Genetic interactions of smc, ftsK, and parB genes in Streptomyces coelicolor and their developmental genome segregation phenotypes

Abstract

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK' truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.

FIG. 1.

Physical maps of regions of chromosomal DNA containing smc, ftsK, or scpAB. In each case, the flanking genes are in the same orientation as the genes of interest. Bar: 1 kb. (A) The smc region of the chromosome. SCO5576 is predicted to encode an acyl phosphatase and glcP1 is involved in glucose uptake. For mutagenic plasmid pHJ2, the region deleted and replaced with a hygromycin resistance gene is shown (dashed line). The insert in complementation plasmid pHW35 is shown (bracketed line). (B) The ftsK region of the chromosome. osaB is part of a two-component regulator involved in osmoadaptation and SCO5751 is predicted to encode a hypothetical membrane protein. For mutagenic plasmid pJR148, the region of ftsK deleted and replaced with a neomycin resistance gene is shown (dashed line). The insert in complementation plasmid pRMD6 is shown (bracketed line). (C) The scpAB region of the chromosome. SCO1768, SCO1771, and SCO1767 are predicted to encode a pseudouridine synthase, a hypothetical protein found in actinomycetes, and a possible DNA hydrolase, respectively. For mutagenic cosmids pRMD7, -8, and -9, respectively, the regions of SCO1768, scpA, and scpAB deleted and replaced with an apramycin resistance gene are shown (dashed lines). The insert in complementation plasmid pRMD11 is shown (bracketed line); the DNA insert ends 1 base after SCO1768. (D) Single, double, and triple mutant strains macroscopically resemble the wild type. Strains were grown on MS agar for 4 days at 30°C. Mutant strains formed robust, gray-pigmented, aerial mycelia with no obvious growth or developmental delays. Strains M145 (wild type), J2537 (ΔparB), JM148 (ΔftsK), HJ2 (Δsmc), RMD3 (Δsmc ΔftsK), RMD4 (Δsmc ΔparB), RMD5 (ΔparB ΔftsK), and RMD6 (Δsmc ΔparB ΔftsK) are shown.

FIG. 2.

Confocal microscope images showing sporulation and DNA segregation phenotypes of the wild type and of single, double, and triple mutant strains. Four-day-old cultures grown on MS agar were sampled on coverslips, fixed, and stained with propidium iodide (red). In each panel, phenotypes of representative aerial hyphae are shown as phase-contrast and fluorescent image pairs, followed by a merged image. (A) M145 (wild type). (B) JM148 (ΔftsK). (C) HJ2 (Δsmc). (D) J2537 (ΔparB). (E) RMD3 (Δsmc ΔftsK). (F) RMD4 (Δsmc ΔparB). (G) RMD5 (ΔparB ΔftsK). (H) RMD6 (Δsmc ΔparB ΔftsK). Bar: 7 μm.

FIG. 3.

FtsK-EGFP and SMC-EGFP fusion protein localization in vegetative and reproductive hyphae. Coverslips were inoculated and hyphae grown for 1 to 3 days on MS agar, fixed, and stained with propidium iodide. In each panel, representative hyphae are shown as phase-contrast and fluorescent images as observed by scanning laser confocal microscopy. From left to right, panels contain phase-contrast, EGFP, and DNA images and a merge of the two fluorescent images. (A) FtsK-EGFP localized in a vegetative filament newly emerging from a spore (lower right; 1 day of growth). (B) FtsK-EGFP localized in a regular pattern in a predivisional aerial filament just prior to prespore formation (long, central filament oriented with long axis of panel; 2 days of growth). (C) The SMC-EGFP fusion protein had an irregular punctuate pattern of localization in a predivisional aerial filament (2 to 3 days of growth). SMC-EGFP foci are not uniformly distributed, nor are they associated with every nucleoid. Bar: 7 μm.

FIG. 4.

The scpA and scpAB mutants have a bilobed nucleoid phenotype. Phase-contrast and fluorescent images of the same fields were taken by scanning laser confocal microscopy. Coverslips were inoculated and grown for 1 to 3 days on MS agar, fixed, and stained with propidium iodide. In each panel, phenotypes of representative aerial hyphae are shown as phase-contrast and fluorescent image pairs, followed by a merged image. For the merged images, the areas in the white boxes are enlarged and a cartoon interpretation is provided. (A) Images of ΔscpA (RMD18) showing spores with DNA appearing bilobed, unlike wild-type spores. (B) Images of ΔscpAB spores (RMD19) also appearing to have a bilobed DNA pattern. Bar: 7 μm.