RNA Preparation and RNA-Seq Bioinformatics for Comparative Transcriptomics.

The principal transcriptome analysis is the determination of differentially expressed genes across experimental conditions. For this, the next-generation sequencing of RNA (RNA-seq) has several advantages over other techniques, such as the capability of detecting all the transcripts in one assay over RT-qPCR, such as its higher accuracy and broader dynamic range over microarrays or the ability to detect novel transcripts, including non-coding RNA molecules, at nucleotide-level resolution over both techniques. Despite these advantages, many microbiology laboratories have not yet applied RNA-seq analyses to their investigations. The high cost of the equipment for next-generation sequencing is no longer an issue since this intermediate part of the analysis can be provided by commercial or central services. Here, we detail a protocol for the first part of the analysis, the RNA extraction and an introductory protocol to the bioinformatics analysis of the sequencing data to generate the differential expression results.

Characterization of Microbial Diversity in Decayed Wood from a Spanish Forest: An Environmental Source of Industrially Relevant Microorganisms.

Rotting wood is inhabited by a large diversity of bacteria, fungi, and insects with complex environmental relationships. The aim of this work was to study the composition of the microbiota (bacteria and fungi) in decaying wood from a northwest Spanish forest as a source of industrially relevant microorganisms. The analyzed forest is situated in a well-defined biogeographic area combining Mediterranean and temperate macrobioclimates. Bacterial diversity, determined by metagenome analyses, was higher than fungal heterogeneity. However, a total of 194 different cultivable bacterial isolates (mainly Bacillaceae, Streptomycetaceae, Paenibacillaceae, and Microbacteriaceae) were obtained, in contrast to 343 fungal strains (mainly Aspergillaceae, Hypocreaceae, and Coniochaetaceae). Isolates traditionally known as secondary metabolite producers, such as Actinobacteria and members of the Penicillium genus, were screened for their antimicrobial activity by the detection of antibiotic biosynthetic clusters and competitive bioassays against fungi involved in wood decay. In addition, the ability of Penicillium isolates to degrade cellulose and release ferulic acid from wood was also examined. These results present decaying wood as an ecologically rich niche and a promising source of biotechnologically interesting microorganisms.

Screening of Antibiotic Gene Clusters in Microorganisms Isolated from Wood.

The biosphere of Earth is made up of a variety of ecosystems governed by complex biological interactions, some of them mediated by microbial bioactive secondary metabolites. These metabolites such as antibiotics (e.g., polyketides and nonribosomal peptides) have been receiving increasing attention, due to their multiple pharmaceutical uses. Besides, antibiotic resistance is on the rise, and it is currently regarded as one of the greatest threats to global human health. The screening of novel antimicrobial polyketides and nonribosomal peptides in poorly studied ecosystems is an interesting alternative to address the problem of antibiotic resistance. This chapter updates a molecular method to identify antibiotics gene clusters and their subsequent production and activity validation. On the one hand, a PCR method based on degenerated primers for nonribosomal peptide synthases (NRPS) and the polyketide synthases (PKS) genes is used as an initial fast screening. On the other hand, a bioassay-based method is the protocol selected for the production confirmation and antibacterial effect estimation. These methods are applied to screen Actinobacteria and Penicillium species as main antibiotic producers isolated from wood.

Coupled Transcriptomics for Differential Expression Analysis and Determination of Transcription Start Sites: Design and Bioinformatics.

The term coupled transcriptomics is coined to describe a design of an RNA-seq experiment intended for both differential expression analysis and genome-wide determination of the transcription start sites (TSS). The minimal requirements for the first analysis are two experimental conditions with at least two biological replicates enabling statistical tests. The second analysis involves the bioinformatics comparison of the data generated from a control RNA-seq library with another library enriched in primary transcripts using Terminator™ 5'-phosphate-dependent exonuclease, in an experiment denominated differential RNA-seq (dRNA-seq). Usually, dRNA-seq is carried out with specific protocols for library construction, different of those used for common differential expression analysis. Our experimental design allows to use the same data for both analyses, reducing the number of libraries to be generated and sequenced. This is a guide for designing a coupled transcriptomics experiment and for the subsequent bioinformatics procedures. The proposed methods can be applied to the detection and study of small RNA genes.

Self-control of the PHO regulon: the PhoP-dependent protein PhoU controls negatively expression of genes of PHO regulon in Streptomyces coelicolor.

Phosphate control of the biosynthesis of secondary metabolites in Streptomyces is mediated by the two component system PhoR-PhoP. Linked to the phoR-phoP cluster, and expressed in the opposite orientation, is a phoU-like encoding gene with low identity to the phoU gene of Escherichia coli. Expression of this phoU-like gene is strictly dependent on PhoP activation. We have isolated a PhoU-null mutant and used transcriptomic and RNA-sequencing (RNA-seq) procedures to identify its transcription start site and regulation. RNA-seq studies identified two transcription start sites, one upstream of phoU and the second upstream of the mptA gene. Whereas transcription of PhoU is entirely dependent on PhoP, expression of the downstream mtpA gene is only partially dependent on PhoP activation. The phoU mutant grows more slowly than the parental strain, sporulates poorly and the spores lack pigmentation. Production of actinorhodin and undecylprodigiosin decreased in the phoU mutant, indicating that PhoU has a positive modulating effect on production of these antibiotics. Indeed, transcriptional studies of expression of the actII-ORF4 and redD genes indicated that the PhoU protein activates expression of these antibiotic regulators. Using the glpQ1 promoter as in vivo reporter of the activity of the PHO regulon genes, we observed that expression of glpQ1 is negatively modulated by PhoU. These results were confirmed by reverse transcription-PCR studies of three genes of the PHO regulon; that is, glpQ1, pstS and phoR. In conclusion, PhoU acts as a negative modulator of expression of the PHO regulon genes and as phoU expression is strictly dependent on PhoP activation, this mechanism appears to work as a feed-back control mechanism (self-regulation).The Journal of Antibiotics advance online publication, 1 November 2017; doi:10.1038/ja.2017.130.

Genome Sequencing of Steroid Producing Bacteria Using Ion Torrent Technology and a Reference Genome.

The Next-Generation Sequencing technology has enormously eased the bacterial genome sequencing and several tens of thousands of genomes have been sequenced during the last 10 years. Most of the genome projects are published as draft version, however, for certain applications the complete genome sequence is required.In this chapter, we describe the strategy that allowed the complete genome sequencing of Mycobacterium neoaurum NRRL B-3805, an industrial strain exploited for steroid production, using Ion Torrent sequencing reads and the genome of a close strain as the reference. This protocol can be applied to analyze the genetic variations between closely related strains; for example, to elucidate the point mutations between a parental strain and a random mutagenesis-derived mutant.

RNA-Seq-Based Comparative Transcriptomics: RNA Preparation and Bioinformatics.

The major transcriptome analysis is the determination of differentially expressed genes across experimental conditions. For this, the next-generation sequencing of RNA (RNA-seq) is an increasingly cost-effective technology for the analysis of transcriptomes with several advantages over gene expression microarrays, such as its higher sensitivity and accuracy, broader dynamic range, and the ability to detect novel transcripts, including noncoding RNA molecules, at nucleotide-level resolution. Although these advantages, many microbiology laboratories have not yet applied RNA-seq analyses to their investigations. The high cost of the equipment for next-generation sequencing is no longer an issue, since this intermediate part of the analysis can be provided by commercial or central services. Here, we detail a protocol for the first part of the analysis, the RNA extraction, and an introductory protocol to the bioinformatics analysis of the sequencing data that generates the differential expression results.

Intra- and Extra-cellular Proteome Analyses of Steroid-Producer Mycobacteria.

The importance of the pathogenic mycobacteria has mainly focused the omic analyses on different aspects of their clinical significance. In contrast, those industrially relevant mycobacteria have received less attention, even though the steroids market sales in 2011, in example, were estimated in $8 billion.The extra-cellular proteome, due to its relevance in the sterols processing and uptake; as well as the intra-cellular proteome, because of its role in steroids bioconversion, are the core of the present chapter. As a proof of concept, the obtaining methods for both sub-proteomes of Mycobacterium neoaurum NRRL B-3805, a relevant industrial strain involved in steroids production, have been developed. Thus, procedures and relevant key points of these proteomes analyses are fully described.

Complete genome sequence of 'Mycobacterium neoaurum' NRRL B-3805, an androstenedione (AD) producer for industrial biotransformation of sterols.

Microbial bioconversion of sterols into high value steroid precursors, such as 4-androstene-3,17-dione (AD), is an industrial challenge. Genes and enzymes involved in sterol degradation have been proposed, although the complete pathway is not yet known. The genome sequencing of the AD producer strain 'Mycobacterium neoaurum' NRRL B-3805 (formerly Mycobacterium sp. NRRL B-3805) will serve to elucidate the critical steps for industrial processes and will provide the basis for further genetic engineering. The genome comprises a circular chromosome (5 421 338bp), is devoid of plasmids and contains 4844 protein-coding genes.

Transcriptional control of the F0F1-ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values.

Corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. Its F(0)F(1)-ATPase operon (atpBEFHAGDC) is expressed optimally at pH 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpB gene. Expression of this operon is controlled by the SigmaH factor. The sigmaH gene (sigH) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. A mutant deleted in the sigH gene expressed the atpBEFHAGDC operon optimally at pH 7.0 at difference of the wild-type strain (optimal expression at pH 9.0). These results suggested that the SigmaH factor is involved in pH control of expression of the F(0) F(1) ATPase operon. The SigmaH protein was expressed in Escherichia coli fused to the GST (glutathione-S-transferase) and purified to homogeneity by affinity chromatography on a GSTrap HP column. The fused protein was identified by immunodetection with anti-GST antibodies. DNA-binding studies by electrophoretic mobility shift assays showed that the SigH protein binds to a region of the atpB promoter containing the sigmaH recognition sequence (-35)TTGGAT…18nt…GTTA(-10). SigmaH plays an important role in the cascade of control of pH stress in Corynebacterium.

Competition between the GlnR and PhoP regulators for the glnA and amtB promoters in Streptomyces coelicolor.

Interaction of regulatory networks is a subject of great interest in systems biology of bacteria. Phosphate control of metabolism in Streptomyces is mediated by the two-component system PhoR-PhoP. Similarly, the utilization of different nitrogen sources is controlled by the regulator GlnR. Transcriptomic and biochemical analysis revealed that glnA (encoding a glutamine synthetase), glnR and other nitrogen metabolism genes are under PhoP control. DNA-binding experiments showed that PhoP binds to other nitrogen-regulated genes (SCO0255, SCO01863 and ureA). Using the glnA promoter as model, we observed that PhoP and GlnR compete for binding to the same promoter region, showing GlnR a higher affinity. Using a total of 14 GlnR-binding sites (50 direct repeat units) we established two information-based models that describe the GlnR box as consisting of two 11-nt direct repeats each with clear differences to PHO box. DNA-binding studies with different mutant sequences of glnA promoter revealed that the sequence recognized by GlnR is found in the coding strand whereas that recognized by PhoP is overlapping in the non-coding strand. In amtB promoter PhoP and GlnR boxes are not totally overlapping and both proteins bind simultaneously. PhoP control of nitrogen metabolism genes helps to balance the cellular P/N equilibrium.

Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506).

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.

Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor.

Phosphate controls the biosynthesis of many classes of secondary metabolites that belong to different biosynthetic groups, indicating that phosphate control is a general mechanism governing secondary metabolism. We refer in this article to the molecular mechanisms of regulation, mediated by the two-component system PhoR-PhoP, of the primary metabolism and the biosynthesis of antibiotics. The two-component PhoR-PhoP system is conserved in all Streptomyces and related actinobacteria sequenced so far, and involves a third component PhoU that modulates the signal transduction cascade. The PhoP DNA-binding sequence is well characterized in Streptomyces coelicolor. It comprises at least two direct repeat units of 11 nt, the first seven of which are highly conserved. Other less conserved direct repeats located adjacent to the core ones can also be bound by PhoP through cooperative protein-protein interactions. The phoR-phoP operon is self-activated and requires phosphorylated PhoP to mediate the full response. About 50 up-regulated PhoP-dependent genes have been identified by comparative transcriptomic studies between the parental S. coelicolor M145 and the ΔphoP mutant strains. The PhoP regulation of several of these genes has been studied in detail using EMSA and DNase I footprinting studies as well as in vivo expression studies with reporter genes and RT-PCR transcriptomic analyses.

The RNA polymerase omega factor RpoZ is regulated by PhoP and has an important role in antibiotic biosynthesis and morphological differentiation in Streptomyces coelicolor.

The RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ßß' core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZ strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZ mutant with the wild-type rpoZ allele restored both phenotype and antibiotic production. Interestingly, the rpoZ gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that rpoZ promoter activity was increased in a ΔphoP background, it can be concluded that rpoZ is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in Streptomyces.

Cross-talk of global nutritional regulators in the control of primary and secondary metabolism in Streptomyces.

Limitation of different nutrients in Streptomyces coelicolor A3(2) triggers nutrient-stress responses, mediated by PhoP, GlnR, AfsR and other regulators, that are integrated at the molecular level and control secondary metabolite biosynthesis and differentiation. In addition, utilization of chitin or N-acetylglucosamine regulates secondary metabolite biosynthesis by a mechanism mediated by DasR. Phosphate control of primary and secondary metabolism in Streptomyces species is mediated by the two-component PhoR-PhoP system. In S. coelicolor, PhoP controls secondary metabolism by binding to a PHO box in the afsS promoter overlapping with the AfsR binding site. Therefore, the afsS promoter serves to integrate the PhoP-mediated response to phosphate limitation and AfsR-mediated responses to other unknown environmental stimuli. Interestingly, phosphate control oversees nitrogen regulation but not vice versa. In ΔphoP mutants, expression of some nitrogen metabolism genes including glnA, glnII and glnK is increased. Phosphate control of these genes is exerted through binding of PhoP to the promoters of glnR (the global nitrogen regulator), glnA, glnII and the amtB-glnK-glnD operon. This regulation allows a 'metabolic homeostasis' of phosphate and nitrogen utilization pathways, preventing nutritional unbalances. Similar mechanisms of interaction between phosphate control and carbon catabolite regulation or between phosphate and DasR-mediated N-acetylglucosamine regulation appear to exist. Transport of N-acetylglucosamine by the NagE2 permease and, therefore, regulation of secondary metabolism, is dependent upon the balance of phosphorylated/dephosphorylated proteins of the N-acetylglucosamine phosphotransferase system. These findings provide the bases for understanding the mechanisms underlying systems biology of Streptomyces species.

Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP.

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. 'Information theory' quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.

Cross-talk between two global regulators in Streptomyces: PhoP and AfsR interact in the control of afsS, pstS and phoRP transcription.

The regulatory proteins AfsR and PhoP control expression of the biosynthesis of actinorhodin and undecylprodigiosin in Streptomyces coelicolor. Electrophoretic mobility shift assays showed that PhoP(DBD) does not bind directly to the actII-ORF4, redD and atrA promoters, but it binds to the afsS promoter, in a region overlapping with the AfsR operator. DNase I footprinting studies revealed a PhoP protected region of 26 nt (PHO box; two direct repeats of 11 nt) that overlaps with the AfsR binding sequence. Binding experiments indicated a competition between AfsR and PhoP; increasing concentrations of PhoP(DBD) resulted in the disappearance of the AfsR-DNA complex. Expression studies using the reporter luxAB gene coupled to afsS promoter showed that PhoP downregulates afsS expression probably by a competition with the AfsR activator. Interestingly, AfsR binds to other PhoP-regulated promoters including those of pstS (a component of the phosphate transport system) and phoRP (encoding the two component system itself). Analysis of the AfsR-protected sequences in each of these promoters allowed us to distinguish the AfsR binding sequence from the overlapping PHO box. The reciprocal regulation of the phoRP promoter by AfsR and of afsS by PhoP suggests a fine interplay of these regulators on the control of secondary metabolism.

Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor.

Expression of genes belonging to the pho regulon in Streptomyces coelicolor is positively regulated (as shown by comparing the wild-type and a DeltaphoP mutant) by binding of the response regulator PhoP to 11-nt direct repeats (DRus). These sequences have been found in over 100 genes of Streptomyces coelicolor; 20 of them were cloned and the binding of PhoP(DBD) to most of their promoters has been shown by electrophoretic mobility shift assays. Deletion experiments showed that at least two DRus are required for proper binding of PhoP(DBD). Deletion of 1 nt leaving a 10-nt direct repeat reduced drastically binding of PhoP(DBD). Three different types of operators have been identified. Complex operators (class III) contain up to six DRus, some of them with poor conservation of the 11-nt consensus sequence, which however were protected by PhoP(DBD) in footprinting analyses. A cooperative binding of PhoP(DBD) molecules initiated at conserved core DRus appears to be the mechanism involved in binding of several PhoP(DBD) monomers to those complex operators. The information theory-based model that incorporates the positive or negative contribution to the binding of PhoP(DBD) of adjacent sequences has been used to deduce the structure of PHO boxes and the relevance of each DRu.

Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions.

Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of Streptomyces coelicolor by homology with the amino acid sequence obtained from the PhoA protein of Streptomyces griseus. PhoA and PhoC correspond to broad-spectrum alkaline phosphatases whereas PhoD is similar to a Ca(2+)-dependent phospholipase D of Streptomyces chromofuscus. The phoA and phoD genes were efficiently expressed in R5 medium under phosphate-limited conditions, as shown by studies using the xylE reporter gene, whereas phoC was poorly transcribed under the same conditions. Expression of phoA was clearly PhoP-dependent since it was not transcribed in the S. coelicolor DeltaphoP mutant and was strongly activated under low phosphate concentrations. Similarly, expression of phoD was PhoP-dependent and highly sensitive to phosphate availability. By contrast, expression of phoC was not PhoP-dependent. Electrophoretic mobility shift assays showed that PhoP binds to the phoA and phoD promoters, but not to that of phoC. Footprinting studies with GST-PhoP revealed the presence of a PHO box (two direct 11 nt repeats) in the phoA promoter and two PHO boxes in the promoter of phoD. The transcription start points of the three promoters were identified by primer extension. The transcription start point of phoD coincides with the G of its translation start codon, indicating that this gene is transcribed as a leaderless mRNA. The deduced -10 and -35 regions of phoD (but not those of phoA) overlapped with the PHO boxes in this promoter, suggesting that an excess of PhoP interferes with binding of the RNA polymerase to this promoter. In summary, the three promoters showed clear differences in the modulation of their expression by PhoP.

Genome-wide transcriptomic and proteomic analysis of the primary response to phosphate limitation in Streptomyces coelicolor M145 and in a DeltaphoP mutant.

Phosphate limitation in Streptomyces and in other bacteria triggers expression changes of a large number of genes. This response is mediated by the two-component PhoR-PhoP system. A Streptomyces coelicolor DeltaphoP mutant (lacking phoP) has been obtained by gene replacement. A genome-wide analysis of the primary response to phosphate limitation using transcriptomic and proteomic studies has been made in the parental S. coelicolor M145 and in the DeltaphoP mutant strains. Statistical analysis of the contrasts between the four sets of data generated (two strains under two phosphate conditions) allowed the classification of all genes into 12 types of profiles. The primary response to phosphate limitation involves upregulation of genes encoding scavenging enzymes needed to obtain phosphate from different phosphorylated organic compounds and overexpression of the high-affinity phosphate transport system pstSCAB. Clear interactions have been found between phosphate metabolism and expression of nitrogen-regulated genes and between phosphate and nitrate respiration genes. PhoP-dependent repressions of antibiotic biosynthesis and of the morphological differentiation genes correlated with the observed DeltaphoP mutant phenotype. Bioinformatic analysis of the presence of PHO boxes (PhoP-binding sequences) in the upstream regions of PhoP-controlled genes were validated by binding of PhoP, as shown by electrophoretic mobility shift assays.