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.

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.

A conserved ClpP-like protease involved in spore outgrowth in Bacillus subtilis.

Germination and outgrowth of endospores of the Gram-positive bacterium Bacillus subtilis involves the degradation and conversion to free amino acids of abundant proteins located in the spore core known as small acid-soluble proteins (SASP). This degradation is mediated primarily by the germination protease Gpr. Here we show that YmfB, a distant homologue of ClpP serine proteases that is highly conserved among endospore-forming bacteria, contributes to SASP degradation but that its function is normally masked by Gpr. Spores from a ymfB gpr double mutant were more delayed in spore outgrowth and more impaired in SASP degradation than were spores from a gpr single mutant. The activity of YmfB relied on three putative active-site residues as well as on the product of a small gene ylzJ located immediately downstream of, and overlapping with, ymfB. We propose that YmfB is an orphan ClpP protease that is dedicated to the degradation of a specialized family of small protein substrates.

A novel RNA polymerase-binding protein controlling genes involved in spore germination in Bacillus subtilis.

A growing class of proteins regulates transcription through interaction with DNA-dependent RNA polymerase. Here we report that a recently identified, highly conserved sporulation gene ylyA encodes a novel RNA polymerase-binding protein that influences the expression of genes under the control of the late-acting, sporulation sigma factor σ(G) in Bacillus subtilis. Spores from a ylyA mutant exhibited defects in germination corresponding to changes in the levels of membrane receptors for spore germinants and a protein channel governing the release of dipicolinic acid and hydration of the spore core during germination. Purified YlyA interacted with RNA polymerase and stimulated transcription from promoters dependent on σ(G) but not promoters dependent on the housekeeping sigma factor σ(A) . YlyA is a previously unrecognized RNA polymerase-binding protein that is dedicated to modulating the expression of genes involved in spore germination.

Gene conservation among endospore-forming bacteria reveals additional sporulation genes in Bacillus subtilis.

The capacity to form endospores is unique to certain members of the low-G+C group of Gram-positive bacteria (Firmicutes) and requires signature sporulation genes that are highly conserved across members of distantly related genera, such as Clostridium and Bacillus. Using gene conservation among endospore-forming bacteria, we identified eight previously uncharacterized genes that are enriched among endospore-forming species. The expression of five of these genes was dependent on sporulation-specific transcription factors. Mutants of none of the genes exhibited a conspicuous defect in sporulation, but mutants of two, ylxY and ylyA, were outcompeted by a wild-type strain under sporulation-inducing conditions, but not during growth. In contrast, a ylmC mutant displayed a slight competitive advantage over the wild type specific to sporulation-inducing conditions. The phenotype of a ylyA mutant was ascribed to a defect in spore germination efficiency. This work demonstrates the power of combining phylogenetic profiling with reverse genetics and gene-regulatory studies to identify unrecognized genes that contribute to a conserved developmental process.

Do mycobacteria produce endospores?

The genus Mycobacterium, which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae. A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette-Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. We conclude that it is unlikely that Mycobacterium is capable of endospore formation.

Transfer-messenger RNA controls the translation of cell-cycle and stress proteins in Streptomyces.

The transfer-messenger RNA (tmRNA)-mediated trans-translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic-producing soil bacterium Streptomyces coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His(8)-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A, elongation factor Tu3, and the cell-cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA-tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress.

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 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.

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.

The secreted signaling protein factor C triggers the A-factor response regulon in Streptomyces griseus: overlapping signaling routes.

Members of the prokaryotic genus Streptomyces produce over 60% of all known antibiotics and a wide range of industrial enzymes. A leading theme in microbiology is which signals are received and transmitted by these organisms to trigger the onset of morphological differentiation and antibiotic production. The small gamma-butyrolactone A-factor is an important autoregulatory signaling molecule in streptomycetes, and A-factor mutants are blocked in development and antibiotic production. In this study we showed that heterologous expression of the 324-amino acid secreted regulatory protein Factor C resulted in restoration of development and enhanced antibiotic production of an A-factor-deficient bald mutant of Streptomyces griseus, although the parental strain lacks an facC gene. Proteome analysis showed that in the facC transformant the production of several secreted proteins that belong to the A-factor regulon was restored. HPLC-MS/MS analysis indicated that this was due to restoration of A-factor production to wild-type levels in the transformant. This indicates a connection between two highly divergent types of signaling molecules and possible interplay between their regulatory networks.

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.

Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering.

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.

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.

GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3(2).

We provide a functional and regulatory analysis of glcP, encoding the major glucose transporter of Streptomyces coelicolor A3(2). GlcP, a member of the Major Facilitator Superfamily (MFS) of bacterial and eucaryotic sugar permeases, was found to be encoded twice at two distinct loci, glcP1 and glcP2, located in the central core and in the variable right arm of the chromosome respectively. Heterologous expression of GlcP in Escherichia coli led to the full restoration of glucose fermentation to mutants lacking glucose transport activity. Biochemical analysis revealed an affinity constant in the low-micromolar range and substrate specificity for glucose and 2-deoxyglucose. Deletion of glcP1 but not glcP2 led to a drastic reduction in growth on glucose reflected by the loss of glucose uptake. This correlated with transcriptional analyses, which showed that glcP1 transcription was strongly inducible by glucose, while glcP2 transcripts were barely detectable. In conclusion, GlcP, which is the first glucose permease from high G+C Gram-positive bacteria characterized at the molecular level, represents the major glucose uptake system in S. coelicolor A3(2) that is indispensable for the high growth rate on glucose. It is anticipated that the activity of GlcP is linked to other glucose-mediated phenomena such as carbon catabolite repression, morphogenesis and antibiotic production.

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 ).