Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor

Abstract

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants-which fail to properly localise FtsZ-could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades.

Fig. 1

FtsZ levels in the whi mutants and restoration by pSCF7. Western analysis using polyclonal antibodies against FtsZ demonstrating FtsZ protein levels in protein extracts of S. coelicolor M145 (wild-type strain) as well as its sporulation mutant derivatives carrying complete deletions of the whiA, whiB, whiG, whiH, whiI, whiJ or ssgB genes. Note that introduction of pSCF7 restores FtsZ to levels similar to those found in the wild-type strain, except for whiH and whiJ mutants, which show enhanced FtsZ protein levels

Fig. 2

Expression of FtsZ from a constitutive promoter rescues the sporulation block of whi mutants. The image shows the respective whi mutants (whiA, whiB, whiG, whiH, whiI, or whiJ) with control plasmid (left), and the mutants carrying pSCF7 (right), which expresses ftsZ from the constitutive ermE promoter. The ssgB null mutant is presented as a control. Note that grey pigmentation is restored to the whi mutants but only partially to the ssgB mutant (see Fig. 3 and Fig. S3 for cryo-SEM images). Single colonies of the whiJ mutant reproducibly showed delayed aerial development. Strains were grown for 4 days on SFM agar plates at 30°C

Fig. 3

Cryo-scanning electron micrographs of mature spore chains of the whi mutants expressing FtsZ. Column 1, S. coelicolor M145 (parental strain) and its whiA, whiB and whiG mutants harbouring control plasmid; column 2, same strains as shown in column 1 but now containing plasmid pSCF7, which expresses ftsZ from the constitutive ermE promoter; column 3, whiH, whiI, whiJ and ssgB mutants containing control plasmid; column 4, same strains as shown in column 3, but now containing plasmid pSCF7 (or pSCF7B for the ssgB mutant). Note that the constitutive expression of ftsZ restores sporulation to all whi mutants, while ssgB mutants produce spore-like bodies with highly variable sizes (see Fig. 5), most likely due to incorrect localization of FtsZ. For high-resolution TEM images of the spore chains see Fig. 5. All strains were grown for 5 days on SFM agar plates at 30°C. See Fig. S3 for lower magnification. All images presented at the same scale. Bar (top left), 1 μm

Fig. 4

Viability and formation of septal ladders during sporulation of sporulation-restored whi mutants. Live/dead staining (left) and septum staining (right) is shown for the whi and ssgB mutants harbouring pSCF7. FM145 was used as the control. Live cells were identified with syto-82, dead cells with propidium iodide. Septa were highlighted by the membrane stain FM5-95. Note that several spores of the whiG and ssgB mutants were not viable (red; dark in b/w), fitting with the viability count and the incomplete restoration of sporulation. Note the multi-lobed DNA produced by whiA and whiG mutants (see Fig. S5 for deconvolution images). Transformants were grown against microscopy cover slips for 3 days on SFM agar plates. Bar, 5 μm

Fig. 5

Transmission electron micrographs of sporulation-restored whi mutants. Thin sections of spore chains of pSCF7 transformants of S. coelicolor M145 and its whi and ssgB mutants were analysed at high resolution by transmission electron microscopy. For each of the transformants a representative spore chain is presented. Arrows indicate multi-lobed chromosomes in whiA and whiG transformants. Insert in the image for the ssgB transformant shows magnification of the thin cell wall of the spore-like bodies produced by the transformants. Bar = 1 μm