CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches.

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

Role for a Lytic Polysaccharide Monooxygenase in Cell Wall Remodeling in Streptomyces coelicolor.

Peptidoglycan is a major constituent of the bacterial cell wall and an important determinant for providing protection to cells. In addition to peptidoglycan, many bacteria synthesize other glycans that become part of the cell wall. Streptomycetes grow apically, where they synthesize a glycan that is exposed at the outer surface, but how it gets there is unknown. Here, we show that deposition of the apical glycan at the cell surface of Streptomyces coelicolor depends on two key enzymes, the glucanase CslZ and the lytic polysaccharide monooxygenase LpmP. Activity of these enzymes allows localized remodeling and degradation of the peptidoglycan, and we propose that this facilitates passage of the glycan. The absence of both enzymes not only prevents morphological development but also sensitizes strains to lysozyme. Given that lytic polysaccharide monooxygenases are commonly found in microbes, this newly identified biological role in cell wall remodeling may be widespread. IMPORTANCE Lytic polysaccharide monooxygenases are used in industry for the efficient degradation of recalcitrant polysaccharide substrates. Only recently, we have begun to appreciate some of their important biological roles. In this article, we provide evidence that these enzymes are involved in remodeling peptidoglycan, which is a conserved component of the bacterial cell wall. Given that lytic polysaccharide monooxygenases are commonly found in microbes, this newly identified biological role in cell wall remodeling may be widespread.

Carbohydrate Binding Modules: Diversity of Domain Architecture in Amylases and Cellulases From Filamentous Microorganisms.

Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.

Profile Comparer Extended: phylogeny of lytic polysaccharide monooxygenase families using profile hidden Markov model alignments.

Insight into the inter- and intra-family relationship of protein families is important, since it can aid understanding of substrate specificity evolution and assign putative functions to proteins with unknown function. To study both these inter- and intra-family relationships, the ability to build phylogenetic trees using the most sensitive sequence similarity search methods (e.g. profile hidden Markov model (pHMM)-pHMM alignments) is required. However, existing solutions require a very long calculation time to obtain the phylogenetic tree. Therefore, a faster protocol is required to make this approach efficient for research. To contribute to this goal, we extended the original Profile Comparer program (PRC) for the construction of large pHMM phylogenetic trees at speeds several orders of magnitude faster compared to pHMM-tree. As an example, PRC Extended (PRCx) was used to study the phylogeny of over 10,000 sequences of lytic polysaccharide monooxygenase (LPMO) from over seven families. Using the newly developed program we were able to reveal previously unknown homologs of LPMOs, namely the PFAM Egh16-like family. Moreover, we show that the substrate specificities have evolved independently several times within the LPMO superfamily. Furthermore, the LPMO phylogenetic tree, does not seem to follow taxonomy-based classification.

A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P1-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

The discovery of novel LPMO families with a new Hidden Markov model.

BACKGROUND: Renewable biopolymers, such as cellulose, starch and chitin are highly resistance to enzymatic degradation. Therefore, there is a need to upgrade current degradation processes by including novel enzymes. Lytic polysaccharide mono-oxygenases (LPMOs) can disrupt recalcitrant biopolymers, thereby enhancing hydrolysis by conventional enzymes. However, novel LPMO families are difficult to identify using existing methods. Therefore, we developed a novel profile Hidden Markov model (HMM) and used it to mine genomes of ascomycetous fungi for novel LPMOs. RESULTS: We constructed a structural alignment and verified that the alignment was correct. In the alignment we identified several known conserved features, such as the histidine brace and the N/Q/E-X-F/Y motif and previously unidentified conserved proline and glycine residues. These residues are distal from the active site, suggesting a role in structure rather than activity. The multiple protein alignment was subsequently used to build a profile Hidden Markov model. This model was initially tested on manually curated datasets and proved to be both sensitive (no false negatives) and specific (no false positives). In some of the genomes analyzed we identified a yet unknown LPMO family. This new family is mostly confined to the phyla of Ascomycota and Basidiomycota and the class of Oomycota. Genomic clustering indicated that at least some members might be involved in the degradation of ß-glucans, while transcriptomic data suggested that others are possibly involved in the degradation of pectin. CONCLUSIONS: The newly developed profile hidden Markov Model was successfully used to mine fungal genomes for a novel family of LPMOs. However, the model is not limited to bacterial and fungal genomes. This is illustrated by the fact that the model was also able to identify another new LPMO family in Drosophila melanogaster. Furthermore, the Hidden Markov model was used to verify the more distant blast hits from the new fungal family of LPMOs, which belong to the Bivalves, Stony corals and Sea anemones. So this Hidden Markov model (Additional file 3) will help the broader scientific community in identifying other yet unknown LPMOs.

Active-site maturation and activity of the copper-radical oxidase GlxA are governed by a tryptophan residue.

GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best characterised. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic sites in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper-co-ordinating tyrosine ligand and houses a radical. In GlxA and Gox, a second co-ordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes, creating a marked difference in the π-π stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here, we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development.

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases.

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.

New approaches to achieve high level enzyme production in Streptomyces lividans.

BACKGROUND: Actinomycetes are saprophytic soil bacteria, and a rich source of industrial enzymes. While some of these enzymes can be produced using well-characterized production platforms such as Escherichia coli or Bacillus subtilis, Streptomyces lividans may be the preferred host for proper folding and efficient secretion of active enzymes. A combination of promoters, signal peptides and hosts were tested in order to obtain the best protein expression in this actinomycete. The xylanase, Xys1, from S. halstedii, the α-amylase, Amy, from S. griseus and the small laccase, SLAC, from S. coelicolor were used as reporters. RESULTS: The promoters xysAp from S. halstedii JM8 and pstSp from S. lividans were the most efficient among those tested. An improvement of 17 % was obtained in xylanase activity when the signal peptide of the α-amylase protein (Amy) of S. griseus IMRU3570 was used to direct its secretion. Enhanced expression of SsgA, a protein that plays a role in processes that require cell-wall remodelling, resulted in a improvement of 40 and 70 % of xylanase and amylase production, respectively. Deletion of genes SLI7232 and SLI4452 encoding putative repressors of xysAp provided improvement of production up to 70 % in the SLI7232 deletion strain. However, full derepression of this promoter activity was not obtained under the conditions assayed. CONCLUSIONS: Streptomyces lividans is a frequently used platform for industrial enzyme production and a rational strain-development approach delivered significant improvement of protein production by this host.

The DyP-type peroxidase DtpA is a Tat-substrate required for GlxA maturation and morphogenesis in Streptomyces.

The filamentous bacterium Streptomyces lividans depends on the radical copper oxidase GlxA for the formation of reproductive aerial structures and, in liquid environments, for the formation of pellets. Incorporation of copper into the active site is essential for the formation of a cross-linked tyrosyl-cysteine cofactor, which is needed for enzymatic activity. In this study, we show a crucial link between GlxA maturation and a group of copper-related proteins including the chaperone Sco and a novel DyP-type peroxidase hereinafter called DtpA. Under copper-limiting conditions, the sco and dtpA deletion mutants are blocked in aerial growth and pellet formation, similarly to a glxA mutant. Western blot analysis showed that GlxA maturation is perturbed in the sco and dtpA mutants, but both maturation and morphology can by rescued by increasing the bioavailability of copper. DtpA acts as a peroxidase in the presence of GlxA and is a substrate for the twin-arginine translocation (Tat) translocation pathway. In agreement, the maturation status of GlxA is also perturbed in tat mutants, which can be compensated for by the addition of copper, thereby partially restoring their morphological defects. Our data support a model wherein a copper-trafficking pathway and Tat-dependent secretion of DtpA link to the GlxA-dependent morphogenesis pathway.

GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of Streptomyces lividans.

Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drastically changed morphology both on solid substrates and in liquid-grown environments, a scenario similarly observed in the absence of the neighbouring glycan synthase CslA (cellulase synthase-like protein). In addition the glxA mutant has lost the stimulation of development by copper, supporting a model whereby the enzymatic action of GlxA on the glycan is required for development and morphology. From a biotechnology perspective, the open mycelium morphology observed with the glxA mutant in submerged culture has implications for use as an enzyme production host.

Copper trafficking in the CsoR regulon of Streptomyces lividans.

In the actinobacterium Streptomyces lividans copper homeostasis is controlled through the action of the metalloregulator CsoR. Under copper stress, cuprous ions bind to apo-CsoR resulting in the transcriptional derepression of genes encoding for copper efflux systems involving CopZ-like copper chaperones and CopA-like P-type ATPases. Whether CsoR obtains copper via a protein-protein mediated trafficking mechanism is unknown. In this study we have characterised the copper trafficking properties of two S. lividans CopZ proteins (SLI_1317 and SLI_3079) under the transcriptional control of a CsoR (SLI_4375). Our findings indicate that both CopZ-proteins have cysteine residues in the Cu(i) binding MX1CX2X3C motif with acid-base properties that are modulated for a high cuprous ion affinity and favourable Cu(i)-exchange with a target. Using electrophoretic mobility shift assays transfer of Cu(i) is shown to occur in a unidirectional manner from the CopZ to the CsoR. This transfer proceeds via a shallow thermodynamic affinity gradient and is also kinetically favoured through the modulation of the acid-base properties of the cysteine residues in the Cys2His cuprous ion binding motif of CsoR. Using RNA-seq coupled with the mechanistic insights of Cu(i) transfer between CopZ and CsoR in vitro, we propose a copper trafficking pathway for the CsoR regulon that initially involves the buffering of cytosolic copper by three CopZ chaperones followed by transfer of Cu(i) to CsoR to illicit a transcriptional response.

Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in Streptomyces lividans.

In Streptomyces lividans an extracytoplasmic copper-binding Sco protein plays a role in two unlinked processes: (i) initiating a morphological development switch and (ii) facilitating the co-factoring of the CuA domain of CcO (cytochrome c oxidase). How Sco obtains copper once secreted to the extracytoplasmic environment is unknown. In the present paper we report on a protein possessing an HX6MX21HXM motif that binds a single cuprous ion with subfemtomolar affinity. High-resolution X-ray structures of this extracytoplasmic copper chaperone-like protein (ECuC) in the apo- and Cu(I)-bound states reveal that the latter possesses a surface-accessible cuprous-ion-binding site located in a dish-shaped region of ß-sheet structure. A cuprous ion is transferred under a favourable thermodynamic gradient from ECuC to Sco with no back transfer occurring. The ionization properties of the cysteine residues in the Cys86xxxCys9° copper-binding motif of Sco, together with their positional locations identified from an X-ray structure of Sco, suggests a role for Cys86 in initiating an inter-complex ligand-exchange reaction with Cu(I)-ECuC. Generation of the genetic knockouts, Δsco, Δecuc and Δsco/ecuc, and subsequent in vivo assays lend support to the existence of a branched extracytoplasmic copper-trafficking pathway in S. lividans. One branch requires both Sco and to a certain extent ECuC to cofactor the CuA domain, whereas the other uses only Sco to deliver copper to a cuproenzyme to initiate morphological development.

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans.

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR(Sl)) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR(Sl) binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5'-TAC/GTA-3' inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR(Sl)-DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoR(Sl) tetramers. Finally, differential binding modes may exist in operator sites having more than one 5'-TAC/GTA-3' inverted repeat with implications in vivo for a mechanism of modular control.

The role of the Cys-X-X-X-Cys motif on the kinetics of cupric ion loading to the Streptomyces lividans Sco protein.

The mechanisms and spectroscopic properties generated by intermediate states upon cupric ion binding to flexible peptide motifs in proteins are of considerable interest. One such motif is the Cys-X-X-X-Cys motif characteristic to members of the Sco family of proteins. In the antibiotic producing bacterium, Streptomyces lividans, a role for its Sco protein (Sco(Sl)) as a cupric metallochaperone to the extracytoplasmic CuA domain of cytochrome c oxidase has been revealed. Stopped-flow kinetic studies have revealed a mechanism of cupric ion capture by Sco(Sl), which passes through a monothiolate intermediate, with distinct spectral features. In the present study we have used two site directed mutants of Sco(Sl), C86A and C90A, to determine which Cys in the CXXXC motif acts as the capture ligand. Comparison of kinetic and thermodynamic parameters obtained from cupric ion binding to the C86A and C90A mutants clearly indicate that Cys86 is the capture ligand and this finding can be reconciled with structural data. At subsaturating levels of cupric ions both mutants bind copper rapidly, but the absorbance properties are distinctly different from wild type Sco(Sl). This is most extreme for the C86A mutant where the Cys90 thiolate bond is considered to be weaker than the Cys86 thiolate bond in the C90A mutant. We put forward an explanation for this behaviour whereby we propose that the cupric ion is moving to a second site with no thiolate coordination.

The genome sequence of Streptomyces lividans 66 reveals a novel tRNA-dependent peptide biosynthetic system within a metal-related genomic island.

The complete genome sequence of the original isolate of the model actinomycete Streptomyces lividans 66, also referred to as 1326, was deciphered after a combination of next-generation sequencing platforms and a hybrid assembly pipeline. Comparative analysis of the genomes of S. lividans 66 and closely related strains, including S. coelicolor M145 and S. lividans TK24, was used to identify strain-specific genes. The genetic diversity identified included a large genomic island with a mosaic structure, present in S. lividans 66 but not in the strain TK24. Sequence analyses showed that this genomic island has an anomalous (G + C) content, suggesting recent acquisition and that it is rich in metal-related genes. Sequences previously linked to a mobile conjugative element, termed plasmid SLP3 and defined here as a 94 kb region, could also be identified within this locus. Transcriptional analysis of the response of S. lividans 66 to copper was used to corroborate a role of this large genomic island, including two SLP3-borne "cryptic" peptide biosynthetic gene clusters, in metal homeostasis. Notably, one of these predicted biosynthetic systems includes an unprecedented nonribosomal peptide synthetase--tRNA-dependent transferase biosynthetic hybrid organization. This observation implies the recruitment of members of the leucyl/phenylalanyl-tRNA-protein transferase family to catalyze peptide bond formation within the biosynthesis of natural products. Thus, the genome sequence of S. lividans 66 not only explains long-standing genetic and phenotypic differences but also opens the door for further in-depth comparative genomic analyses of model Streptomyces strains, as well as for the discovery of novel natural products following genome-mining approaches.

Morphological development and cytochrome c oxidase activity in Streptomyces lividans are dependent on the action of a copper bound Sco protein.

Copper has an important role in the life cycle of many streptomycetes, stimulating the developmental switch between vegetative mycelium and aerial hyphae concomitant with the production of antibiotics. In streptomycetes, a gene encoding for a putative Sco-like protein has been identified and is part of an operon that contains two other genes predicted to handle cellular copper. We report on the Sco-like protein from Streptomyces lividans (Sco(Sl)) and present a series of experiments that firmly establish a role for Sco(Sl) as a copper metallochaperone as opposed to a role as a thiol-disulphide reductase that has been assigned to other bacterial Sco proteins. Under low copper concentrations, a Δsco mutant in S. lividans displays two phenotypes; the development switch between vegetative mycelium and aerial hyphae stalls and cytochrome c oxidase (CcO) activity is significantly decreased. At elevated copper levels, the development and CcO activity in the Δsco mutant are restored to wild-type levels and are thus independent of Sco(Sl). A CcO knockout reveals that morphological development is independent of CcO activity leading us to suggest that Sco(Sl) has at least two targets in S. lividans. We establish that one Sco(Sl) target is the dinuclear Cu(A) domain of CcO and it is the cupric form of Sco(Sl) that is functionally active. The mechanism of cupric ion capture by Sco(Sl) has been investigated, and an important role for a conserved His residue is identified.

Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR).

A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoR(Sl)) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and Cu(I)-CsoR(Sl) to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoR(Sl) reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P(1)-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ΔcsoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon.

SapB and the rodlins are required for development of Streptomyces coelicolor in high osmolarity media.

Streptomyces coelicolor produces spore-forming aerial hyphae after a period of vegetative growth. These aerial structures are decorated with a hydrophobic coating of rodlets consisting of chaplins and rodlins. Here, we show that rodlins and the surface-active peptide SapB are essential for development during growth in a medium with high osmolarity. To this end, both vegetative and aerial hyphae secrete SapB, whereas rodlins are only secreted by the spore-forming aerial hyphae.