Search for additional replication terminators in the Bacillus subtilis 168 chromosome

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• The Bacillus subtilis 168 chromosome contains multiple DNA replication terminators, primarily in the terminus region.
• These terminators are crucial for preventing replication fork collisions and ensuring genome stability.

Purpose of the Study:

• To identify novel DNA replication terminators in the Bacillus subtilis chromosome.
• To characterize the functional and sequence properties of identified terminators, particularly in terminus and STer site regions.
• To compare the characteristics of terminators from different chromosomal locations.

Main Methods:

• Low-stringency hybridization using a degenerate DNA probe for consensus terminator sequences.
• Cloning and characterization of hybridizing DNA fragments.
• Database searching (SubtiList) for terminator sequences.
• Replication termination protein (RTP) binding assays.
• In vitro replication fork movement assays using plasmids.

Main Results:

• An additional functional terminator, binding RTP and blocking replication forks, was identified in the terminus region near glnA.
• Low-stringency hybridization did not detect terminus region-type terminators in STer site regions.
• Two candidate terminators from STer site regions showed sequence similarity but did not bind RTP or arrest replication forks, indicating they lack typical terminator features.

Conclusions:

• The Bacillus subtilis chromosome harbors additional functional DNA replication terminators in the terminus region.
• Terminators located in STer site regions, unlike those in the terminus region, do not possess the canonical A+B site structure and lack typical terminator activity.
• This suggests distinct mechanisms or sequence requirements for termination at different chromosomal locations.