The replication checkpoint control in Bacillus subtilis: identification ofa novel RTP-binding sequence essential for the replication fork arrest after induction of the stringent response

We have shown previously that induction of the stringent response in Bacill us subtilis resulted in the arrest of chromosomal replication between 100 a nd 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively, This replication checkpoint was also shown to involve the RTP protein, normally active at th e chromosomal terminus. In this study, we show that the replication block i s absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP, DNA-DNA hybridizati on data indicated that one or more such LSTer sites mapped to gene yxcC (-1 28 kb from oriC). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with r espect to replication, as in the chromosome. This LSTer2 site was further m apped to a 3.65 kb fragment overlapping the next40 probe, Remarkably, this fragment contains a 17 bp sequence (B'-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This B'-1 se quence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting, Importantly, preci se deletion of this sequence abolished the replication arrest. We propose t hat this modified a site is an essential constituent of the LSTer2 site, Th e differences between arrest at the normal chromosomal terminus and arrest at LSTer2 site are discussed.