Summary auto-generated
Streptomyces bacteria possess unique features including a large, linear, GC-rich chromosome and a mycelial life cycle. This study compared the chromosomal replication origins (oriC) of three Streptomyces species: S. antibioticus, S. chrysomallus, and S. lividans. The researchers identified 19 conserved DnaA boxes within each oriC region, determining a consensus DnaA box sequence. Unlike Escherichia coli, Streptomyces oriC lacks AT-rich 13-mer direct repeats but contains scattered short AT-rich sequences among the DnaA boxes. Using surface plasmon resonance, the researchers measured DnaA protein binding to individual DnaA boxes, finding dissociation constants ranging from 12 to 78 nanomolar. The DNA-binding domain of S. lividans DnaA selectively bound to oriC fragments from all three species. Attempts to observe DnaA-mediated unwinding of Streptomyces oriC were unsuccessful, suggesting that additional accessory proteins may be required to assist in DNA unwinding during replication initiation.
Key findings
- Streptomyces oriC regions contain 19 conserved DnaA boxes with consensus sequence (T/C)(T/C)(G/A/C)TCCACA, with identical positioning across three species
- DnaA protein exhibits variable binding affinity to individual DnaA boxes (12-78 nM dissociation constants) and selectively binds Streptomyces oriC fragments
- Streptomyces oriC lacks the AT-rich 13-mer direct repeats found in E. coli but contains five short AT-rich sequences distributed throughout the region
- DnaA-mediated unwinding of Streptomyces oriC could not be demonstrated, indicating putative accessory proteins may be required for replication initiation
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Abstract
Streptomycetes differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large, linear, GC-rich chromosome. To deduce structural features of the Streptomyces origin of chromosomal replication, the oriC sequences of three Streptomyces species (S. antibioticus, S. chrysomallus and S. lividans) were compared. In Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved. The consensus sequence of the DnaA box identified within Streptomyces oriC is (T/C)(T/C)(G/A/C)TCCACA (preferred bases underlined). The interactions of DnaA with DNA fragments containing single, two or three DnaA boxes were studied using surface plasmon resonance. The dissociation constant (KD) for specific binding of individual DnaA boxes varied between 12 and 78 nM. Streptomyces oriC does not contain the three AT-rich 13-mer direct repeats present in the 5' part of the Escherichia coli oriC region. However, short AT-rich sequences are distributed among the DnaA boxes of Streptomyces oriC. Repeated attempts to unwind Streptomyces oriC have been unsuccessful. It remains to be elucidated whether DnaA interacts with putative accessory proteins which help in unwinding Streptomyces oriC.