Sporulation-specific Cell Division Research Articles

Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

The SepF-like proteins SflA and SflB prevent ectopic localization of FtsZ and DivIVA during sporulation of Streptomyces coelicolor

Bacterial cytokinesis starts with the polymerization of the tubulin-like FtsZ, which forms the cell division scaffold. SepF aligns FtsZ polymers and also acts as a membrane anchor for the Z-ring. While in most bacteria cell division takes place at midcell, during sporulation of Streptomyces many septa are laid down almost simultaneously in multinucleoid aerial hyphae. The genomes of streptomycetes encode two additional SepF paralogs, SflA and SflB, which can interact with SepF. Here we show that the sporogenic aerial hyphae of sflA and sflB mutants of Streptomyces coelicolor frequently branch, a phenomenon never seen in the wild-type strain. The branching coincided with ectopic localization of DivIVA along the lateral wall of sporulating aerial hyphae. Constitutive expression of SflA and SflB largely inhibited hyphal growth, further correlating SflAB activity to that of DivIVA. SflAB localized in foci prior to and after the time of sporulation-specific cell division, while SepF co-localized with active septum synthesis. Foci of FtsZ and DivIVA frequently persisted between adjacent spores in spore chains of sflA and sflB mutants, at sites occupied by SflAB in wild-type cells. Taken together, our data show that SflA and SflB play an important role in the control of growth and cell division during Streptomyces development.

Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

SepG coordinates sporulation-specific cell division and nucleoid organization in Streptomyces coelicolor.

Bacterial cell division is a highly complex process that requires tight coordination between septum formation and chromosome replication and segregation. In bacteria that divide by binary fission a single septum is formed at mid-cell, a process that is coordinated by the conserved cell division scaffold protein FtsZ. In contrast, during sporulation-specific cell division in streptomycetes, up to a hundred rings of FtsZ (Z rings) are produced almost simultaneously, dividing the multinucleoid aerial hyphae into long chains of unigenomic spores. This involves the active recruitment of FtsZ by the SsgB protein, and at the same time requires sophisticated systems to regulate chromosome dynamics. Here, we show that SepG is required for the onset of sporulation and acts by ensuring that SsgB is localized to future septum sites. Förster resonance energy transfer imaging suggests direct interaction between SepG and SsgB. The beta-lactamase reporter system showed that SepG is a transmembrane protein with its central domain oriented towards the cytoplasm. Without SepG, SsgB fails to localize properly, consistent with a crucial role for SepG in the membrane localization of the SsgB–FtsZ complex. While SsgB remains associated with FtsZ, SepG re-localizes to the (pre)spore periphery. Expanded doughnut-shaped nucleoids are formed in sepG null mutants, suggesting that SepG is required for nucleoid compaction. Taken together, our work shows that SepG, encoded by one of the last genes in the conserved dcw cluster of cell division and cell-wall-related genes in Gram-positive bacteria whose function was still largely unresolved, coordinates septum synthesis and chromosome organization in Streptomyces.

Transcriptional analysis of the cell division-related ssg genes in Streptomyces coelicolor reveals direct control of ssgR by AtrA

SsgA-like proteins are a family of actinomycete-specific regulatory proteins that control cell division and spore maturation in streptomycetes. SsgA and SsgB together activate sporulation-specific cell division by controlling the localization of FtsZ. Here we report the identification of novel regulators that control the transcription of the ssgA-like genes. Transcriptional regulators controlling ssg gene expression were identified using a DNA-affinity capture assay. Supporting transcriptional and DNA binding studies showed that the ssgA activator gene ssgR is controlled by the TetR-family regulator AtrA, while the γ-butyrolactone-responsive AdpA (SCO2792) and SlbR (SCO0608) and the metabolic regulator Rok7B7 (SCO6008) were identified as candidate regulators for the cell division genes ssgA, ssgB and ssgG. Transcription of the cell division gene ssgB depended on the sporulation genes whiA and whiH, while ssgR, ssgA and ssgD were transcribed independently of the whi genes. Our work sheds new light on the mechanisms by which sporulation-specific cell division is controlled in Streptomyces.Electronic supplementary materialThe online version of this article (doi:10.1007/s10482-015-0479-2) contains supplementary material, which is available to authorized users.

A novel taxonomic marker that discriminates between morphologically complex actinomycetes

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.

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

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10482-011-9678-7) contains supplementary material, which is available to authorized users.

Structural and Functional Characterizations of SsgB, a Conserved Activator of Developmental Cell Division in Morphologically Complex Actinomycetes

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 A resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic "whirly" single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.

The SsgA-like proteins in actinomycetes: small proteins up to a big task.

Several unique protein families have been identified that play a role in the control of developmental cell division in streptomycetes. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes, affecting cell wall-related events such as septum localization and synthesis, thickening of the spore wall and autolytic spore separation. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been succesfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. Recent observations suggest that SsgB most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. Here we review 10 years of research on the SsgA-like proteins in actinomycetes and discuss the most interesting regulatory, functional, phylogenetic and applied aspects of this relatively unknown protein family.Electronic supplementary materialThe online version of this article (doi:10.1007/s10482-008-9225-3) contains supplementary material, which is available to authorized users.

Loss of the controlled localization of growth stage‐specific cell‐wall synthesis pleiotropically affects developmental gene expression in an ssgA mutant of Streptomyces coelicolor

Members of the family of SsgA-like proteins (SALPs) are found exclusively in sporulating actinomycetes, and SsgA itself activates sporulation-specific cell division. We previously showed that SALPs play a chaperonin-like role in supporting the function of enzymes involved in peptidoglycan maintenance (PBPs and autolysins). Here we show that SsgA localizes dynamically during development, and most likely marks the sites where changes in local cell-wall morphogenesis are required, in particular septum formation and germination. In sporogenic aerial hyphae, SsgA initially localizes as strong foci to the growing tips, followed by distribution as closely spaced foci in a pattern similar to an early stage of FtsZ assembly. Spore septa formed in these hyphae colocalize with single SsgA-GFP foci, and when the maturing spores are separated, these foci are distributed symmetrically, resulting in two foci per mature spore. Evidence is provided that SsgA also controls the correct localization of germination sites. Transcriptome analysis revealed that expression of around 300 genes was significantly altered in mutants in ssgA and its regulatory gene ssgR. The list includes surprisingly many known developmental genes, most of which were upregulated, highlighting SsgA as a key player in the control of Streptomyces development.

SsgA‐like proteins determine the fate of peptidoglycan during sporulation of Streptomyces coelicolor

During developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome condensation and segregation. Several unique protein families are involved in the control of this process in actinomycetes, including that of the SsgA-like proteins (SALPs). Mutants for each of the individual SALP genes were obtained, and high-resolution and fluorescence imaging revealed that each plays an important and highly specific role in the control of the sporulation process, and their function relates to the build-up and degradation of septal and spore-wall peptidoglycan. While SsgA and SsgB are essential for sporulation-specific cell division in Streptomyces coelicolor, SsgC-G are responsible for correct DNA segregation/condensation (SsgC), spore wall synthesis (SsgD), autolytic spore separation (SsgE, SsgF) or exact septum localization (SsgG). Our experiments paint a picture of a novel protein family that acts through timing and localization of the activity of penicillin-binding proteins and autolysins, thus controlling important steps during the initiation and the completion of sporulation in actinomycetes.

Transcription of the sporulation gene ssgA is activated by the IclR-type regulator SsgR in a whi-independent manner in Streptomyces coelicolor A3(2).

SsgA plays an important role in the control of sporulation-specific cell division and morphogenesis of streptomycetes, and ssgA null mutants have a rare conditionally non-sporulating phenotype. In this paper we show that transcription of ssgA and of the upstream-located ssgR, an iclR-type regulatory gene, is developmentally regulated in Streptomyces coelicolor and activated towards the onset of sporulation. A constructed ssgR null mutant was phenotypically very similar to the ssgA mutant. The absence of ssgA transcription in this mutant is probably the sole cause of its sporulation deficiency, as wild-type levels of sporulation could be restored by the SsgR-independent expression of ssgA from the ermE promoter. Binding of a truncated version of SsgR to the ssgA promoter region showed that ssgA transcription is directly activated by SsgR; such a dependence of ssgA on SsgR in S. coelicolor is in clear contrast to the situation in S. griseus, where ssgA transcription is activated by A-factor, and its control by the SsgR orthologue, SsfR, is far less important. Our failure to complement the ssgR mutant with S. griseus ssfR suggests functional differences between the genes. These observations may explain some of the major differences in developmental control between the phylogenetically divergent species S. coelicolor and S. griseus, highlighted in a recent microreview (Chater and Horinouchi (2003) Mol Microbiol 48: 9-15). Surprisingly, transcription of ssgA and ssgR is not dependent on the early whi genes (whiA, whiB, whiG, whiH, whiI and whiJ ).

The Streptomyces coelicolor ssgB gene is required for early stages of sporulation

ssgB was identified as a novel early sporulation gene in Streptomyces coelicolor. An ssgB deletion mutant failed to sporulate, over-produced actinorhodin, and its colonies were significantly larger than those of the parental strain, suggesting an important role for the ssgB gene product in the process of growth cessation prior to sporulation-specific cell division. This places ssgB temporally before the paralogous sporulation gene ssgA. Analysis of ssgB mutant hyphae by electron microscopy and by confocal fluorescence microscopy showed that it was defective in the initiation of sporulation, as no sporulation septa could be identified, and DNA segregation had not yet been initiated in the mutant.

The stress-response sigma factor sigma(H) controls the expression of ssgB, a homologue of the sporulation-specific cell division gene ssgA, in Streptomyces coelicolor A3(2).

By using a previously established method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, we identified a promoter in Streptomyces coelicolor A3(2) that is recognized by a heterologous RNA polymerase containing the late sporulation-specific sigma factor sigma(F). The promoter directed the expression of a gene named ssgB, which is related to the sporulation-specific cell division gene ssgA. These genes, together with three others, constitute a new family of paralogous genes specific for Streptomyces. S1-nuclease mapping using RNA prepared from an Escherichia coli strain that expresses ssgB under the control of sigma(F), and from S. coelicolor A3(2) at various developmental stages, identified identical transcription start points in both strains, corresponding to the promoter ssgBp. The promoter is developmentally regulated in S. coelicolor: it is induced at the time of aerial mycelium formation and is most active during sporulation. However, the level of the ssgB transcript was unaffected in a sigF mutant of S. coelicolor A3(2). Interestingly, the level of the transcript was substantially reduced in an S. coelicolor strain that was mutant for the sigH gene, which encodes a stress-response sigma factor (sigma(H)) that is essential for sporulation in S. coelicolor A3(2). This dependence of ssgB upon sigH was confirmed by an in vitro transcription assay, in which sigma(H), in the presence of the S. coelicolor core RNA polymerase, was able to recognize ssgBp. These results suggest that the S. coelicolor ssgB gene is under the control of the stress-response sigma(H). Transcription of ssgB was investigated in S. coelicolor A3(2) mutants with lesions in each of six known early whi genes required for sporulation septation. Expression of ssgB was deregulated in three of the mutants ( whiA, whiI, and whiJ). Based on these data, it is proposed that the ssgB gene product plays a role in the developmental process in S. coelicolor A3(2).