Identification and characterization of cis- and trans-acting elements involved in prophage induction in Streptococcus thermophilusJ34

The genetic switch region of temperate Streptococcus thermophilus phage TP-J34 contains two divergently oriented promoters and several predicted operator sites. It separates lytic cycle-promoting genes from those promoting lysogeny. A polycistronic transcript comprises the genes coding for repressor Crh, metalloproteinase-motif protein Rir and superinfection exclusion lipoprotein Ltp. Weak promoters effecting monocistronic transcripts were localized for ltp and int (encoding integrase) by Northern blot and 5-RACE-PCR. These transcripts appeared in lysogenic as well as lytic state. A polycistronic transcript comprising genes coh (encoding Cro homolog), ant (encoding putative antirepressor), orf7, orf8 and orf9 was only detected in the lytic state. Four operator sites, of which three were located in the intergenic regions between crh and coh, and one between coh and ant, were identified by competition electromobility shift assays. Cooperative binding of Crh to two operator sites immediately upstream of coh could be demonstrated. Coh was shown to bind to the operator closest to crh only. Oligomerization was proven by cross-linking Crh by glutaraldehyde. Knock-out of rir revealed a key role in prophage induction. Rir and Crh were shown to form a complex in solution and Rir prevented binding of Crh to its operator sites.