Sporulation Septum Research Articles

Importance and regulation of inositol biosynthesis during growth and differentiation of Streptomyces

Unusually among bacteria, actinobacteria possess myo-inositol 1-phosphate synthase (mIPS). In the developmentally complex Streptomyces coelicolor, the mIPS-encoding gene (inoA) is cotranscribed with a putative regulatory gene (inoR). The inoRA transcript was more abundant in an inoR in-frame deletion mutant, and InoR formed different complexes in vitro with an extensive region around the inoRA promoter. Binding was relieved by adding glucose 6-phosphate. Thus, InoR is a metabolite-sensitive autorepressor that influences inoA expression, and hence the level of inositol, by controlling transcription from P(inoRA) . Disruption of inoA resulted in inositol-dependent growth and development, with full phenotypic correction at 0.1 mM inositol: at lower inositol concentrations differentiation was arrested at intermediate stages. This pattern may partly reflect increased demand for membrane phospholipids during sporulation septation. A corresponding sharp upregulation of inoRA transcription coincident with sporulation was dependent on a developmental regulator, WhiI. A truncated form of WhiI could bind two sites downstream of P(inoRA) , and one of the WhiI-binding sites overlapped the InoR-binding site. The combined action of a metabolic regulator and a developmental regulator at the simple P(inoRA) promoter is a previously undescribed strategy for the differential provision of developmentally appropriate levels of a substance required during the formation of spore chains.

The MreB-Like Protein Mbl ofStreptomyces coelicolorA3(2) Depends on MreB for Proper Localization and Contributes to Spore Wall Synthesis

Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ΔmreB Δmbl double mutant was viable. Δsco6166 had a wild-type phenotype. ΔmreB, Δmbl, and ΔmreB Δmbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth.

Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation.

SpoIIIE is an FtsK-related protein that transports the forespore chromosome across the Bacillus subtilis sporulation septum. We use membrane photobleaching and protoplast assays to demonstrate that SpoIIIE is required for septal membrane fission in the presence of trapped DNA, and that DNA is transported across separate daughter cell membranes, suggesting that SpoIIIE forms a channel that partitions the daughter cell membranes. Our results reveal a close correlation between septal membrane fission and the assembly of a stable SpoIIIE translocation complex at the septal midpoint. Time-lapse epifluorescence, total internal reflection fluorescence (TIRF) microscopy, and live-cell photoactivation localization microscopy (PALM) demonstrate that the SpoIIIE transmembrane domain mediates dynamic localization to active division sites, whereas the assembly of a stable focus also requires the cytoplasmic domain. The transmembrane domain fails to completely separate the membrane, and it assembles unstable foci. TIRF microscopy and biophysical modeling of fluorescence recovery after photobleaching (FRAP) data suggest that this unstable protein transitions between disassembled and assembled oligomeric states. We propose a new model for the role of SpoIIIE assembly in septal membrane fission that has strong implications for how the chromosome terminus crosses the septum.

SpoIID-Mediated Peptidoglycan Degradation Is Required throughout Engulfment during Bacillus subtilis Sporulation

SpoIID is a membrane-anchored enzyme that degrades peptidoglycan and is essential for engulfment and sporulation in Bacillus subtilis. SpoIID is targeted to the sporulation septum, where it interacts with two other proteins required for engulfment: SpoIIP and SpoIIM. We changed conserved amino acids in SpoIID to alanine to determine whether there was a correlation between the effect of each substitution on the in vivo and in vitro activities of SpoIID. We identified one amino acid substitution, E88A, that eliminated peptidoglycan degradation activity and one, D210A, that reduced it, as well as two substitutions that destabilized the protein in B. subtilis (R106A and K203A). Using these mutants, we show that the peptidoglycan degradation activity of SpoIID is required for the first step of engulfment (septal thinning), as well as throughout membrane migration, and we show that SpoIID levels are substantially above the minimum required for engulfment. The inactive mutant E88A shows increased septal localization compared to the wild type, suggesting that the degradation cycle of the SpoIID/SpoIIP complex is accompanied by the activity-dependent release of SpoIID from the complex and subsequent rebinding. This mutant is also capable of moving SpoIIP across the sporulation septum, suggesting that SpoIID binding, but not peptidoglycan degradation activity, is needed for relocalization of SpoIIP. Finally, the mutant with reduced activity (D210A) causes uneven engulfment and time-lapse microscopy indicates that the fastest-moving membrane arm has greater concentrations of SpoIIP than the slower-moving arm, demonstrating a correlation between SpoIIP protein levels and the rate of membrane migration.

Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double-stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum.

Streptomyces coelicolor Dps‐like proteins: differential dual roles in response to stress during vegetative growth and in nucleoid condensation during reproductive cell division

The Dps protein, a member of the ferritin family, contributes to DNA protection during oxidative stress and plays a central role in nucleoid condensation during stationary phase in unicellular eubacteria. Genome searches revealed the presence of three Dps-like orthologues within the genome of the Gram-positive bacterium Streptomyces coelicolor. Disruption of the S. coelicolor dpsA, dpsB and dpsC genes resulted in irregular condensation of spore nucleoids in a gene-specific manner. These irregularities are correlated with changes to the spacing between sporulation septa. This is the first example of these proteins playing a role in bacterial cell division. Translational fusions provided evidence for both developmental control of DpsA and DpsC expression and their localization to sporogenic compartments of aerial hyphae. In addition, various stress conditions induced expression of the Dps proteins in a stimulus-dependent manner in vegetative hyphae, suggesting stress-induced, protein-specific protective functions in addition to their role during reproductive cell division. Unlike in other bacteria, the S. coelicolor Dps proteins are not induced in response to oxidative stress.

FtsW Is a Dispensable Cell Division Protein Required for Z-Ring Stabilization during Sporulation Septation in Streptomyces coelicolor

The conserved rodA and ftsW genes encode polytopic membrane proteins that are essential for bacterial cell elongation and division, respectively, and each gene is invariably linked with a cognate class B high-molecular-weight penicillin-binding protein (HMW PBP) gene. Filamentous differentiating Streptomyces coelicolor possesses four such gene pairs. Whereas rodA, although not its cognate HMW PBP gene, is essential in these bacteria, mutation of SCO5302 or SCO2607 (sfr) caused no gross changes to growth and septation. In contrast, disruption of either ftsW or the cognate ftsI gene blocked the formation of sporulation septa in aerial hyphae. The inability of spiral polymers of FtsZ to reorganize into rings in aerial hyphae of these mutants indicates an early pivotal role of an FtsW-FtsI complex in cell division. Concerted assembly of the complete divisome was unnecessary for Z-ring stabilization in aerial hyphae as ftsQ mutants were found to be blocked at a later stage in cell division, during septum closure. Complete cross wall formation occurred in vegetative hyphae in all three fts mutants, indicating that the typical bacterial divisome functions specifically during nonessential sporulation septation, providing a unique opportunity to interrogate the function and dependencies of individual components of the divisome in vivo.

SpoIIQ Anchors Membrane Proteins on Both Sides of the Sporulation Septum in Bacillus subtilis

During the process of spore formation in Bacillus subtilis, many membrane proteins localize to the polar septum where they participate in morphogenesis and signal transduction. The forespore membrane protein SpoIIQ plays a central role in anchoring several mother-cell membrane proteins in the septal membrane. Here, we report that SpoIIQ is also responsible for anchoring a membrane protein on the forespore side of the sporulation septum. Co-immunoprecipitation experiments reveal that SpoIIQ resides in a complex with the polytopic membrane protein SpoIIE. During the early stages of sporulation, SpoIIE participates in the switch from medial to polar division and co-localizes with FtsZ at the polar septum. We show that after cytokinesis, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane on the forespore side. Importantly, the re-localization of SpoIIE to the engulfing septum requires SpoIIQ. These results indicate that SpoIIQ is required to anchor membrane proteins on both sides of the division septum. Moreover, our data suggest that forespore membrane proteins can localize to the septal membrane by diffusion-and-capture as has been described for membrane proteins in the mother cell. Finally, our results raise the intriguing possibility that SpoIIE has an uncharacterized function at a late stage of sporulation.

FtsK and SpoIIIE: the tale of the conserved tails

During Bacillus subtilis sporulation, the SpoIIIE DNA translocase moves a trapped chromosome across the sporulation septum into the forespore. The preferential assembly of SpoIIIE complexes in the mother cell provided the idea that SpoIIIE functioned as a DNA exporter, which ensured translocation orientation. In this issue of Molecular Microbiology, Becker and Pogliano reinvestigate the molecular mechanisms that orient the activity of SpoIIIE. Their findings indicate that SpoIIIE reads the polarity of DNA like its Escherichia coli homologue, FtsK.

An Unusual Response Regulator Influences Sporulation at Early and Late Stages inStreptomyces coelicolor

WhiI, a regulator required for efficient sporulation septation in the aerial mycelium of Streptomyces coelicolor, resembles response regulators of bacterial two-component systems but lacks some conserved features of typical phosphorylation pockets. Four amino acids of the abnormal "phosphorylation pocket" were changed by site-directed mutagenesis. Unlike whiI null mutations, these point mutations did not interfere with sporulation septation but had various effects on spore maturation. Transcriptome analysis was used to compare gene expression in the wild-type strain, a D27A mutant (pale gray spores), a D69E mutant (wild-type spores), and a null mutant (white aerial mycelium, no spores) (a new variant of PCR targeting was used to introduce the point mutations into the chromosomal copy of whiI). The results revealed 45 genes that were affected by the deletion of whiI. Many of these showed increased expression in the wild type at the time when aerial growth and development were taking place. About half of them showed reduced expression in the null mutant, and about half showed increased expression. Some, but not all, of these 45 genes were also affected by the D27A mutation, and a few were affected by the D69E mutation. The results were consistent with a model in which WhiI acts differently at sequential stages of development. Consideration of the functions of whiI-influenced genes provides some insights into the physiology of aerial hyphae. Mutation of seven whiI-influenced genes revealed that three of them play roles in spore maturation.

Role of an FtsK-Like Protein in Genetic Stability inStreptomyces coelicolorA3(2)

Streptomyces coelicolor A3(2) does not have a canonical cell division cycle during most of its complex life cycle, yet it contains a gene (ftsK(SC)) encoding a protein similar to FtsK, which couples the completion of cell division and chromosome segregation in unicellular bacteria such as Escherichia coli. Here, we show that various constructed ftsK(SC) mutants all grew apparently normally and sporulated but upon restreaking gave rise to many aberrant colonies and to high frequencies of chloramphenicol-sensitive mutants, a phenotype previously associated with large terminal deletions from the linear chromosome. Indeed, most of the aberrant colonies had lost large fragments near one or both chromosomal termini, as if chromosome ends had failed to reach their prespore destination before the closure of sporulation septa. A constructed FtsK(SC)-enhanced green fluorescent protein fusion protein was particularly abundant in aerial hyphae, forming distinctive complexes before localizing to each sporulation septum, suggesting a role for FtsK(SC) in chromosome segregation during sporulation. Use of a fluorescent reporter showed that when ftsK(SC) was deleted, several spore compartments in most spore chains failed to express the late-sporulation-specific sigma factor gene sigF, even though they contained chromosomal DNA. This suggested that sigF expression is autonomously activated in each spore compartment in response to completion of chromosome transfer, which would be a previously unknown checkpoint for late-sporulation-specific gene expression. These results provide new insight into the genetic instability prevalent among streptomycetes, including those used in the industrial production of antibiotics.

Bacterial Division: Another Way to Box in the Ring

Proper placement of the cell division site in some rod-shaped bacteria requires two different negative regulatory systems, nucleoid occlusion and the Min proteins. Caulobacter crescentus lacks these systems, but recent work has uncovered a novel regulator that achieves the same goals.

Developmental control of a parAB promoter leads to formation of sporulation-associated ParB complexes in Streptomyces coelicolor.

The Streptomyces coelicolor partitioning protein ParB binds to numerous parS sites in the oriC-proximal part of the linear chromosome. ParB binding results in the formation of large complexes, which behave differentially during the complex life cycle (D. Jakimowicz, B. Gust, J. Zakrzewska-Czerwinska, and K. F. Chater, J. Bacteriol. 187:3572-3580, 2005). Here we have analyzed the transcriptional regulation that underpins this developmentally specific behavior. Analysis of promoter mutations showed that the irregularly spaced complexes present in vegetative hyphae are dependent on the constitutive parABp(1) promoter, while sporulation-specific induction of the promoter parABp(2) is required for the assembly of arrays of ParB complexes in aerial hyphae and thus is necessary for efficient chromosome segregation. Expression from parABp(2) depended absolutely on two sporulation regulatory genes, whiA and whiB, and partially on two others, whiH and whiI, all four of which are needed for sporulation septation. Because of this pattern of dependence, we investigated the transcription of these four whi genes in whiA and whiB mutants, revealing significant regulatory interplay between whiA and whiB. A strain in which sporulation septation (but not vegetative septation) was blocked by mutation of a sporulation-specific promoter of ftsZ showed close to wild-type induction of parABp(2) and formed fairly regular ParB-enhanced green fluorescent protein foci in aerial hyphae, ruling out strong morphological coupling or checkpoint regulation between septation and DNA partitioning during sporulation. A model for developmental regulation of parABp(2) expression is presented.

Evidence that the SpoIIIE DNA translocase participates in membrane fusion during cytokinesis and engulfment

During Bacillus subtilis sporulation, SpoIIIE is required for translocation of the trapped forespore chromosome across the sporulation septum, for compartmentalization of cell-specific gene expression, and for membrane fusion after engulfment. We isolated mutations within the SpoIIIE membrane domain that block localization and function. One mutant protein initially localizes normally and completes DNA translocation, but shows reduced membrane fusion after engulfment. Fluorescence recovery after photobleaching experiments demonstrate that in this mutant the sporulation septum remains open, allowing cytoplasmic contents to diffuse between daughter cells, suggesting that it blocks membrane fusion after cytokinesis as well as after engulfment. We propose that SpoIIIE catalyses these topologically opposite fusion events by assembling or disassembling a proteinaceous fusion pore. Mutants defective in SpoIIIE assembly also demonstrate that the ability of SpoIIIE to provide a diffusion barrier is directly proportional to its ability to assemble a focus at the septal midpoint during DNA translocation. Thus, SpoIIIE mediates compartmentalization by two distinct mechanisms: the SpoIIIE focus first provides a temporary diffusion barrier during DNA translocation, and then mediates the completion of membrane fusion after division to provide a permanent diffusion barrier. SpoIIIE-like proteins might therefore serve to couple the final step in cytokinesis, septal membrane fusion, to the completion of chromosome segregation.

Control of the Expression and Compartmentalization of σ G Activity during Sporulation of Bacillus subtilis by Regulators of σ F and σ E

During formation of spores by Bacillus subtilis the RNA polymerase factor sigma(G) ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of sigma(G) ordinarily requires prior activity of sigma(F) in the prespore and sigma(E) in the mother cell. Here we report that in spoIIA mutants lacking both sigma(F) and the anti-sigma factor SpoIIAB and in which sigma(E) is not active, sigma(G) nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of sigma(G) activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for sigma(G), from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to sigma(G). When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, sigma(G) became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature sigma(G) activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of sigma(G) activity, and pro-sigma(E) (but not sigma(E)) can counteract this effect of SpoIIGA. We find that in strains lacking sigma(F) and SpoIIAB and engineered to produce active sigma(E) in the mother cell without the need for SpoIIGA, sigma(G) also becomes active in the mother cell.

The interplay of glycogen metabolism and differentiation provides an insight into the developmental biology of Streptomyces coelicolor

Mycelial colonies of the developmentally complex actinomycete Streptomyces coelicolor growing on solid medium contain glycogen in two distinct locations. Phase I deposits are found in a substrate mycelium region bordering the developing aerial mycelium. Their production involves GlgBI, one of two glycogen branching enzyme isoforms. Phase II deposits occur in the upper regions of aerial hyphae, in long tip cells that are dividing, or have just divided, into unigenomic prespore compartments. Their formation involves a second branching enzyme isoform, GlgBII. To find out if the gene for the second isoform, glgBII, is regulated by any of the well-studied whiA, B, G, H or I genes needed for sporulation septation, glgBI or glgBII was disrupted in a set of whi mutants, and the glycogen phenotypes examined by transmission electron microscopy. In the whiG mutants, deposits were found throughout the aerial mycelium and the adjacent region of the substrate mycelium, but the morphology of all the deposits, i.e. whether they were in the form of granules of branched glycogen or large blobs of unbranched glycan, depended solely on GlgBI. In contrast, the whiA, B, H and I mutations had no obvious effect on the pattern of glycogen deposition, or on the spatial specificity of the branching enzyme isoforms (though phase II glycogen deposits were reduced in size and abundance in the whiA and B mutants, and increased in the whiH mutant). These results indicate that glgBII is regulated (directly or indirectly) by whiG, and not by any of the other whi genes tested, and that the aerial hyphae of a whiG mutant are atypical in being physiologically similar to the substrate hyphae from which they emerge. A new role for aerial hyphae is proposed.

Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.

Zipper-like interaction between proteins in adjacent daughter cells mediates protein localization.

Protein localization is crucial for cellular morphogenesis and intracellular signal transduction cascades. Here we describe an interaction between two membrane proteins expressed in different cells of the Bacillus subtilis sporangium, the mother cell protein SpoIIIAH and the forespore protein SpoIIQ. We used affinity chromatography, coimmunoprecipitation, and the yeast two-hybrid system to demonstrate that the extracellular domains of these proteins interact, tethering SpoIIIAH to the sporulation septum, and directing its assembly with SpoIIQ into helical arcs and foci around the forespore. We also demonstrate that this interaction can direct proteins made in the same cell to active division sites, as when SpoIIQ is made in the mother cell, it localizes to nascent septa in a SpoIIIAH-dependent manner. Both SpoIIIAH and SpoIIQ are necessary for activation of the second forespore-specific transcription factor (sigma(G)) after engulfment, and we propose that the SpoIIIAH-SpoIIQ complex contributes to a morphological checkpoint coupling sigma(G) activation to engulfment. In keeping with this hypothesis, SpoIIIAH localization depends on the first step of engulfment, septal thinning. The SpoIIQ-SpoIIIAH complex reaches from the mother cell cytoplasm to the forespore cytoplasm and is ideally positioned to govern the activity of engulfment-dependent transcription factors.

PBP1 is a component of the Bacillus subtilis cell division machinery.

Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell division. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell division proteins. This study proves that PBP1 forms part of the B. subtilis cell division machinery.

Septal localization of forespore membrane proteins during engulfment in Bacillus subtilis.

In Bacillus subtilis, many membrane proteins localize to the sporulation septum, where they play key roles in spore morphogenesis and cell-specific gene expression, but the mechanism for septal targeting is not well understood. SpoIIQ, a forespore-expressed protein, is involved in engulfment and forespore-specific gene expression. We find that SpoIIQ dynamically localizes to the sporulation septum, tracks the engulfing mother cell membrane, assembles into helical arcs around the forespore and is finally degraded. Retention of SpoIIQ in the septum requires one or more mother cell-expressed proteins. We also observed that any forespore-expressed membrane protein initially localizes to the septum and later spreads throughout the forespore membrane, suggesting that membrane protein insertion occurs at the forespore septal region. This possibility provides an attractive mechanism for how activation of mother cell-specific gene expression is restricted to adjacent sister cells, since direct insertion of the signaling protein SpoIIR into the septum would spatially restrict its activity. In keeping with this hypothesis, we find that SpoIIR localizes to the septum and is transiently expressed.