Minimal requirements of the Streptomyces lividans 66 oriC region and its transcriptional and translational activities

Abstract

Deletion analysis of a previously constructed minichromosome revealed that a stretch of DNA which is longer than 623 bp but shorter than 837 bp is required for autonomous replication of the Streptomyces lividans chromosome. Each of the dnaA and dnaN genes flanking the oriC region is individually transcribed from two promoters. Within the intergenic, nontranslatable region between the dnaA and dnaN genes, five main transcripts and several less abundant transcripts of various lengths as well as one of the promoters were identified. The introduction of additional DnaA boxes in S. lividans led to a significant increase in dnaA gene transcripts and to an enhanced level of the DnaA (73-kDa) protein. In summary, the data suggest that dnaA gene transcription is autoregulated and that initiation of the S. lividans chromosome is tightly controlled.The replication of several bacterial chromosomes was found to be initiated at the origin of replication, oriC. The features of oriC and, with a few exceptions, the arrangement of genes in its vicinity are remarkably similar among eubacteria (25, 38). Sequence analyses revealed that the origins of various eubacteria contain highly conserved segments which are separated by variable spacer regions. The DnaA box motifs (TTATC/AC AC/AA) (36) constituting the binding sites for the initiator protein DnaA are prominent in the conserved parts of oriC regions from various bacteria. The only relevant variation is an A-to-G exchange at the third position of the DnaA box motif within GC-rich DNA present in gram-positive bacteria such as Micrococcus luteus (10) and Streptomyces lividans (42). Most eubacteria contain a block of genes, dnaA-dnaN-recF-gyrB (10, 38), encoding the initiator protein DnaA, the ␤-subunit of DNA polymerase III holoenzyme, a product for recombination, and the ␤-subunit of DNA gyrase, respectively. Within most bacteria, oriC is situated close to the dnaA gene. In Escherichia coli, the arrangement of the dnaA region matches that in other bacteria; however, this region is located about 42 kb away from oriC (35).Procaryotic chromosomal replication has been best studied in E. coli. Its DnaA protein is a basic protein of 52 kDa to which ATP binds. About 10 to 40 ATP-DnaA molecules interact with four DnaA boxes within oriC (19,36). According to a current model (14), the newly synthesized DnaA protein is titrated by these DnaA boxes. As soon as the number of DnaA molecules exceeds the number of boxes, initiation is supposed to occur. A high number of GATC sites which can be methylated by the Dam methyltransferase have been found in the oriC region of E. coli, as well as in other members of the family Enterobacteriaceae (44), and GANTC sites are present in the Caulobacter crescentus oriC (43).Contrary to many bacteria, Streptomyces species grow as substrate mycelia which differentiate to aerial mycelia and spores upon depletion of nutrients. Depending on the species, the size of the GC-rich Streptomyces chromosome ranges between 6.6 and 8 Mb (18,32). Recently, we identified the...

Replisome Localization in Vegetative and Aerial Hyphae of Streptomyces coelicolor

“…1B) (the level of DnaN-EGFP remained constant in the cells not treated with novobiocin [data not shown]). This suggests that inhibition of replication may increase the level of dnaN transcription; notably, the two dnaN promoters (27) are located within the oriC region. Thus, these data show clearly that the observed fluorescent foci represent replisomes but not "storage structures" of nonactive ␤ subunits; formation of such structures should be expected in nonreplicative cells (particularly in nonreplicative cells "overexpressing" DnaN-EGFP) rather than in replication-active cells.…”

Purification and characterization of the Streptomyces lividans initiator protein DnaA

“…The chromosomal replication origin (oriC) of Streptomyces lividans was identified as an autonomously replicating fragment of approximately 0.7 kb (36,38). It carries 17 DnaA boxes which are arranged in two clusters.…”

“…The presence of the DnaA protein was verified by Western blot analysis (Fig. 1C) using previously described polyclonal antibodies raised against a glutathione S-transferase-DnaA fusion protein (38). Fractions containing the eluted DnaA protein were collect- ed, and proteins were precipitated with ammonium sulfate (20 to 35% saturation).…”

Characterization of the oriC region of Mycobacterium smegmatis

“…Together these data suggest that M. smegmatis oriC plasmids are stably maintained. oriC plasmids from many other bacteria have been shown to be unstable (17,27,30). It is not clear why the M. smegmatis oriC plasmids are stably maintained.…”

“…The oriC plasmids of many bacteria, including B. subtilis, Streptomyces coelicolor, and Pseudomonas putida, exist in low copy number (17,27,30). To determine the copy number of M. smegmatis oriC plasmids relative to the chromosomal oriC, total DNA from cells containing oriC plasmids was extracted, digested with PvuII, gel electrophoresed, transferred to nitrocellulose membranes, probed with radiolabeled oriC-specific DNA fragments (24), and visualized by autoradiography (Fig.…”