Vitamins D3 and D2 have marked but different global effects on gene expression in a rat oligodendrocyte precursor cell line.

BACKGROUND: Vitamin D deficiency increases the risk of developing multiple sclerosis (MS) but it is unclear whether vitamin D supplementation improves the clinical course of MS, and there is uncertainty about the dose and form of vitamin D (D2 or D3) to be used. The mechanisms underlying the effects of vitamin D in MS are not clear. Vitamin D3 increases the rate of differentiation of primary oligodendrocyte precursor cells (OPCs), suggesting that it might help remyelination in addition to modulating the immune response. Here we analyzed the transcriptome of differentiating rat CG4 OPCs treated with vitamin D2 or with vitamin D3 at 24 h and 72 h following onset of differentiation. METHODS: Gene expression in differentiating CG4 cells in response to vitamin D2 or D3 was quantified using Agilent DNA microarrays (n = 4 replicates), and the transcriptome data were processed and analysed using the R software environment. Differential expression between the experimental conditions was determined using LIMMA, applying the Benjamini and Hochberg multiple testing correction to p-values, and significant genes were grouped into co-expression clusters by hierarchical clustering. The functional significance of gene groups was explored by pathway enrichment analysis using the clusterProfiler package. RESULTS: Differentiation alone changed the expression of about 10% of the genes at 72 h compared to 24 h. Vitamin D2 and D3 exerted different effects on gene expression, with D3 influencing 1272 genes and D2 574 at 24 h. The expression of the vast majority of these genes was either not changed in differentiating cells not exposed to vitamin D or followed the same trajectory as the latter. D3-repressed genes were enriched for Gene Ontology (GO) categories including transcription factors and the Notch pathway, while D3-induced genes were enriched for the Ras pathway. CONCLUSIONS: This study shows that vitamin D3, compared with D2, changes the expression of a larger number of genes in OLs. Identification of genes affected by D3 in OLs should help to identify mechanisms mediating its action in MS.

Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor.

Stress-induced adaptations require multiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and 'translatome'; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as 'potentiation'. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors were more highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures.

Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor.

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.

Exploiting human and mouse transcriptomic data: Identification of circadian genes and pathways influencing health.

The power of the application of bioinformatics across multiple publicly available transcriptomic data sets was explored. Using 19 human and mouse circadian transcriptomic data sets, we found that NR1D1 and NR1D2 which encode heme-responsive nuclear receptors are the most rhythmic transcripts across sleep conditions and tissues suggesting that they are at the core of circadian rhythm generation. Analyzes of human transcriptomic data show that a core set of transcripts related to processes including immune function, glucocorticoid signalling, and lipid metabolism is rhythmically expressed independently of the sleep-wake cycle. We also identify key transcripts associated with transcription and translation that are disrupted by sleep manipulations, and through network analysis identify putative mechanisms underlying the adverse health outcomes associated with sleep disruption, such as diabetes and cancer. Comparative bioinformatics applied to existing and future data sets will be a powerful tool for the identification of core circadian- and sleep-dependent molecules.

Mistimed sleep disrupts circadian regulation of the human transcriptome.

Circadian organization of the mammalian transcriptome is achieved by rhythmic recruitment of key modifiers of chromatin structure and transcriptional and translational processes. These rhythmic processes, together with posttranslational modification, constitute circadian oscillators in the brain and peripheral tissues, which drive rhythms in physiology and behavior, including the sleep-wake cycle. In humans, sleep is normally timed to occur during the biological night, when body temperature is low and melatonin is synthesized. Desynchrony of sleep-wake timing and other circadian rhythms, such as occurs in shift work and jet lag, is associated with disruption of rhythmicity in physiology and endocrinology. However, to what extent mistimed sleep affects the molecular regulators of circadian rhythmicity remains to be established. Here, we show that mistimed sleep leads to a reduction of rhythmic transcripts in the human blood transcriptome from 6.4% at baseline to 1.0% during forced desynchrony of sleep and centrally driven circadian rhythms. Transcripts affected are key regulators of gene expression, including those associated with chromatin modification (methylases and acetylases), transcription (RNA polymerase II), translation (ribosomal proteins, initiation, and elongation factors), temperature-regulated transcription (cold inducible RNA-binding proteins), and core clock genes including CLOCK and ARNTL (BMAL1). We also estimated the separate contribution of sleep and circadian rhythmicity and found that the sleep-wake cycle coordinates the timing of transcription and translation in particular. The data show that mistimed sleep affects molecular processes at the core of circadian rhythm generation and imply that appropriate timing of sleep contributes significantly to the overall temporal organization of the human transcriptome.

Effects of insufficient sleep on circadian rhythmicity and expression amplitude of the human blood transcriptome.

Insufficient sleep and circadian rhythm disruption are associated with negative health outcomes, including obesity, cardiovascular disease, and cognitive impairment, but the mechanisms involved remain largely unexplored. Twenty-six participants were exposed to 1 wk of insufficient sleep (sleep-restriction condition 5.70 h, SEM = 0.03 sleep per 24 h) and 1 wk of sufficient sleep (control condition 8.50 h sleep, SEM = 0.11). Immediately following each condition, 10 whole-blood RNA samples were collected from each participant, while controlling for the effects of light, activity, and food, during a period of total sleep deprivation. Transcriptome analysis revealed that 711 genes were up- or down-regulated by insufficient sleep. Insufficient sleep also reduced the number of genes with a circadian expression profile from 1,855 to 1,481, reduced the circadian amplitude of these genes, and led to an increase in the number of genes that responded to subsequent total sleep deprivation from 122 to 856. Genes affected by insufficient sleep were associated with circadian rhythms (PER1, PER2, PER3, CRY2, CLOCK, NR1D1, NR1D2, RORA, DEC1, CSNK1E), sleep homeostasis (IL6, STAT3, KCNV2, CAMK2D), oxidative stress (PRDX2, PRDX5), and metabolism (SLC2A3, SLC2A5, GHRL, ABCA1). Biological processes affected included chromatin modification, gene-expression regulation, macromolecular metabolism, and inflammatory, immune and stress responses. Thus, insufficient sleep affects the human blood transcriptome, disrupts its circadian regulation, and intensifies the effects of acute total sleep deprivation. The identified biological processes may be involved with the negative effects of sleep loss on health, and highlight the interrelatedness of sleep homeostasis, circadian rhythmicity, and metabolism.

A terD domain-encoding gene (SCO2368) is involved in calcium homeostasis and participates in calcium regulation of a DosR-like regulon in Streptomyces coelicolor.

Although Streptomyces coelicolor is not resistant to tellurite, it possesses several TerD domain-encoding (tdd) genes of unknown function. To elucidate the function of tdd8, the transcriptomes of S. coelicolor strain M145 and of a tdd8 deletion mutant derivative (the Δtdd8 strain) were compared. Several orthologs of Mycobacterium tuberculosis genes involved in dormancy survival were upregulated in the deletion mutant at the visual onset of prodiginine production. These genes are organized in a putative redox stress response cluster comprising two large loci. A binding motif similar to the dormancy survival regulator (DosR) binding site of M. tuberculosis has been identified in the upstream sequences of most genes in these loci. A predicted role for these genes in the redox stress response is supported by the low NAD(+)/NADH ratio in the Δtdd8 strain. This S. coelicolor gene cluster was shown to be induced by hypoxia and NO stress. While the tdd8 deletion mutant (the Δtdd8 strain) was unable to maintain calcium homeostasis in a calcium-depleted medium, the addition of Ca(2+) in Δtdd8 culture medium reduced the expression of several genes of the redox stress response cluster. The results shown in this work are consistent with Tdd8 playing a significant role in calcium homeostasis and redox stress adaptation.

A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing.

Streptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. Escherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E. coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E. coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression.

Deciphering the regulon of Streptomyces coelicolor AbrC3, a positive response regulator of antibiotic production.

The atypical two-component system (TCS) AbrC1/C2/C3 (encoded by SCO4598, SCO4597, and SCO4596), comprising two histidine kinases (HKs) and a response regulator (RR), is crucial for antibiotic production in Streptomyces coelicolor and for morphological differentiation under certain nutritional conditions. In this study, we demonstrate that deletion of the RR-encoding gene, abrC3 (SCO4596), results in a dramatic decrease in actinorhodin (ACT) and undecylprodiginine (RED) production and delays morphological development. In contrast, the overexpression of abrC3 in the parent strain leads to a 33% increase in ACT production in liquid medium. Transcriptomic analysis and chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis of the ΔabrC3 mutant and the parent strain revealed that AbrC3 directly controls ACT production by binding to the actII-ORF4 promoter region; this was independently verified by in vitro DNA-binding assays. This binding is dependent on the sequence 5'-GAASGSGRMS-3'. In contrast, the regulation of RED production is not due to direct binding of AbrC3 to either the redZ or redD promoter region. This study also revealed other members of the AbrC3 regulon: AbrC3 is a positive autoregulator which also binds to the promoter regions of SCO0736, bdtA (SCO3328), absR1 (SCO6992), and SCO6809. The direct targets share the 10-base consensus binding sequence and may be responsible for some of the phenotypes of the ΔabrC3 mutant. The identification of the AbrC3 regulon as part of the complex regulatory network governing antibiotic production widens our knowledge regarding TCS involvement in control of antibiotic synthesis and may contribute to the rational design of new hyperproducer host strains through genetic manipulation of such systems.

Diverse control of metabolism and other cellular processes in Streptomyces coelicolor by the PhoP transcription factor: genome-wide identification of in vivo targets.

Streptomycetes sense and respond to the stress of phosphate starvation via the two-component PhoR-PhoP signal transduction system. To identify the in vivo targets of PhoP we have undertaken a chromatin-immunoprecipitation-on-microarray analysis of wild-type and phoP mutant cultures and, in parallel, have quantified their transcriptomes. Most (ca. 80%) of the previously in vitro characterized PhoP targets were identified in this study among several hundred other putative novel PhoP targets. In addition to activating genes for phosphate scavenging systems PhoP was shown to target two gene clusters for cell wall/extracellular polymer biosynthesis. Furthermore PhoP was found to repress an unprecedented range of pathways upon entering phosphate limitation including nitrogen assimilation, oxidative phosphorylation, nucleotide biosynthesis and glycogen catabolism. Moreover, PhoP was shown to target many key genes involved in antibiotic production and morphological differentiation, including afsS, atrA, bldA, bldC, bldD, bldK, bldM, cdaR, cdgA, cdgB and scbR-scbA. Intriguingly, in the PhoP-dependent cpk polyketide gene cluster, PhoP accumulates substantially at three specific sites within the giant polyketide synthase-encoding genes. This study suggests that, following phosphate limitation, Streptomyces coelicolor PhoP functions as a 'master' regulator, suppressing central metabolism, secondary metabolism and developmental pathways until sufficient phosphate is salvaged to support further growth and, ultimately, morphological development.

The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in streptomyces coelicolor.

Members of the ROK family of proteins are mostly transcriptional regulators and kinases that generally relate to the control of primary metabolism, whereby its member glucose kinase acts as the central control protein in carbon control in Streptomyces. Here, we show that deletion of SCO6008 (rok7B7) strongly affects carbon catabolite repression (CCR), growth, and antibiotic production in Streptomyces coelicolor. Deletion of SCO7543 also affected antibiotic production, while no major changes were observed after deletion of the rok family genes SCO0794, SCO1060, SCO2846, SCO6566, or SCO6600. Global expression profiling of the rok7B7 mutant by proteomics and microarray analysis revealed strong upregulation of the xylose transporter operon xylFGH, which lies immediately downstream of rok7B7, consistent with the improved growth and delayed development of the mutant on xylose. The enhanced CCR, which was especially obvious on rich or xylose-containing media, correlated with elevated expression of glucose kinase and of the glucose transporter GlcP. In liquid-grown cultures, expression of the biosynthetic enzymes for production of prodigionines, siderophores, and calcium-dependent antibiotic (CDA) was enhanced in the mutant, and overproduction of prodigionines was corroborated by matrix-assisted laser desorption ionization-time-of-flight analysis. These data present Rok7B7 as a pleiotropic regulator of growth, CCR, and antibiotic production in Streptomyces.

Identification of new developmentally regulated genes involved in Streptomyces coelicolor sporulation.

BACKGROUND: The sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation. RESULTS: We report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity. CONCLUSION: Aerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.

The effects of physiological and injurious hydrostatic pressure on murine ex vivo articular and growth plate cartilage explants: an RNAseq study.

Introduction: Chondrocytes are continuously exposed to loads placed upon them. Physiological loads are pivotal to the maintenance of articular cartilage health, while abnormal loads contribute to pathological joint degradation. Similarly, the growth plate cartilage is subject to various loads during growth and development. Due to the high-water content of cartilage, hydrostatic pressure is considered one of the main biomechanical influencers on chondrocytes and has been shown to play an important role in the mechano-regulation of cartilage. Methods: Herein, we conducted RNAseq analysis of ex vivo hip cap (articular), and metatarsal (growth plate) cartilage cultures subjected to physiological (5 MPa) and injurious (50 MPa) hydrostatic pressure, using the Illumina platform (n = 4 replicates). Results: Several hundreds of genes were shown to be differentially modulated by hydrostatic pressure, with the majority of these changes evidenced in hip cap cartilage cultures (375 significantly upregulated and 322 downregulated in 5 MPa versus control; 1022 upregulated and 724 downregulated in 50 MPa versus control). Conversely, fewer genes were differentially affected by hydrostatic pressure in the metatarsal cultures (5 significantly upregulated and 23 downregulated in 5 MPa versus control; 7 significantly upregulated and 19 downregulated in 50 MPa versus control). Using Gene Ontology annotations for Biological Processes, in the hip cap data we identified a number of pathways that were modulated by both physiological and injurious hydrostatic pressure. Pathways upregulated in response to 50 MPa versus control, included those involved in the generation of precursor metabolites and cellular respiration. Biological processes that were downregulated in this tissue included ossification, connective tissue development, and chondrocyte differentiation. Discussion: Collectively our data highlights the divergent chondrocyte phenotypes in articular and growth plate cartilage. Further, we show that the magnitude of hydrostatic pressure application has distinct effects on gene expression and biological processes in hip cap cartilage explants. Finally, we identified differential expression of a number of genes that have previously been identified as osteoarthritis risk genes, including Ctsk, and Chadl. Together these data may provide potential genetic targets for future investigations in osteoarthritis research and novel therapeutics.

Vitamins D2 and D3 Have Overlapping But Different Effects on the Human Immune System Revealed Through Analysis of the Blood Transcriptome.

Vitamin D is best known for its role in maintaining bone health and calcium homeostasis. However, it also exerts a broad range of extra-skeletal effects on cellular physiology and on the immune system. Vitamins D2 and D3 share a high degree of structural similarity. Functional equivalence in their vitamin D-dependent effects on human physiology is usually assumed but has in fact not been well defined experimentally. In this study we seek to redress the gap in knowledge by undertaking an in-depth examination of changes in the human blood transcriptome following supplementation with physiological doses of vitamin D2 and D3. Our work extends a previously published randomized placebo-controlled trial that recruited healthy white European and South Asian women who were given 15 µg of vitamin D2 or D3 daily over 12 weeks in wintertime in the UK (Nov-Mar) by additionally determining changes in the blood transcriptome over the intervention period using microarrays. An integrated comparison of the results defines both the effect of vitamin D3 or D2 on gene expression, and any influence of ethnic background. An important aspect of this analysis was the focus on the changes in expression from baseline to the 12-week endpoint of treatment within each individual, harnessing the longitudinal design of the study. Whilst overlap in the repertoire of differentially expressed genes was present in the D2 or D3-dependent effects identified, most changes were specific to either one vitamin or the other. The data also pointed to the possibility of ethnic differences in the responses. Notably, following vitamin D3 supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding pathways of the innate and adaptive immune systems, potentially shifting the immune system to a more tolerogenic status. Surprisingly, gene expression associated with type I and type II interferon activity, critical to the innate response to bacterial and viral infections, differed following supplementation with either vitamin D2 or vitamin D3, with only vitamin D3 having a stimulatory effect. This study suggests that further investigation of the respective physiological roles of vitamin D2 and vitamin D3 is warranted.

Elucidation of Focal Adhesion Kinase as a Modulator of Migration and Invasion and as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia.

The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL.

Chemotranscriptomic Profiling Defines Drug-Specific Signatures of the Glycopeptide Antibiotics Dalbavancin, Vancomycin and Chlorobiphenyl-Vancomycin in a VanB-Type-Resistant Streptomycete.

Dalbavancin, vancomycin and chlorobiphenyl-vancomycin share a high degree of structural similarity and the same primary mode of drug action. All inhibit bacterial cell wall biosynthesis through complexation with intermediates in peptidoglycan biosynthesis mediated via interaction with peptidyl-d-alanyl-d-alanine (d-Ala-d-Ala) residues present at the termini of the intermediates. VanB-type glycopeptide resistance in bacteria encodes an inducible reprogramming of bacterial cell wall biosynthesis that generates precursors terminating with d-alanyl-d-lactate (d-Ala-d-Lac). This system in Streptomyces coelicolor confers protection against the natural product vancomycin but not dalbavancin or chlorobiphenyl-vancomycin, which are semi-synthetic derivatives and fail to sufficiently activate the inducible VanB-type sensory response. We used transcriptome profiling by RNAseq to identify the gene expression signatures elucidated in S. coelicolor in response to the three different glycopeptide compounds. An integrated comparison of the results defines both the contribution of the VanB resistance system to the control of changes in gene transcription and the impact at the transcriptional level of the structural diversity present in the glycopeptide antibiotics used. Dalbavancin induces markedly more extensive changes in the expression of genes required for transport processes, RNA methylation, haem biosynthesis and the biosynthesis of the amino acids arginine and glutamine. Chlorobiphenyl-vancomycin exhibits specific effects on tryptophan and calcium-dependent antibiotic biosynthesis and has a stronger repressive effect on translation. Vancomycin predictably has a uniquely strong effect on the genes controlled by the VanB resistance system and also impacts metal ion homeostasis and leucine biosynthesis. Leaderless gene transcription is disfavoured in the core transcriptional up- and down-regulation taking place in response to all the glycopeptide antibiotics, while HrdB-dependent transcripts are favoured in the down-regulated group. This study illustrates the biological impact of peripheral changes to glycopeptide antibiotic structure and could inform the design of future semi-synthetic glycopeptide derivatives.

Metabolic and evolutionary insights into the closely-related species Streptomyces coelicolor and Streptomyces lividans deduced from high-resolution comparative genomic hybridization.

BACKGROUND: Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145. RESULTS: The high coverage and specificity (detection of three nucleotide differences) of the new microarrays used has highlighted the macroscopic genomic differences between two S. lividans strains and S. coelicolor. In a series of case studies we have validated the microarray and have identified subtle changes in genomic structure which occur in the Asp-activating adenylation domains of CDA non-ribosomal peptide synthetase genes which provides evidence of gene shuffling between these domains. We also identify single nucleotide sequence inter-species differences which exist in the actinorhodin biosynthetic gene cluster. As the glyoxylate bypass is non-functional in both S. lividans strains due to the absence of the gene encoding isocitrate lyase it is likely that the ethylmalonyl-CoA pathway functions as the alternative mechanism for the assimilation of C2 compounds. CONCLUSIONS: This study provides evidence for widespread genetic recombination, rather than it being focussed at 'hotspots', suggesting that the previously proposed 'archipelago model' of genomic differences between S. coelicolor and S. lividans is unduly simplistic. The two S. lividans strains investigated differ considerably in genetic complement, with TK24 lacking 175 more genes than its wild-type parent when compared to S. coelicolor. Additionally, we confirm the presence of bldB in S. lividans and deduce that S. lividans 66 and TK24, both deficient in the glyoxylate bypass, possess an alternative metabolic mechanism for the assimilation of C2 compounds. Given that streptomycetes generally display high genetic instability it is envisaged that these high-density arrays will find application for rapid assessment of genome content (particularly amplifications/deletions) in mutational studies of S. coelicolor and related species.

NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolor.

Streptomycetes have a complex morphogenetic programme culminating in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. We here compared whole genome expression of a wild-type colony of Streptomyces coelicolor forming aerial hyphae and spores with that of the chp null mutant that forms few aerial structures. This revealed that expression of 244 genes was significantly altered, among which genes known to be involved in development. One of the genes that was no longer expressed in the DeltachpABCDEFGH mutant was nepA, which was previously shown to be expressed in a compartment connecting the substrate mycelium with the sporulating parts of the aerial mycelium. We here show that expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall. Germination of spores of a nepA deletion mutant was faster and more synchronous, resulting in colonies with an accelerated morphogenetic programme. Crucially, spores of the DeltanepA mutant also germinated in water, unlike those of the wild-type strain. Taken together, NepA is the first bacterial structural cell wall protein that is important for maintenance of spore dormancy under unfavourable environmental conditions.

One of the two genes encoding nucleoid-associated HU proteins in Streptomyces coelicolor is developmentally regulated and specifically involved in spore maturation.

Streptomyces genomes encode two homologs of the nucleoid-associated HU proteins. One of them, here designated HupA, is of a conventional type similar to E. coli HUalpha and HUbeta, while the other, HupS, is a two-domain protein. In addition to the N-terminal part that is similar to that of HU proteins, it has a C-terminal domain that is similar to the alanine- and lysine-rich C termini of eukaryotic linker histones. Such two-domain HU proteins are found only among Actinobacteria. In this phylum some organisms have only a single HU protein of the type with a C-terminal histone H1-like domain (e.g., Hlp in Mycobacterium smegmatis), while others have only a single conventional HU. Yet others, including the streptomycetes, produce both types of HU proteins. We show here that the two HU genes in Streptomyces coelicolor are differentially regulated and that hupS is specifically expressed during sporulation, while hupA is expressed in vegetative hyphae. The developmental upregulation of hupS occurred in sporogenic aerial hyphal compartments and was dependent on the developmental regulators whiA, whiG, and whiI. HupS was found to be nucleoid associated in spores, and a hupS deletion mutant had an average nucleoid size in spores larger than that in the parent strain. The mutant spores were also defective in heat resistance and spore pigmentation, although they possessed apparently normal spore walls and displayed no increased sensitivity to detergents. Overall, the results show that HupS is specifically involved in sporulation and may affect nucleoid architecture and protection in spores of S. coelicolor.

Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 in Streptomyces coelicolor A3(2).

The AbsA1-AbsA2 two component signalling system of Streptomyces coelicolor has long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of a ΔabsA2 deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complemented ΔabsA2 mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deleting absA2 on gene expression and to identify the in vivo AbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions of redZ and actII-orfIV AbsA2 binds to several previously unidentified sites within the cda biosynthetic gene cluster within and/or upstream of SCO3215-SCO3216, SCO3217, SCO3229-SCO3230, and SCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual 'biphasic' ChIP peaks were observed with AbsA2 binding across the regulatory genes actII-orfIV and redZ and the absA2 gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis in Streptomyces coelicolor and the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.