Sporulation Septum Research Articles

The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis.

SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

Structural basis of dual activation of cell division by the actinobacterial transcription factors WhiA and WhiB

Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as Mycobacterium tuberculosis. The WhiA/B regulons and binding sites have been elucidated in Streptomyces venezuelae (Sven), where they coordinate to activate sporulation septation. However, how these factors cooperate at the molecular level is not understood. Here we present cryoelectron microscopy structures of Sven transcriptional regulatory complexes comprising RNA polymerase (RNAP) σA-holoenzyme and WhiA and WhiB, in complex with the WhiA/B target promoter sepX. These structures reveal that WhiB binds to domain 4 of σA (σA4) of the σA-holoenzyme, bridging an interaction with WhiA while making non-specific contacts with the DNA upstream of the -35 core promoter element. The N-terminal homing endonuclease-like domain of WhiA interacts with WhiB, while the WhiA C-terminal domain (WhiA-CTD) makes base-specific contacts with the conserved WhiA GACAC motif. Notably, the structure of the WhiA-CTD and its interactions with the WhiA motif are strikingly similar to those observed between σA4 housekeeping σ-factors and the -35 promoter element, suggesting an evolutionary relationship. Structure-guided mutagenesis designed to disrupt these protein-DNA interactions reduces or abolishes developmental cell division in Sven, confirming their significance. Finally, we compare the architecture of the WhiA/B σA-holoenzyme promoter complex with the unrelated but model CAP Class I and Class II complexes, showing that WhiA/WhiB represent a new mechanism in bacterial transcriptional activation.

DNA damage-induced block of sporulation in Streptomyces venezuelae involves downregulation of ssgB.

DNA damage often causes an arrest of the cell cycle that provides time for genome integrity to be restored. In bacteria, the classical SOS DNA damage response leads to an inhibition of cell division resulting in temporarily filamentous growth. This raises the question as to whether such a response mechanism might similarly function in naturally filamentous bacteria such as Streptomyces. Streptomyces exhibit two functionally distinct forms of cell division: cross-wall formation in vegetative hyphae and sporulation septation in aerial hyphae. Here, we show that the genotoxic agent mitomycin C confers a block in sporulation septation in Streptomyces venezuelae in a mechanism that involves, at least in part, the downregulation of ssgB. Notably, this DNA damage response does not appear to block cross-wall formation and may be independent of canonical SOS and developmental regulators. We also show that the mitomycin C-induced block in sporulation can be partially bypassed by the constitutive expression of ssgB, though this appears to be largely limited to mitomycin C treatment and the resultant spore-like cells have reduced viability.

Preprint Highlight: Hyphal compartmentalization and sporulation in Streptomyces require the conserved cell division protein SepX.

Most bacteria undergo binary division. Streptomyces, filamentous soil bacteria with a multicellular life cycle, have two functionally distinct modes of cell division: vegetative cross-wall formation and sporulation septation. Although sporulation septation is an important survival strategy, the physiological significance of cross-wall formation remains largely unknown. Using genetics and quantitative imaging, the authors identified a previously uncharacterized protein, SepX, essential for cross-wall formation and for efficient sporulation septation. Deletion of sepX caused severe growth defects, demonstrating the necessity for vegetative division. These results provide a genetic basis for future research into the regulation of bacterial multicellular development, including how cross-walls form, how they relate to fitness, and how the two distinct cell types are coordinated.

Influence of core divisome proteins on cell division in Streptomyces venezuelae ATCC 10712.

The sporulating, filamentous soil bacterium Streptomyces venezuelae ATCC 10712 differentiates under submerged and surface growth conditions. In order to lay a solid foundation for the study of development-associated division for this organism, a congenic set of mutants was isolated, individually deleted for a gene encoding either a cytoplasmic (i.e. ftsZ) or core inner membrane (i.e. divIC, ftsL, ftsI, ftsQ, ftsW) component of the divisome. While ftsZ mutants are completely blocked for division, single mutants in the other core divisome genes resulted in partial, yet similar, blocks in sporulation septum formation. Double and triple mutants for core divisome membrane components displayed phenotypes that were similar to those of the single mutants, demonstrating that the phenotypes were not synergistic. Division in this organism is still partially functional without multiple core divisome proteins, suggesting that perhaps other unknown lineage-specific proteins perform redundant functions. In addition, by isolating an ftsZ2p mutant with an altered -10 region, the conserved developmentally controlled promoter was also shown to be required for sporulation-associated division. Finally, microscopic observation of FtsZ-YFP dynamics in the different mutant backgrounds led to the conclusion that the initial assembly of regular Z rings does not per se require the tested divisome membrane proteins, but the stability of Z rings is dependent on the divisome membrane components tested. The observation is consistent with the interpretation that Z ring instability likely results from and further contributes to the observed defects in sporulation septation in mutants lacking core divisome proteins.

Linking the Peptidoglycan Synthesis Protein Complex with Asymmetric Cell Division during Bacillus subtilis Sporulation.

Peptidoglycan is generally considered one of the main determinants of cell shape in bacteria. In rod-shaped bacteria, cell elongation requires peptidoglycan synthesis to lengthen the cell wall. In addition, peptidoglycan is synthesized at the division septum during cell division. Sporulation of Bacillus subtilis begins with an asymmetric cell division. Formation of the sporulation septum requires almost the same set of proteins as the vegetative septum; however, these two septa are significantly different. In addition to their differences in localization, the sporulation septum is thinner and it contains SpoIIE, a crucial sporulation specific protein. Here we show that peptidoglycan biosynthesis is linked to the cell division machinery during sporulation septum formation. We detected a direct interaction between SpoIIE and GpsB and found that both proteins co-localize during the early stages of asymmetric septum formation. We propose that SpoIIE is part of a multi-protein complex which includes GpsB, other division proteins and peptidoglycan synthesis proteins, and could provide a link between the peptidoglycan synthesis machinery and the complex morphological changes required for forespore formation during B. subtilis sporulation.

Megalaria yunnanensis sp. nov. from Yunnan, China

Megalaria yunnanensis from southern China is described as a new species. The lichen is diagnosed by its bi-layered exciple, presence of atranorin, zeorin, and fumarprotocetraric acid in the thallus, ascospore size (20–25 × 5–7.5 μm), and the lack of indentations from the cell lumina into the center of the spore septum.

Asymmetric cell division during Bacillus subtilis sporulation.

Bacillus subtilis is a rod-shaped bacterium which divides precisely at mid-cell during vegetative growth. Unlike Escherichia coli, another model organism used for studying cell division, B. subtilis can also divide asymmetrically during sporulation, the simplest cell differentiation process. The asymmetrically positioned sporulation septum serves as a morphological foundation for establishing differential gene expression in the smaller forespore and larger mother cell. Both vegetative and sporulation septation events are fine-tuned with cell cycle, and placement of both septa are highly precise. We understand in some detail how this is achieved during vegetative growth but have limited information about how the asymmetric septation site is determined during sporulation.

The actinobacterial WhiB-like (Wbl) family of transcription factors.

SummaryThe WhiB‐like (Wbl) family of proteins are exclusively found in Actinobacteria. Wbls have been shown to play key roles in virulence and antibiotic resistance in Mycobacteria and Corynebacteria, reflecting their importance during infection by the human pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Corynebacterium diphtheriae. In the antibiotic‐producing Streptomyces, several Wbls have important roles in the regulation of morphological differentiation, including WhiB, a protein that controls the initiation of sporulation septation and the founding member of the Wbl family. In recent years, genome sequencing has revealed the prevalence of Wbl paralogues in species throughout the Actinobacteria. Wbl proteins are small (generally ~80–140 residues) and each contains four invariant cysteine residues that bind an O2‐ and NO‐sensitive [4Fe–4S] cluster, raising the question as to how they can maintain distinct cellular functions within a given species. Despite their discovery over 25 years ago, the Wbl protein family has largely remained enigmatic. Here I summarise recent research in Mycobacteria, Corynebacteria and Streptomyces that sheds light on the biochemical function of Wbls as transcription factors and as potential sensors of O2 and NO. I suggest that Wbl evolution has created diversity in protein–protein interactions, [4Fe–4S] cluster‐sensitivity and the ability to bind DNA.

The positioning of the asymmetric septum during sporulation in Bacillus subtilis.

Probably one of the most controversial questions about the cell division of Bacillus subtilis, a rod-shaped bacterium, concerns the mechanism that ensures correct division septum placement–at mid-cell during vegetative growth but closer to one end during sporulation. In general, bacteria multiply by binary fission, in which the division septum forms almost exactly at the cell centre. How the division machinery achieves such accuracy is a question of continuing interest. We understand in some detail how this is achieved during vegetative growth in Escherichia coli and B. subtilis, where two main negative regulators, nucleoid occlusion and the Min system, help to determine the division site, but we still do not know exactly how the asymmetric septation site is determined during sporulation in B. subtilis. Clearly, the inhibitory effects of the nucleoid occlusion and Min system on polar division have to be overcome. We evaluated the positioning of the asymmetric septum and its accuracy by statistical analysis of the site of septation. We also clarified the role of SpoIIE, RefZ and MinCD on the accuracy of this process. We determined that the sporulation septum forms approximately 1/6 of a cell length from one of the cell poles with high precision and that SpoIIE, RefZ and MinCD have a crucial role in precisely localizing the sporulation septum. Our results strongly support the idea that asymmetric septum formation is a very precise and highly controlled process regulated by a still unknown mechanism.

GerM is required to assemble the basal platform of the SpoIIIA-SpoIIQ transenvelope complex during sporulation in Bacillus subtilis.

Sporulating Bacillus subtilis cells assemble a multimeric membrane complex connecting the mother cell and developing spore that is required to maintain forespore differentiation. An early step in the assembly of this transenvelope complex (called the A-Q complex) is an interaction between the extracellular domains of the forespore membrane protein SpoIIQ and the mother cell membrane protein SpoIIIAH. This interaction provides a platform onto which the remaining components of the complex assemble and also functions as an anchor for cell-cell signalling and morphogenetic proteins involved in spore development. SpoIIQ is required to recruit SpoIIIAH to the sporulation septum on the mother cell side; however, the mechanism by which SpoIIQ specifically localizes to the septal membranes on the forespore side has remained enigmatic. Here, we identify GerM, a lipoprotein previously implicated in spore germination, as the missing factor required for SpoIIQ localization. Our data indicate that GerM and SpoIIIAH, derived from the mother cell, and SpoIIQ, from the forespore, have reciprocal localization dependencies suggesting they constitute a tripartite platform for the assembly of the A-Q complex and a hub for the localization of mother cell and forespore proteins.

Morphogenic Protein RodZ Interacts with Sporulation Specific SpoIIE in Bacillus subtilis.

The first landmark in sporulation of Bacillus subtilis is the formation of an asymmetric septum followed by selective activation of the transcription factor σF in the resulting smaller cell. How the morphological transformations that occur during sporulation are coupled to cell-specific activation of transcription is largely unknown. The membrane protein SpoIIE is a constituent of the asymmetric sporulation septum and is a crucial determinant of σF activation. Here we report that the morphogenic protein, RodZ, which is essential for cell shape determination, is additionally required for asymmetric septum formation and sporulation. In cells depleted of RodZ, formation of asymmetric septa is disturbed and σF activation is perturbed. During sporulation, we found that SpoIIE recruits RodZ to the asymmetric septum. Moreover, we detected a direct interaction between SpoIIE and RodZ in vitro and in vivo, indicating that SpoIIE-RodZ may form a complex to coordinate asymmetric septum formation and σF activation. We propose that RodZ could provide a link between the cell shape machinery and the coordinated morphological and developmental transitions required to form a resistant spore.

Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum.

SpoIIIE is a membrane-anchored DNA translocase that localizes to the septal midpoint to mediate chromosome translocation and membrane fission during Bacillus subtilis sporulation. Here we use cell-specific protein degradation and quantitative photoactivated localization microscopy in strains with a thick sporulation septum to investigate the architecture and function of the SpoIIIE DNA translocation complex in vivo. We were able to visualize SpoIIIE complexes with approximately equal numbers of molecules in the mother cell and the forespore. Cell-specific protein degradation showed that only the mother cell complex is required to translocate DNA into the forespore, whereas degradation in either cell reverses membrane fission. Our data suggest that SpoIIIE assembles a coaxially paired channel for each chromosome arm comprised of one hexamer in each cell to maintain membrane fission during DNA translocation. We show that SpoIIIE can operate, in principle, as a bi-directional motor that exports DNA.

Synthesis of the spore envelope in the developmental life cycle of Streptomyces coelicolor

Members of the family of Streptomycetaceae, the main producer of antibiotics and other secondary metabolites, are Gram-positive multi-cellular soil bacteria with a complex life cycle. By apical tip extension Streptomyces coelicolor forms a multiply branching vegetative mycelium penetrating the substrate. Upon nutrient limitation, a hydrophobic aerial mycelium is erected, which eventually develops into a regular chain of spores that are able to survive detrimental environmental conditions.Morphological differentiation involves a switch in the peptidoglycan synthesizing machinery. Whereas apical tip extension is directed by the so-called polarisome, sporulation septation and synthesis of the thickened spore wall involves a multi-protein complex, which resembles the elongasome of rod-shaped bacteria. The Streptomyces spore wall synthesizing complex (SSSC) does not only direct synthesis of the peptidoglycan layer but is also involved in the incorporation of anionic spore wall glycopolymers, which contribute to the resistance of spores. The SSSC also contains eukaryotic type serine/threonine kinases which might control its activity by protein-phosphorylation.

In Vivo Assembly and Arrangement of the DNA Translocase SpoIIIE During Chromosome Segregation and Membrane Fission in B. Subtilis

SpoIIIE, a DNA translocase motor protein of the SpoIIIE/FtsK family, is responsible for chromosome segregation during bacterial cell division. In sporulating Bacillus subtilis, SpoIIIE aggregates as a focus at the mid-sporulating septum to export a circular chromosome from the mother into the daughter cell. The architecture of SpoIIIE at the focus and its mechanism of operation for the DNA translocation and the membrane fission are still controversial. Here, we directly visualize the SpoIIIE architecture at the macromolecular scale and developed a novel genetic tool to determine its operational mode during DNA translocation and membrane fission in vivo. Using a combination of these genetic tools together with quantitative single molecule imaging and fluorescence recovery after photobleaching, we demonstrate that SpoIIIE assembles as two complexes (∼40nm size), one in the mother and one in the daughter cell. In these complexes there are enough molecules to form either two or four hexamers. Moreover, we demonstrate that the mother cell-SpoIIIE is able to translocate DNA and accomplish sporulation, even in absence of daughter cell-SpoIIIE. On the other hand, SpoIIIE from both mother and daughter cells mediate the septal membrane fission. Altogether, our results indicate that two hexamers of SpoIIIE assembles into a DNA crossing channel across the sporulation septum for chromosome segregation and septal membrane fission.

A sporulation‐specific, sigF‐dependent protein, SspA, affects septum positioning in Streptomyces coelicolor

The RNA polymerase sigma factor SigF controls late development during sporulation in the filamentous bacterium Streptomyces coelicolor. The only known SigF-dependent gene identified so far, SCO5321, is found in the biosynthetic cluster encoding spore pigment synthesis. Here we identify the first direct target for SigF, the gene sspA, encoding a sporulation-specific protein. Bioinformatic analysis suggests that SspA is a secreted lipoprotein with two PepSY signature domains. The sspA deletion mutant exhibits irregular sporulation septation and altered spore shape, suggesting that SspA plays a role in septum formation and spore maturation. The fluorescent translational fusion protein SspA–mCherry localized first to septum sites, then subsequently around the surface of the spores. Both SspA protein and sspA transcription are absent from the sigF null mutant. Moreover, in vitro transcription assay confirmed that RNA polymerase holoenzyme containing SigF is sufficient for initiation of transcription from a single sspA promoter. In addition, in vivo and in vitro experiments showed that sspA is a direct target of BldD, which functions to repress sporulation genes, including whiG, ftsZ and ssgB, during vegetative growth, co-ordinating their expression during sporulation septation.

Genes required for aerial growth, cell division, and chromosome segregation are targets of WhiA before sporulation in Streptomyces venezuelae.

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacterium Streptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA in Streptomyces development and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus, Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed that whiA is required for the initiation of sporulation septation and chromosome segregation in S. venezuelae, and several genes encoding key proteins of the Streptomyces cell division machinery, such as ftsZ, ftsW, and ftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiG and the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, and filP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.

Peptidoglycan hydrolysis is required for assembly and activity of the transenvelope secretion complex during sporulation in Bacillus subtilis

Sporulating Bacillus subtilis cells assemble a transenvelope secretion complex that connects the mother cell and developing spore. The forespore protein SpoIIQ and the mother-cell protein SpoIIIAH interact across the double membrane septum and are thought to assemble into a channel that serves as the basement layer of this specialized secretion system. SpoIIQ is absolutely required to recruit SpoIIIAH to the sporulation septum on the mother-cell side, however the mechanism by which SpoIIQ is localized has been unclear. Here, we show that SpoIIQ localization requires its partner protein SpoIIIAH and degradation of the septal peptidoglycan (PG) by the two cell wall hydrolases SpoIID and SpoIIP. Our data suggest that PG degradation enables a second mother-cell-produced protein to interact with SpoIIQ. Cells in which both mother-cell anchoring mechanisms have been disabled have a synergistic sporulation defect suggesting that both localization factors function in the secretion complex. Finally, we show that septal PG degradation is critical for the assembly of an active complex. Altogether, these results suggest that the specialized secretion system that links the mother cell and forespore has a complexity approaching those found in Gram-negative bacteria and reveal that the sporulating cell must overcome similar challenges in assembling a transenvelope complex.

Non-sporulating ftsZ mutants in Streptomyces coelicolor reveal amino acid residues critical for FtsZ polymerization dynamics

During sporulation of Streptomyces coelicolor, the cytokinetic protein FtsZ is assembled into dozens of regularly spaced Z rings, which orchestrate the division of aerial hyphae into spores. We have previously found that a missense allele of ftsZ, ftsZ17(Spo), primarily affects sporulation septation rather than formation of cross-walls in vegetative mycelium. To clarify what aspect of FtsZ function is compromised in such non-sporulating mutants, we here use a genetic strategy to identify new ftsZ(Spo) alleles and describe how some of the mutations affect the biochemical properties of FtsZ. We have established a system for purification of recombinant untagged S. coelicolor FtsZ, and shown that it assembles dynamically into single protofilaments, displays a critical concentration indicative of cooperative assembly and has a rate of GTP hydrolysis that is substantially higher than that of the closely related Mycobacterium tuberculosis FtsZ. Of the nine isolated ftsZ(Spo) mutations, four affect the interface between the two main subdomains of FtsZ that is implicated in the assembly-induced conformational changes thought to mediate the GTP/GDP-driven cooperative assembly of FtsZ. We find that all these four mutations affect the polymerization behaviour of FtsZ in vitro. In addition, at least one ftsZ(Spo) mutation at the longitudinal contact surface between subunits in protofilaments strongly affects formation of polymers in vitro. We conclude that the assembly of Z rings during sporulation of S. coelicolor is highly sensitive to disturbances of FtsZ polymerization and therefore constitutes an excellent system for analysis of the elusive properties of FtsZ that mediate its characteristic polymerization dynamics.

Molecular and cytological analysis of the expression ofStreptomycessporulation regulatory genewhiH

The whiH gene is required for the orderly sporulation septation that divides aerial hyphae into spores in Streptomyces coelicolor. Here, we use a whiHp-mCherry transcriptional reporter construct to show that whiHp is active specifically in aerial hyphae, fluorescence being dependent on sporulation sigma factor WhiG. The results show that the promoter is active before the septation event that separates the subapical compartment from the tip compartment destined to become a spore chain. We conclude that WhiG-directed RNA polymerase activity, which is required for whiH transcription, must precede this septation event and is not restricted to apical sporogenic compartment of the aerial hyphae. Further, it is demonstrated that WhiH, a predicted member of the GntR family of transcription factors, is able to bind specifically to a sequence in its own promoter, strongly suggesting that it acts as an autoregulatory transcription factor. Finally, we show by site-directed mutagenesis and a genetic complementation test that whiH is translated from a start codon overlapping with the previously identified transcription start point, implying leaderless transcription.