coli, caused the inhibition of host DNA, RNA and protein synthesis, and ultimately led to cell death. To identify the primary target of e3-induced shutoff, an e3-resistant E. coli mutant was isolated and analyzed. Plasmid libraries of this mutant's genomic DNA, when screened for genes that could protect wild-type E. coli against e3, yielded the rpoB and dksA genes, which specify the RNA polymerase $\beta$ subunit and a suppressor for DnaK, respectively. The wild-type dksA gene, but not the wild-type rpoB gene, was able to protect against e3, suggesting that the primary e3-resistant mutation was in the rpoB gene and that protection by the dksA gene depended upon overexpression from the plasmid. I suggest that e3 acts by distorting the structure of the host RNA polymerase, thus preventing host transcription, and that this distortion can be prevented or reversed by a chaperonin-like activity specified by dksA. The host-shutoff still occurred normally during infection by an SPO1 mutant which lacked e3 activity, and it occurred much more rapidly than that caused by expression of e3 in uninfected cells. Thus, the e3 product must be only one component of the host-shutoff machinery, which must include elements whose function is redundant to that of e3. At high multiplicities of infection, the mutant SPO1 produced more phage progeny than the wild-type SPO1, suggesting that high concentrations of e3 can be inhibitory to phage growth as well as to host function. Perhaps for that reason, expression of both e3 and e22 is shut off after a brief period of high activity.