REPLICATION TERMINATOR PROTEIN-BASED REPLICATION FORK-ARREST SYSTEMS IN VARIOUS BACILLUS SPECIES

The replication terminator protein (RTP) of Bacillus subtilis interact s with its cognate DNA terminators to cause replication fork arrest, t hereby ensuring that the forks approaching one another at the conclusi on of a round of replication meet within a restricted terminus region. .ii similar situation exists in Escherichia coli, but it appears that the folk-arrest systems in these two organisms have evolved independen tly of one another. In the present work RTP homologs in four species c losely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B., vallismortis) have been identified and characteri zed. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of th e rtp genes raise the possibility that these organisms arose within th is branch of the tree by horizontal transfer into a common ancestor af ter their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the impo rtance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region o f RTP emphasizes the importance of its high affinity for the DNA termi nator in its functioning. By coincidence, the single change (E30K) fou nd in the B. mojavensis RTP corresponds exactly to that purposefully i ntroduced hy others into B, subtilis RTP to implicate a crucial role f or E30 in the fork-arrest mechanism. The natural occurrence of this va riant is difficult to reconcile with such an implication, and it was s hown directly that RTP.E30K functions normally in fork arrest in B. su btilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B. subtilis 168 genome sequence to bring the total to nine.