Search for additional replication terminators in the Bacillus subtilis 168 chromosome

Abstract

The Bacillus subtilis 168 chromosome is known to contain at least six DNA replication terminators in the terminus region of the chromosome. By using a degenerate DNA probe for the consensus terminator sequence and low-stringency hybridization conditions, several additional minor hybridizing bands were identified. DNA corresponding to the most intense of these bands was cloned and characterized. Although localized in the terminus region, it could not bind RTP and possibly represents a degenerate terminator. A search of the SubtiList database identified an additional terminator sequence in the terminus region, near glnA. It was shown to bind RTP and to function in blocking replication fork movement in a polar manner. Its orientation conformed to the replication fork trap arrangement of the other terminators. The low-stringency hybridization experiments failed to identify any terminus region-type terminators in the region of the chromosome where postinitiation control sequences (STer sites) are known to reside. The two most likely terminators in STer site regions, in terms of sequence similarity to terminus region terminators, were identified through sequence searching. They were synthesized and were found not to bind RTP under conditions that allowed binding to terminus region terminators. Neither did they elicit fork arrest, when present in a plasmid, under stringent conditions. It is concluded that the STer site terminators, at least the first two to the left of oriC, do not have the typical consensus A؉B site makeup of terminus region terminators.Escherichia coli and Bacillus subtilis both have a system for arresting a replication fork within the terminus region of their circular chromosomes (6). In each case, fork arrest requires interaction between a specific terminator protein (Tus in E. coli, RTP in B. subtilis) and a short DNA sequence known as a terminator. The terminator protein-DNA terminator complexes are polar in their action (i.e., they arrest a replication fork approaching from one particular direction only). Tus and RTP show no recognizable sequence similarity to each other, and neither do their cognate terminators. Tus functions as a monomer and interacts with a terminator sequence of 22 to 23 bp. The interaction that takes place between RTP and its terminator is more complex. A dimer of RTP binds to each of two adjacent and opposed sites (A and B) which make up a minimal terminator of 29 bp (Fig. 1, top section). This sequence encompasses the segment running from nucleotides (nt) Ϫ2 to 27 of the originally identified terminators (3). The 16-bp A and B sites each comprise a trinucleotide (common to both sites [boxed in Fig. 1]) plus a 13-bp sequence which is different between the two sites. Six terminators (TerI to TerVI) have so far been identified in B. subtilis 168, and two terminators have been identified in B. subtilis W23 (5) (the consensus is shown in Fig. 1). The consensus B site (nt 12 to 27) possesses a detectable level of symmetry about nt 21 (note arrows under G15, A17 and T25, C27 in ...

Effects of replication termination mutants on chromosome partitioning in Bacillus subtilis

“…The replication fork duplicating from 360°t o 172°presumably terminates on meeting the fork already halted at TerI. The seven other Ter sites flank TerI and TerII oriented such that they create a replication fork trap in the terminal 10% of the chromosome (12,14). This trap seems to ensure that a fork that passes through TerI is impeded by the subsequent Ter sites (likewise if a fork passes through TerII).…”

“…Nine Ter sites have been identified in B. subtilis (12). TerI and TerII are located 59 bp apart at Ϸ172°on the circular 360°c hromosome (13,14).…”

Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis

“…4 and Supplementary Methods online). The role of SpoIIIE in vegetative growth 10 and the location of dif at the center of the terminus of replication in B. subtilis 30,31 suggest that the dif region is the last chromosomal fragment to be translocated into the forespore during sporulation. We tested six candidate sequences identified with this approach (SRS1-6) using the magnetic tweezers assay for specific DNA sequence recognition.…”

Sequence Analysis of the Bacillus subtilis 168 Chromosome Region between the sspC and odhA Loci (184 -180 )

“…DNA sequence alignment of a potential DNA terminator with the known consensus of B. subtilis DNA terminator. 15 The numbering indicates position of the nucleotide residue. Underline indicates identical nucleotide residues.…”

“…13 There are at least seven DNA replication terminators known to exist in the terminus region of the B. subtilis chromosome. 13 ' 15 One of the known DNA termination sequences (TerV) was found in a non-coding region adjacent to yodU (E5). 13 In addition, 2 new putative DNA replication terminators (TerIX and TerX in Fig.…”