Functional evidence that the principal DNA replication origin of the Streptomyces coelicolor chromosome is close to the dnaA-gyrB region

Abstract

The degree of overrepresentation of selected chromosomal genes in rapidly growing cultures of Streptomyces coelicolor was assessed by quantitative DNA hybridization analysis. The results are consistent with the hypothesis that the principal origin of replication is close to the dnaA-gyrB region, in the center of the linear chromosome, and that replication proceeds bidirectionally.The streptomycete bacteria are among the most complex members of the prokaryotic world and possess large (ca. 8 Mb [11]) linear genomes (12, 13). Since the Streptomyces coelicolor chromosome is linear, it cannot be assumed that its mode of replication will be the same as that of other bacteria, such as Escherichia coli or Bacillus subtilis. Any investigation of the mode of chromosomal replication in Streptomyces species is hampered by a number of methodological and physiological problems which are not encountered with unicellular bacteria. For instance, the mycelial growth habit of streptomycetes means that synchronous cultures are difficult or impossible to prepare, while thymine-requiring mutants, which enable the specific labelling of DNA (15,17), are lacking for this genus (8). In an attempt to circumvent these difficulties, we have used quantitative DNA-DNA hybridization analysis, with specific gene probes from both homologous and heterologous sources, in order to locate the functional origin of DNA replication on the S. coelicolor A3(2) chromosome. This technique was used to compare the relative abundance of particular genes at different locations on the S. coelicolor chromosome in stationary-phase and rapidly growing cultures. This approach is a molecular biological analog of the genetic experiments performed by Masters and Broda (14) to demonstrate the bidirectional replication of the E. coli chromosome from a single fixed site of initiation. It is known that, in bacterial cells which are actively replicating their DNA, genes close to the replication origin will be overrepresented in comparison to those close to the terminus. This effect should be exaggerated in cells growing at, or close to, their maximum specific growth rate, when a number of rounds of chromosome replication may be proceeding at the same time (5). The gyrB gene was chosen as a probe because, in all bacterial genera examined to date, the replication origin has been found to lie within or close to the dnaA-dnaN-recF-gyrB region (16). The other five probes were chosen in order to detect genes which are widely spaced on the chromosome (11).The experiments were performed with continuous-flow cultures of S. coelicolor MT1109, an SCP1-/SCP2-derivative of the wild-type strain 1147 (7) (unpublished data); twofolddiluted Luria broth was used as the growth medium (18). Each chemostat experiment involved several shift-up steps from an initial low growth rate (dilution rate, 0.19 h-1). A steady state was established at each step, until a dilution rate just below the critical dilution rate (0.32 h-1; rate at which dilution exceeds growth) was attained. After establishment ...

Instability of artificially circularized chromosomes of Streptomyces lividans

“…The linear chromosomes (and linear plasmids) of Streptomyces, however, possess a centrally located origin of replication, oriC, near genes that are involved in replication (dnaA, dnaN and gyrBA ) (Calcutt and Schmidt, 1992;Zakrzewska-Czerwinska and Schrempf, 1992). Replication of the Streptomyces chromosome appears to proceed bidirectionally towards the telomeres (Musialowski et al, 1994) and presumably leaves 3Ј single-strand gaps at the termini that may be filled in by TP-primed replication initiated at the telomeres (Chang and Cohen, 1994;Chen, 1996).…”

Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces

“…Recent discoveries suggest that replication of the linear chromosome of Streptomyces coelicolor A3(2) proceeds bidirectionally from the centrally located oriC region toward the ends of the chromosome (12).…”

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2)

“…The corresponding sequences of the S. coelicolor chromosome were found to be flanked by dnaA (encoding the replication initiator protein) and dnaN (for the ␤ subunit of DNA polymerase III) (11). The rpmH gene (for ribosomal protein L34) and rnpA (for the RNase P protein) lie very close upstream of dnaA (11), and gyrB (for the DNA gyrase B subunit) is located 2 to 3 kb downstream of dnaN (30). Functional evidence that oriC is the principal replication origin was obtained by studying the overrepresentation of a sample of chromosomal genes in rapidly growing cultures (30).…”

“…The rpmH gene (for ribosomal protein L34) and rnpA (for the RNase P protein) lie very close upstream of dnaA (11), and gyrB (for the DNA gyrase B subunit) is located 2 to 3 kb downstream of dnaN (30). Functional evidence that oriC is the principal replication origin was obtained by studying the overrepresentation of a sample of chromosomal genes in rapidly growing cultures (30). The location of oriC in the center of what is now known to be a linear chromosome in at least some derivatives of S. coelicolor A3(2) (25) is interesting in comparison with a similar finding for the linear chromosome of Borrelia burgdorferi (12).…”