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.     

Characterization of the sporulation control protein SsgA by use of an efficient method to create and screen random mutant libraries in streptomycetes

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function.     

Characterization of the Sporulation Control Protein SsgA by Use of an Efficient Method To Create and Screen Random Mutant Libraries in Streptomycetes

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function.     

A phosphoglucose isomerase mutant in Aspergillus nidulans is defective in hyphal polarity and conidiation

Upon germination Aspergillus nidulans swoM1 exhibits abnormal development by extending a primary germ tube that quickly reverts to isotropic growth and results in an enlarged, swollen apex with pronounced wall thickenings. Apical lysis occurs in 38% of the germlings. A point mutation in the AN6037.3 gene encoding the only phosphoglucose isomerase in A. nidulans is responsible for the defect. Loss of polarity is bypassed when glucose is replaced with alternate carbon sources but in all cases the mutant is unable to conidiate due to a block in conidiophore development at vesicle formation. In conidiophores SwoM::GFP localizes to multiple punctate, foci within each actively growing cell type, and to multiple foci in mature dormant conidia. In hyphae SwoM::GFP localized to two rings spanning the center of mature septa. In hyphae localization is concentrated at actively growing hyphal tips.     

Influence of CrgA on assembly of the cell division protein FtsZ during development of Streptomyces coelicolor

The product of the crgA gene of Streptomyces coelicolor represents a novel family of small proteins. A single orthologous gene is located close to the origin of replication of all fully sequenced actinomycete genomes and borders a conserved gene cluster implicated in cell growth and division. In S. coelicolor, CrgA is important for coordinating growth and cell division in sporogenic hyphae. In this study, we demonstrate that CrgA is an integral membrane protein whose peak expression is coordinated with the onset of development of aerial hyphae. The protein localizes to discrete foci away from growing hyphal tips. Upon overexpression, CrgA localizes to apical syncytial cells of aerial hyphae and inhibits the formation of productive cytokinetic rings of the bacterial tubulin homolog FtsZ, leading to proteolytic turnover of this major cell division determinant. In the absence of known prokaryotic cell division inhibitors in actinomycetes, CrgA may have an important conserved function influencing Z-ring formation in these bacteria.     

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 successfully 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.     

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 Å 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 mechanisms governing the correct timing and localization of cell division is one of the most studied topics in cell biology. In unicellular bacteria like Escherichia coli, cell division occurs at the mid-cell position, away from the chromosomes (1–3). The key step in this process is the appropriate timing and localization of cell division protein FtsZ to the future septum site, followed by polymerization to the Z-ring and sequential recruitment of the divisome components (1, 4). One of the major advances in our understanding of the cell division process and, in particular, of the function of FtsZ came from elucidation of the three-dimensional structure of FtsZ, which showed striking similarity to the eukaryotic protein tubulin, despite very low sequence similarity (5). Such prokaryotic ancestry was later also revealed for the cytoskeletal proteins MreB and Mbl, which belong to the actin family (6, 7), and underscored the notion of generally conserved principles in cytokinesis.Spore-forming Gram-positive Streptomyces bacteria are an important source of clinically useful antibiotics and anticancer agents (8). In these morphologically complex microorganisms, cell division is distinctly different from that in unicellular bacteria in several ways. For one, they are the only known organisms where FtsZ and MreB are both dispensable for growth (9, 10), which makes streptomycetes ideal for the study of cytokinesis. Streptomycetes have a complex life cycle that is mechanistically very similar to filamentous fungi, in producing a mycelium and propagating by sporulation (11, 12). During sporulation, the cell division machinery produces up to 100 septa simultaneously, spaced at around 1 μm, resulting in long chains of uniform and unigenomic spores (10, 13, 14). Besides the simultaneous production of multiple septa, cell division in mycelial actinomycetes also differs from that in other bacteria at the molecular level; actinomycetes lack orthologues of MinC and MinE for septum site localization (15, 16) as well as the nucleoid occlusion system Noc and Z-ring anchoring proteins, such as FtsA and ZipA (1, 6). Instead, several unique protein families have been identified that play a role in the control of cell division, including CrgA and the SsgA-like proteins (17–19). However, molecular details of their mode of action are so far lacking.The SsgA (sporulation of Streptomyces griseus)-like proteins (SALPs)³ are small (around 130–140 residues), actinomycete-specific proteins, which control sporulation-related processes in streptomycetes (17, 20). Streptomyces coelicolor contains seven SALP paralogues (SsgA to SsgG). SsgA and SsgB are essential for sporulation of S. coelicolor (21, 22). SALPs are involved in the control of cell wall-related events, such as septum localization and synthesis, thickening of the spore wall, and autolytic spore separation (17, 20), and SsgA itself directly activates sporulation-specific cell division (22, 23). The morphological complexity of actinomycetes apparently correlates to the number of SALP homologues in each organism, with one paralogue in single spore-forming actinomycetes (e.g. Salinispora or Thermobifida) and up to seven in multispore formers (two in erythromycin producer Saccharopolyspora erythraea, 3–5 in Frankia, and 6–7 in Streptomyces) (17). Most SALPs can be assigned to three subfamilies (SsgA, SsgBG, and SsgDE) based on phylogenetic analysis (17). At present, there are no functional homologues for the SALPs, and structural information is lacking. To advance our understanding of how SALPs function at the molecular level and to provide a structural template for a unique protein family without obvious relatives in any other organism, we selected the single SALP homologue from the thermophilic soil bacterium Thermobifida fusca (a major degrader of plant cell walls used in waste remediation (24)) for detailed structural analysis by x-ray crystallography, as part of our structural genomics program.In this work, we show that SsgB is most likely the archetypical SALP that occurs in morphologically complex actinomycetes, with an evolutionarily conserved function in the control of development. The three-dimensional structure of the SsgB orthologue from T. fusca was determined and revealed significant structural similarity to a eukaryotic family of ssDNA/gRNA-binding proteins. However, the structure and experimental data both suggest that SALPs probably interact with protein ligands through a hydrophobic region on their surface.     

A Cajal body-independent pathway for telomerase trafficking in mice

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.     

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.     

Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.     

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.     

Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51

Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitro and in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that the SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed.     

BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatin

Breast cancer-associated protein 1 (BRCA1) forms foci at sites of induced DNA damage, but any significance of these normal S-phase foci is unknown. BRCA1 distribution does not simply mirror or overlap that of replicating DNA; however, BRCA1 foci frequently abut sites of BrdU incorporation, mostly at mid-to-late S phase. Although BRCA1 does not overlap XIST RNA across the inactive X chromosome, BRCA1 foci position overwhelmingly in heterochromatic regions, particularly the nucleolar periphery where many centromeres reside. In humans and mice, including early embryonic cells, BRCA1 commonly associates with interphase centromere-kinetochore complexes, including pericentric heterochromatin. Proliferating cell nuclear antigen or BrdU labeling demonstrates that BRCA1 localizes adjacent to, or "paints," major satellite blocks as chromocenters replicate, where topoisomerase is also enriched. BRCA1 loss is often associated with proliferative defects, including postmitotic bridges enriched with satellite DNA. These findings implicate BRCA1 in replication-linked maintenance of centric/pericentric heterochromatin and suggest a novel means whereby BRCA1 loss may contribute to genomic instability and cancer.     

Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.