A 'Gram-negative-type' DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2)

The Streptomyces coelicolor dnaE gene, encoding the catalytic alpha-subunit of DNA polymerase III (pol III) was isolated by genetic complementation of a temperature-sensitive DNA replication mutant, S. coelicolor ts-38, The d educed protein sequence (1179 residues) is highly similar to the Escherichi a coli-type pol III alpha-subunit, rather than to the PolC-type alpha-subun it that is known to be essential for replication in the "low G + C' Gram-po sitive bacteria such as Bacillus subtilis. The dnaE gene is able to restore replication to a 'slow stop' mutant (ts-38) and a 'fast stop' mutant (ts-1 14); the dnaE gene of ts-38 carries a single amino acid substitution (Glu-8 02 to Lys), and the mutation in ts-114 has been mapped between codons 697 a nd 1062 of dnaE, Mutant ts-38 is considered to be defective in assembly of the multisubunit pol III holoenzyme and, hence, in initiation of replicatio n, whereas ts-114 is defective in chain elongation. This study provides the first evidence that a DnaE-type pol III is essential for replication in a Gram-positive bacterium. In addition, the complementation studies suggest t hat the C-terminal 117 residues are not essential for DnaE function in S, c oelicolor, When integrated at a distant site on the chromosome, a fragment containing the 3' half of dnaE(codons 697-1179) is capable of rescuing ts-3 8 (but not ts-114) at the restrictive temperature; it was demonstrated that homogenotization was responsible for this phenomenon.