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The bacterial protein WhiD belongs to the Wbl family of iron–sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer–dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd) for the SvWhiD–σHrdB4 complex as ~0.7 μM, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8–10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.

Significance Diverse bacterial lifestyle transitions are controlled by the nucleotide second messenger c-di-GMP, including virulence, motility, and biofilm formation. To control such fundamentally distinct processes, the set of genes under c-di-GMP control must have gone through several shifts during bacterial evolution. Here we show that the same σ–(c-di-GMP)–anti-σ switch has been co-opted during evolution to regulate distinct biological functions in unicellular and filamentous bacteria, controlling type IV pilus production in the genus Rubrobacter and the differentiation of reproductive hyphae into spores in Streptomyces. Moreover, we show that the anti-σ likely originated as a homodimer and evolved to become a monomer through an intragenic duplication event. This study thus describes the structural and functional evolution of a c-di-GMP regulatory switch., Filamentous actinobacteria of the genus Streptomyces have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The Streptomyces venezuelae RsiG–(c-di-GMP)2–WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)3S(X)2R(X)3Q(X)3D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a Rubrobacter radiotolerans (RsiG)2–(c-di-GMP)2–WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium R. radiotolerans, the (RsiG)2–(c-di-GMP)2–WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%)7,8, suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods.\ud \ud

Streptomyces are filamentous bacteria with a complex developmental life cycle characterized by the formation of spore-forming aerial hyphae. Transcription of the chaplin and rodlin genes, which are essential for aerial hyphae production, is directed by the extracytoplasmic function (ECF) σ factor BldN, which is in turn controlled by an anti-σ factor, RsbN. RsbN shows no sequence similarity to known anti-σ factors and binds and inhibits BldN in an unknown manner. Here we describe the 2.23 Å structure of the RsbN–BldN complex. The structure shows that BldN harbors σ2 and σ4 domains that are individually similar to other ECF σ domains, which bind −10 and −35 promoter regions, respectively. The anti-σ RsbN consists of three helices, with α3 forming a long helix embraced between BldN σ2 and σ4 while RsbN α1–α2 dock against σ4 in a manner that would block −35 DNA binding. RsbN binding also freezes BldN in a conformation inactive for simultaneous −10 and −35 promoter interaction and RNAP binding. Strikingly, RsbN is structurally distinct from previously solved anti-σ proteins. Thus, these data characterize the molecular determinants controlling a central Streptomyces developmental switch and reveal RsbN to be the founding member of a new structural class of anti-σ factor.

Streptomycetes are notable for their complex life cycle and production of most clinically important antibiotics. A key factor that controls entry into development and the onset of antibiotic production is the 68-residue protein, BldC. BldC is a putative DNA-binding protein related to MerR regulators, but lacks coiled-coil dimerization and effector-binding domains characteristic of classical MerR proteins. Hence, the molecular function of the protein has been unclear. Here we show that BldC is indeed a DNA-binding protein and controls a regulon that includes other key developmental regulators. Intriguingly, BldC DNA-binding sites vary significantly in length. Our BldC-DNA structures explain this DNA-binding capability by revealing that BldC utilizes a DNA-binding mode distinct from MerR and other known regulators, involving asymmetric head-to-tail oligomerization on DNA direct repeats that results in dramatic DNA distortion. Notably, BldC-like proteins radiate throughout eubacteria, establishing BldC as the founding member of a new structural family of regulators., BldC regulates the onset of differentiation in Streptomycetes by a yet unknown molecular mechanism. Using a combination of structural, biochemical and in vivo approaches, the authors show that BldC controls the transcription of several developmental regulators and unravel its DNA binding mode.

Streptomyces are ubiquitous soil bacteria that undergo a complex developmental transition coinciding with their production of antibiotics. This transition is controlled by binding of a novel tetrameric form of the second messenger, 3΄-5΄ cyclic diguanylic acid (c-di-GMP) to the master repressor, BldD. In all domains of life, nucleotide-based second messengers allow a rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions. c-di-GMP can assume alternative oligomeric states to effect different functions, binding to effector proteins as monomers, intercalated dimers or, uniquely in the case of BldD, as a tetramer. However, at physiological concentrations c-di-GMP is a monomer and little is known about how higher oligomeric complexes assemble on effector proteins and if intermediates in assembly pathways have regulatory significance. Here, we show that c-di-GMP binds BldD using an ordered, sequential mechanism and that BldD function necessitates the assembly of the BldD2-(c-di-GMP)4 complex.

BldD-(c-di-GMP) sits on top of the regulatory network that controls differentiation in Streptomyces, repressing a large regulon of developmental genes when the bacteria are growing vegetatively. In this way, BldD functions as an inhibitor that blocks the initiation of sporulation. Here, we report the identification and characterisation of BldO, an additional developmental repressor that acts to sustain vegetative growth and prevent entry into sporulation. However, unlike the pleiotropic regulator BldD, we show that BldO functions as the dedicated repressor of a single key target gene, whiB, and that deletion of bldO or constitutive expression of whiB is sufficient to induce precocious hypersporulation.

Proteins that regulate transcription often also play an architectural role in the genome. Thus, it has been difficult to define with precision the distinctions between transcription factors and nucleoid-associated proteins (NAPs). Anachronistic descriptions of NAPs as 'histone-like' implied an organizational function in a bacterial chromatin-like complex. Definitions based on protein abundance, regulatory mechanisms, target gene number, or the features of their DNA-binding sites are insufficient as marks of distinction, and trying to distinguish transcription factors and NAPs based on their ranking within regulatory hierarchies or positions in gene-control networks is also unsatisfactory. The terms 'transcription factor' and 'NAP' are ad hoc operational definitions with each protein lying along a spectrum of structural and functional features extending from highly specific actors with few gene targets to those with a pervasive influence on the transcriptome. The Streptomyces BldC protein is used to illustrate these issues.

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood

Simocyclinones are antibiotics produced by Streptomyces and Kitasatospora species that inhibit the validated drug target DNA gyrase in a unique way, and they are thus of therapeutic interest. Structural approaches have revealed their mode of action, the inducible-efflux mechanism in the producing organism, and given insight into one step in their biosynthesis. The crystal structures of simocyclinones bound to their target (gyrase), the transcriptional repressor SimR and the biosynthetic enzyme SimC7 reveal fascinating insight into how molecular recognition is achieved with these three unrelated proteins., Simocyclinones are actinomycete natural products that target bacterial DNA gyrase; structural work has revealed their molecular mode of action, aspects of their biosynthesis and the mechanism underlying their inducible export.

Understanding the mechanisms that drive bacterial morphogenesis depends on the dissection of the regulatory networks that underpin the cell biological processes involved. Recently, Streptomyces venezuelae has emerged as an attractive model system for the study of morphological differentiation in Streptomyces. This has led to significant progress in identifying the genes controlled by the transcription factors that regulate aerial mycelium formation (Bld regulators) and sporulation (Whi regulators). Taking advantage of S. venezuelae, we used ChIP-seq coupled with RNA-seq to identify the genes directly under the control of BldC. Because S. venezuelae sporulates in liquid culture, the complete spore-to-spore life cycle can be examined using time-lapse microscopy, and we applied this technique to the bldC mutant. These combined approaches reveal BldC to be a member of an emerging class of Bld regulators that function principally to repress key sporulation genes, thereby extending vegetative growth and blocking the onset of morphological differentiation., Streptomycetes are filamentous bacteria that differentiate by producing spore-bearing reproductive structures called aerial hyphae. The transition from vegetative to reproductive growth is controlled by the bld (bald) loci, and mutations in bld genes prevent the formation of aerial hyphae, either by blocking entry into development (typically mutations in activators) or by inducing precocious sporulation in the vegetative mycelium (typically mutations in repressors). One of the bld genes, bldC, encodes a 68-residue DNA-binding protein related to the DNA-binding domain of MerR-family transcription factors. Recent work has shown that BldC binds DNA by a novel mechanism, but there is less insight into its impact on Streptomyces development. Here we used ChIP-seq coupled with RNA-seq to define the BldC regulon in the model species Streptomyces venezuelae, showing that BldC can function both as a repressor and as an activator of transcription. Using electron microscopy and time-lapse imaging, we show that bldC mutants are bald because they initiate development prematurely, bypassing the formation of aerial hyphae. This is consistent with the premature expression of BldC target genes encoding proteins with key roles in development (e.g., whiD, whiI, sigF), chromosome condensation and segregation (e.g., smeA-sffA, hupS), and sporulation-specific cell division (e.g., dynAB), suggesting that BldC-mediated repression is critical to maintain a sustained period of vegetative growth prior to sporulation. We discuss the possible significance of BldC as an evolutionary link between MerR family transcription factors and DNA architectural proteins.

Bacterial dynamins were discovered ∼10 y ago and the explosion in genome sequencing has shown that they radiate throughout the bacteria, being present in >1,000 species. In eukaryotes, dynamins play critical roles in the detachment of endocytic vesicles from the plasma membrane, the division of chloroplasts and peroxisomes, and both the fusion and fission of mitochondria. However, in evolutionary terms, dynamins are of bacterial origin, and yet the biological functions of bacterial dynamins remain poorly understood. Here we demonstrate a critical role for dynamins in bacterial cytokinesis, reminiscent of the essential role of eukaryotic dynamins in the division of chloroplasts and mitochondria.

During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein–protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry., IMPORTANCE In all sigma factor-antisigma factor regulatory switches, the relative abundance of the two proteins is critical to the proper functioning of the system. Many sigma-antisigma operons are cotranscribed and translationally coupled, leading to a generic assumption that the sigma and antisigma factors are produced in a fixed 1:1 ratio. In the case of sigR-rsrA, we show instead that the antisigma factor is produced in excess over the sigma factor, providing a buffer to prevent spurious release of sigma activity. This excess arises in part because sigR has an extremely rare noncanonical GTC start codon, and as a result, SigR translation initiation is repressed by IF3. This finding highlights the potential significance of noncanonical start codons, very few of which have been characterized experimentally. It also emphasizes the limitations of predicting start codons using bioinformatic approaches, which rely heavily on the assumption that ATG, GTG, and TTG are the only permissible start codons.

Summary BldD‐(c‐di‐GMP) sits on top of the regulatory network that controls differentiation in Streptomyces, repressing a large regulon of developmental genes when the bacteria are growing vegetatively. In this way, BldD functions as an inhibitor that blocks the initiation of sporulation. Here, we report the identification and characterisation of BldO, an additional developmental repressor that acts to sustain vegetative growth and prevent entry into sporulation. However, unlike the pleiotropic regulator BldD, we show that BldO functions as the dedicated repressor of a single key target gene, whiB, and that deletion of bldO or constitutive expression of whiB is sufficient to induce precocious hypersporulation.

Summary Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development., Graphical Abstract, Highlights • c-di-GMP controls development in the multicellular bacterium Streptomyces • c-di-GMP mediates complex formation between sporulation σ, σWhiG, and anti-σ, RsiG • RsiG uses two novel E(X)3S(X)2R(X)3Q(X)3D signature motifs to bind two c-di-GMPs • When c-di-GMP levels drop, σWhiG is released to activate late sporulation regulators, In the antibiotic-producing bacterium Streptomyces, an unusual dimer of the cyclic dinucleotide signaling molecule c-di-GMP mediates effective complex formation between a sporulation-specific σ and its cognate anti-σ to control differentiation of the reproductive hyphae into spores. The anti-σ binds c-di-GMP using two copies of a novel E(X)3S(X)2R(X)3Q(X)3D signature motif.

SummaryThe cyclic dinucleotide c-di-GMP is a signaling molecule with diverse functions in cellular physiology. Here, we report that c-di-GMP can assemble into a tetramer that mediates the effective dimerization of a transcription factor, BldD, which controls the progression of multicellular differentiation in sporulating actinomycete bacteria. BldD represses expression of sporulation genes during vegetative growth in a manner that depends on c-di-GMP-mediated dimerization. Structural and biochemical analyses show that tetrameric c-di-GMP links two subunits of BldD through their C-terminal domains, which are otherwise separated by ∼10 Å and thus cannot effect dimerization directly. Binding of the c-di-GMP tetramer by BldD is selective and requires a bipartite RXD-X8-RXXD signature. The findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions.

The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.

The regulation of disulphide stress in actinomycetes such as Streptomyces coelicolor is known to involve the zinc-containing anti-sigma factor RsrA that binds and inactivates the redox-regulated sigma factor sigmaR. However, it is not known how RsrA senses disulphide stress nor what role the metal ion plays. Using in vitro assays, we show that while zinc is not required for sigmaR binding it is required for functional anti-sigma factor activity, and that it plays a critical role in modulating the reactivity of RsrA cysteine thiol groups towards oxidation. Apo-RsrA is easily oxidised and, while the Zn-bound form is relatively resistant, the metal ion is readily expelled when the protein is treated with strong oxidants such as diamide. We also show, using a combination of proteolysis and mass spectrometry, that the first critical disulphide to form in RsrA involves Cys11 and one of either Cys41 or Cys44, all previously implicated in metal binding. Circular dichroism spectroscopy was used to follow structural changes during oxidation of RsrA, which indicated that concomitant with formation of this critical disulphide bond is a major restructuring of the protein where its alpha-helical content increases. Our data demonstrate that RsrA can only bind sigmaR in the reduced state and that this state is stabilised by zinc. Redox stress induces disulphide bond formation amongst zinc-ligating residues, expelling the metal ion and stabilising a structure incapable of binding the sigma factor.

WhiB is the founding member of a family of proteins (the WhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. In Streptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) in Streptomyces venezuelae. In the first demonstration of in vivo genome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes of whiA and whiB mutants. Using ChIP-seq, we demonstrated that in vivo DNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiA in vivo., IMPORTANCE Despite the central importance of WhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularly in vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function of Streptomyces WhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNA in vivo. Our data provide the most compelling in vivo evidence to date that WhiB, and, by extension, probably all Wbl proteins, function as transcription factors. Further, we show that WhiB does not act independently but rather coregulates its regulon of sporulation genes with a partner transcription factor, WhiA.

The chaplin and rodlin proteins together constitute the major components of the hydrophobic sheath that coats the aerial hyphae and spores in Streptomyces, and mutants lacking the chaplins are unable to erect aerial hyphae and differentiate on minimal media. We have gained insight into the developmental regulation of the chaplin (chp) and rodlin (rdl) genes by exploiting a new model species, Streptomyces venezuelae, which sporulates in liquid culture. Using microarrays, the chaplin and rodlin genes were found to be highly induced during submerged sporulation in a bldN-dependent manner. Using σ(BldN) ChIP-chip, we show that this dependence arises because the chaplin and rodlin genes are direct biochemical targets of σ(BldN) . sven3186 (here named rsbN for regulator of sigma BldN), the gene lying immediately downstream of bldN, was also identified as a target of σ(BldN) . Disruption of rsbN causes precocious sporulation and biochemical experiments demonstrate that RsbN functions as a σ(BldN) -specific anti-sigma factor.

BldD is a transcriptional regulator essential for morphological development and antibiotic production in Streptomyces coelicolor. Here we identify the BldD regulon by means of chromatin immunoprecipitation-microarray analysis (ChIP-chip). The BldD regulon encompasses ~167 transcriptional units, of which more than 20 are known to play important roles in development (e.g. bldA, bldC, bldH/adpA, bldM, bldN, ssgA, ssgB, ftsZ, whiB, whiG, smeA-ssfA) and/or secondary metabolism (e.g. nsdA, cvn9, bldA, bldC, leuA). Strikingly, 42 BldD target genes (~25% of the regulon) encode regulatory proteins, stressing the central, pleiotropic role of BldD. Almost all BldD binding sites identified by ChIP-chip are present in the promoters of the target genes. An exception is the tRNA gene bldA, where BldD binds within the region encoding the primary transcript, immediately downstream of the position corresponding to the processed, mature 3 end of the tRNA. Through gene overexpression, we identified a novel BldD target gene (cdgA) that influences differentiation and antibiotic production. cdgA encodes a GGDEF domain protein, implicating c-di-GMP in the regulation of Streptomyces development. Sequence analysis of the upstream regions of the complete regulon identified a 15 bp inverted repeat that functions as a high-affinity binding site for BldD, as was shown by electrophoretic mobility shift assays and DNase I footprinting analysis. High-scoring copies of the BldD binding site were found at relevant positions in the genomes of other bacteria containing a BldD homologue, suggesting the role of BldD is conserved in sporulating actinomycetes.

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained approximately 4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g approximately 2.06, 1.94) consistent with a one-electron reduction of a [4Fe-4S](2+) cluster to a [4Fe-4S](1+) state with electron spin of S = (1/2). The anaerobically reconstituted [4Fe-4S] cluster was oxygen sensitive. Upon exposure to air, absorption at 410 and 505 nm first increased and then showed a steady decrease with time until the protein was colorless in the near UV/visible region. These changes are consistent with an oxygen-induced change from a [4Fe-4S] to a [2Fe-2S] cluster, followed by complete loss of cluster from the protein. Each of the four conserved cysteine residues, Cys-23, -53, -56, and -62, was essential for WhiD function in vivo.

SapB is a morphogenetic peptide that is important for aerial mycelium formation by the filamentous bacterium Streptomyces coelicolor . Production of SapB commences during aerial mycelium formation and depends on most of the genes known to be required for the morphogenesis of aerial hyphae. Furthermore, the application of purified SapB to mutants blocked in morphogenesis restores their capacity to form aerial hyphae. Here, we present evidence that SapB is a lantibiotic-like peptide that is derived by posttranslational modification from the product of a gene ( ramS ) in the four-gene ram operon, which is under the control of the regulatory gene ramR . We show that the product of another gene in the operon ( ramC ) contains a region that is similar to enzymes involved in the biosynthesis of lantibiotics, suggesting that it might be involved in the posttranslational processing of RamS. We conclude that SapB is derived from RamS through proteolytic cleavage and the introduction of four dehydroalanine residues and two lanthionine bridges. We provide an example of a morphogenetic role for an antibiotic-like molecule.

The filamentous bacteriumStreptomyces coelicolordifferentiates by forming specialized, spore-bearing aerial hyphae that grow into the air. Using microarrays, we identified genes that are down-regulated in a mutant unable to erect aerial hyphae. Through this route, we identified a previously unknown layer of aerial mycelium surface proteins (the “chaplins”). The chaplins share a hydrophobic domain of ∼40 residues (the “chaplin domain”), and all have a secretion signal. The five short chaplins (ChpD,E,F,G,H) have one chaplin domain, whereas the three long chaplins (ChpA,B,C) have two chaplin domains and a C-terminal “sorting signal” that targets them for covalent attachment to the cell wall by sortase enzyme. Expression of the two chaplin genes examined (chpE, chpH) depended on aerial hyphae formation but not sporulation, andegfpfusions showed their expression localized to aerial structures. Mass spectrometry of cell wall extracts confirmed that the short chaplins localized to the cell surface. Deletion of chaplin genes caused severe delays in aerial hyphae formation, a phenotype rescued by exogenous application of chaplin proteins. These observations implicate the chaplins in aerial mycelium formation, and suggest that coating of the envelope by the chaplins is required for aerial hyphae to grow out of the aqueous environment of the substrate mycelium into the air.

bldN is one of a set of genes required for the formation of specialized, spore-bearing aerial hyphae during differentiation in the mycelial bacterium Streptomyces coelicolor . Previous analysis (M. J. Bibb et al., J. Bacteriol. 182:4606-4616, 2000) showed that bldN encodes a member of the extracytoplasmic function subfamily of RNA polymerase σ factors and that translation from the most strongly predicted start codon (GTG 1 ) would give rise to a σ factor having an unusual N-terminal extension of ca. 86 residues. Here, by using a combination of site-directed mutagenesis and immunoblot analysis, we provide evidence that all bldN translation arises from initiation at GTG 1 and that the primary translation product is a proprotein (pro-σ BldN ) that is proteolytically processed to a mature species (σ BldN ) by removal of most of the unusual N-terminal extension. A time course taken during differentiation of the wild type on solid medium showed early production of pro-σ BldN and the subsequent appearance of mature σ BldN , which was concomitant with aerial mycelium formation and the disappearance of pro-σ BldN . Two genes encoding members of a family of metalloproteases that are involved in the regulated proteolytic processing of transcription factors in other organisms were identified in the S. coelicolor genome, but their disruption did not affect differentiation or pro-σ BldN processing.

The extracytoplasmic function (ECF) sigma factor sigma(R) is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sigma(R) dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on sigma(R) or how it suppresses sigma(R)-dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, approximately 10kDa domain (sigma(RN)) that encompasses region 2 of sigma(R) that represents the major RsrA binding site. We show that sigma(RN) inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits sigma(R) activity is by blocking this association. We also report the 2.4A resolution crystal structure of sigma(RN) that reveals extensive structural conservation with the equivalent region of sigma(70) from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A. sigma(RN) has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.

whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although bldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for bldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. bldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.

The orphan, atypical response regulators BldM and WhiI each play critical roles in Streptomyces differentiation. BldM is required for the formation of aerial hyphae, and WhiI is required for the differentiation of these reproductive structures into mature spores. To gain insight into BldM function, we defined the genome-wide BldM regulon using ChIP-Seq and transcriptional profiling. BldM target genes clustered into two groups based on their whi gene dependency. Expression of Group I genes depended on bldM but was independent of all the whi genes, and biochemical experiments showed that Group I promoters were controlled by a BldM homodimer. In contrast, Group II genes were expressed later than Group I genes and their expression depended not only on bldM but also on whiI and whiG (encoding the sigma factor that activates whiI). Additional ChIP-Seq analysis showed that BldM Group II genes were also direct targets of WhiI and that in vivo binding of WhiI to these promoters depended on BldM and vice versa. We go on to demonstrate that BldM and WhiI form a functional heterodimer that controls Group II promoters, serving to integrate signals from two distinct developmental pathways. The BldM-WhiI system thus exemplifies the potential of response regulator heterodimer formation as a mechanism to expand the signaling capabilities of bacterial cells., Author Summary Two-component signal transduction systems are a primary means of regulating gene expression in bacteria. Recognizing the diversity of mechanisms associated with these systems is therefore critical to understanding the full signaling potential of bacterial cells. We have analyzed the behavior of two orphan, atypical response regulators that play key roles in controlling morphological differentiation in the filamentous bacteria Streptomyces-BldM and WhiI. We demonstrate that BldM activates its Group I target promoters as a homodimer, but that it subsequently activates its Group II target promoters by forming a functional heterodimer with WhiI. BldM-WhiI heterodimer formation thus represents an unusual mechanism for the coactivation of target genes and the integration of regulatory signals at promoters, enhancing the known repertoire of signaling capabilities associated with two-component systems.

The colony structure and life cycle of the grampositive, soil-dwelling bacterium Streptomyces coelicolor provide a fascinating exception to the view of bacteria as simple unicellular microorganisms. Mutations in genes involved in morphogenesis alter colony appearance but do not usually compromise viability. The majority of genes identified as being important for aerial hypha formation encode regulatory proteins; however, recent work has resulted in the characterization of two classes of structural molecules that are necessary for aerial development: the SapB surfactant peptide (specified by the ram gene cluster) and eight chaplin proteins (ChpA through H). It has been found that while Streptomyces has many of the conventional genes that are necessary for these processes to occur, Streptomyces cells are organized very differently from other bacteria and these differences are highly relevant to colony development and spore formation. In Streptomyces, however, DivIVA is an essential protein that does not seem to be associated with cell division but rather is crucial for coordinating cell wall growth. The basic mechanism of Z-ring formation appears to be shared between S. coelicolor and other prokaryotes; however, there are important differences in how Streptomyces employs and executes cell division. Streptomyces has no homologues of the Bacillus subtilis or Escherichia coli MinC or MinD proteins and uses its DivIVA for functions apparently unrelated to cell division.

During a search for new differentiation factors in Streptomyces coelicolor A3(2), a locus at 11 o'clock on the S. coelicolor map was identified which harbours several genes that show extensive similarity to cell division and differentiation genes from Escherichia coli and Bacillus subtilis. From the sequence data it was concluded that the region contains the genes mireB, mreC, mreD (murein formation gene cluster E), pbp83 (high-molecular-weight penicillin-binding protein) and sfr (member of the spoVE/ftsW/rodA family). Mre gene products are reported to be responsible for determining cell shape in E. coli and Bacillus. The S. coelicolor mreC gene was inactivated by gene disruption, resulting in mutants which showed significant growth retardation in comparison to the wild type. Inactivation of the mreB gene was incompatible with viability, and thus mreB represents a Streptomyces cell division gene that is essential for survival. Promoter-probe experiments led to the identification of an operon structure, with promoters located upstream of mreB, pbp83 and sfr. Detailed studies of mreB transcription revealed the existence of three promoters; two of them are constitutively transcribed, whereas the third is developmentally regulated.

The extracytoplasmic function (ECF) sigma factor, sigmaE, is required for normal cell wall integrity in Streptomyces coelicolor. We have investigated the regulation of sigmaE through a transcriptional and mutational analysis of sigE and the surrounding genes. Nucleotide sequencing identified three genes located downstream of sigE; orf202, cseB and cseC (cse, control of sigE ). cseB and cseC encode a putative response regulator and a putative transmembrane sensor histidine protein kinase respectively. Although most sigE transcription appeared to be monocistronic, sigE was also transcribed as part of a larger operon, including at least orf202. sigE null mutants are sensitive to cell wall lytic enzymes, have an altered peptidoglycan muropeptide profile, and on medium deficient in Mg2+ they overproduce actinorhodin, sporulate poorly and form crenellated colonies. A constructed cseB null mutant appeared to have the same phenotype as a sigE null mutant, which was accounted for by the observed absolute dependence of the sigE promoter on cseB. It is likely that the major role of cseB is to regulate sigE transcription because expression of sigE alone from a heterologous promoter suppressed the cseB mutation. Mg2+ suppresses the CseB/SigE phenotype, probably by stabilizing the cell envelope, and sigE transcript levels were consistently higher in Mg2+-deficient cultures than in high Mg2+-grown cultures. We propose a model in which the CseB/CseC two-component system modulates activity of the sigE promoter in response to signals from the cell envelope.

whiE is a complex locus that specifies the polyketide spore pigment in Streptomyces coelicolor A3(2). Two divergently oriented promoters, whiEP1 and whiEP2 , were identified in the whiE gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated; whiEP1 and whiEP2 transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known “early” whi genes required for sporulation septum formation ( whiA , - B , - G , - H , - I , and - J ). Mutation of the late sporulation-specific sigma factor gene, sigF , had no effect on the activity of whiEP1 but blocked transcription from whiEP2 . However, ς F -containing holoenzyme was not sufficient to direct transcription of whiEP2 in vitro. The whiEP2 promoter controls expression of whiE ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of whiE ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779–2791, 1995). Consistent with these observations, construction of a sigF null mutant of S. coelicolor M145 caused the same change in spore color, showing that disruption of sigF in S. coelicolor changes the nature of the spore pigment rather than preventing its synthesis altogether.

In the non-motile mycelial organism Streptomyces coelicolor A3(2), the sporulation gene whiG encodes a protein that closely resembles RNA polymerase sigma factors such as sigma D of Bacillus subtilis, which mainly control motility and chemotaxis genes. Here, we show that the whiG gene product, purified from an Escherichia coli strain carrying an expression construct, could activate E. coli core RNA polymerase in vitro to transcribe a sigma D-dependent motility-related promoter from B. subtilis. Such RNA polymerase holoenzyme preparations could also transcribe from an S. coelicolor promoter, PTH4, previously shown to require an intact whiG gene for in-vivo transcription. The in-vivo dependence on whiG was therefore shown to be direct. Unusually, the initiation of PTH4 transcription in vitro depended on the provision of appropriate dinucleotides. The whiG-dependent PTH4 transcription unit consisted of a single gene, orfTH4. Sequence comparisons suggested that the gene product was a member of a small group of proteins that include the B. subtilis and E. coli ProX proteins. Though none of these proteins shared more than about 30% of extended primary sequence identity, they had similar size and hydropathy profiles, and could be aligned end to end to reveal a mosaic of similarities. The ProX proteins of B. subtilis and E. coli are implicated in glycine betaine transport in response to hyperosmotic stress. However, disruption of orfTH4 did not cause any obvious phenotypic changes in growth or development on media of varying osmotic strengths.

The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus. The phsA promoter strikingly resembles a putative Streptomyces sigma E cognate promoter, and purified E sigma E holoenzyme transcribed the phsA promoter in vitro. However, the phsA promoter was still active in an S. antibioticus sigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10-fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.

WhiA 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., IMPORTANCE Since the initial identification of the genetic loci required for Streptomyces development, all of the bld and whi developmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction of Streptomyces venezuelae as a new model system for the genus, a species that sporulates in liquid culture. Taking advantage of S. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild-type and sporulation-defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at all time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post-transcriptionally. Transcription of whiG did not depend upon any of the six known 'early' whi genes required for sporulation septum formation (whiA, B, G, H, I and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for sigma WhiG-dependent promoters and E sigma WhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network.

The filamentous bacteria Streptomyces grow by tip extension and through the initiation of new branches, and this apical growth is directed by a polarisome-like complex involving the essential polarity protein DivIVA. New branch sites must be marked de novo and, until recently, there was no understanding of how these new sites are selected. Equally, hyphal branching patterns are affected by environmental conditions, but there was no insight into how polar growth and hyphal branching might be regulated in response to external or internal cues. This review focuses on recent discoveries that reveal the principal mechanism of branch site selection in Streptomyces, and the first mechanism to be identified that regulates polarisome behaviour to modulate polar growth and hyphal branching.

The chaplin and rodlin proteins together constitute the major components of the hydrophobic sheath that coats the aerial hyphae and spores in Streptomyces, and mutants lacking the chaplins are unable to erect aerial hyphae and differentiate on minimal media. We have gained insight into the developmental regulation of the chaplin (chp) and rodlin (rdl) genes by exploiting a new model species, Streptomyces venezuelae, which sporulates in liquid culture. Using microarrays, the chaplin and rodlin genes were found to be highly induced during submerged sporulation in a bldN-dependent manner. Using σ(BldN) ChIP-chip, we show that this dependence arises because the chaplin and rodlin genes are direct biochemical targets of σ(BldN) . sven3186 (here named rsbN for regulator of sigma BldN), the gene lying immediately downstream of bldN, was also identified as a target of σ(BldN) . Disruption of rsbN causes precocious sporulation and biochemical experiments demonstrate that RsbN functions as a σ(BldN) -specific anti-sigma factor.

We describe the identification and functional characterization of cdgB (SCO4281), a recently discovered target of BldD, a key regulator of morphological differentiation and antibiotic production in the mycelial bacteria of the genus Streptomyces . cdgB ( c yclic d imeric G MP [c-di-GMP] B ) encodes a GGDEF-containing protein that has diguanylate cyclase activity in vitro . Consistent with this enzymatic activity, heterologous expression of cdgB in Escherichia coli resulted in increased production of extracellular matrix in colonies and enhanced surface attachment of cells in standing liquid cultures. In Streptomyces coelicolor , both overexpression and deletion of cdgB inhibited aerial-mycelium formation, and overexpression also inhibited production of the antibiotic actinorhodin, implicating c-di-GMP in the regulation of developmental processes in Streptomyces .

The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2). We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.

WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster, and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from Escherichia coli as a soluble protein containing a single [4Fe-4S](2+) cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and pH 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance. [4Fe-4S](2+) WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apoprotein destabilized the protein fold significantly but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay, demonstrating that it does not function as a general protein disulfide reductase.

A gene ( bar ) was identified adjacent to the hrdD sigma factor gene in Streptomyces coelicolor A3(2). The predicted bar product showed 32.2% and 30.4% identity to those of the pat and bar genes of the bialaphos (Bp) producers Streptomyces viridochromogenes and Streptomyces hygroscopicus , respectively; these genes encode phosphinothricin (PPT) N -acetyltransferases that function as enzymes in the Bp biosynthetic pathway and as resistance determinants. The S. coelicolor bar gene conferred high-level resistance to Bp when cloned in S. coelicolor on a high-copy-number vector. Enzymic assay showed that the S. coelicolor bar gene product inactivates PPT by transfer of acetyl groups from acetyl CoA. The S. coelicolor bar gene appears to be expressed from two promoters ( p 1 and p 2) and is divergently transcribed with respect to hrdD . The downstream ( barp2 ) transcript overlaps the hrdDp1 transcript and the upstream ( barp1 ) transcript overlaps both the hrdDp1 and hrdDp2 transcripts. Inactivation of hrdD did not prevent transcription from either bar promoter, indicating that σ hrdD is not essential for recognition of these sequences.