Replisome localization in vegetative and aerial hyphae of Streptomyces coelicolor

Abstract

Using a functional fusion of DnaN to enhanced green fluorescent protein, we examined the subcellular localization of the replisome machinery in the vegetative mycelium and aerial mycelium of the multinucleoid organism Streptomyces coelicolor. Chromosome replication took place in many compartments of both types of hypha, with the apical compartments of the aerial mycelium exhibiting the highest replication activity. Within a single compartment, the number of "current" ongoing DNA replications was lower than the expected chromosome number, and the appearance of fluorescent foci was often heterogeneous, indicating that this process is asynchronous within compartments and that only selected chromosomes undergo replication.

FIG. 1.

Analysis of Streptomyces strains expressing DnaN-EGFP. (A) Time course of DnaN-EGFP production in S. coelicolor (left) and expression of DnaN-EGFP in S. lividans TK24 after treatment with novobiocin (right). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated total proteins were scanned with a phosphorimager. Strains were grown on MM agar containing 1% mannitol (S. coelicolor) or in YEME-TSB liquid medium (S. lividans) at 30°C (14). Lane WT, wild-type strain; lane M, molecular weight markers; lane egfp, S. coelicolor M145(pIJ8641) expressing unfused EGFP (27 kDa). (B) Effect of DNA replication on the presence of replisome foci. S. lividans TK24 dnaN-egfp cultures were treated with novobiocin (final concentration, 200 μg/ml) for 120 min (middle panels) or with kanamycin (final concentration, 50 μg/ml) for 120 min (right panels). The left panels contained untreated cells. The strain was grown in 2× YT medium (13) at 30°C. Scale bars, 5 μm.

FIG. 2.

Localization of replicative DNA polymerase in the vegetative mycelium and aerial mycelium of S. coelicolor. S. coelicolor dnaN-egfp was grown for 26 h (vegetative mycelium) or for 42 to 66 h (aerial mycelium). The images show overlays of the DnaN-EGFP (green) fluorescence and cell walls stained with WGA (red) or DNA stained with 7-aminoactinomycin D (only in the case of spore chains). Scale bars, 5 μm. The diagram in the center shows the positions of the different types of compartments analyzed in the vegetative mycelium and aerial mycelium. (A) Germinating spores; (B) vegetative hyphae; (C) aerial hyphae; (D) apical compartments of aerial hyphae; (E) prespores and spores. The numbered arrowheads indicate examples of different types of fluorescence, as follows: arrowhead 1, bright foci; arrowhead 2, diffuse foci; arrowhead 3, dispersed fluorescence; arrowhead 4, lack of fluorescence. Red and yellow arrowheads indicate cross walls and tips of hyphae, respectively.

FIG. 3.

Types of DnaN-EGFP fluorescence in the vegetative and aerial hyphae of S. coelicolor. In the analysis 256 and 183 compartments of the vegetative mycelium and the aerial mycelium, respectively, were examined.