Comparative analysis of non-coding RNAs in the antibiotic-producing Streptomyces bacteria

Moody, Matthew J.; Young, Rachel; Jones, Stephanie E.; Elliot, Marie A.

doi:10.1186/1471-2164-14-558

Cited by 56 publications

(75 citation statements)

References 97 publications

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“…Such effects have been suggested for antibiotic production in Streptomyces, with many ncRNAs being specified within antibiotic biosynthesis clusters (73). Taking into account available data about ncRNAs in S. coelicolor (73)(74)(75), we identified two inserts in intergenic small RNA (sRNA) regions (scr3437 and scr3580) that influenced RED production.…”

Section: Discussionmentioning

confidence: 99%

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

Wang

Chater

et al. 2017

Appl Environ Microbiol

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Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genomewide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.KEYWORDS genome-wide, transposition mutagenesis, Streptomyces coelicolor, antibiotic biosynthesis, prodiginine

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Section: Discussionmentioning

confidence: 99%

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

Wang

Chater

et al. 2017

Appl Environ Microbiol

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“…Samples were frozen at Ϫ80°C for 3 to 6 days. Total RNA was extracted by mechanical disruption, and coextracted DNA was digested using Turbo DNase (Ambion), as previously described by Moody et al (22). Total RNA was quantified using a NanoDrop ND-1000, and RNA integrity was confirmed by size fractionating 2 g of total RNA on an agarose gel.…”

Section: Methodsmentioning

confidence: 99%

Resuscitation-Promoting Factors Are Cell Wall-Lytic Enzymes with Important Roles in the Germination and Growth of Streptomyces coelicolor

et al. 2015

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Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage. Streptomyces species are Gram-positive bacteria that abound in soil environments. They are renowned for their secondary metabolic capabilities; they produce a multitude of compounds that have found utility in both medicine and agriculture, including a vast array of antibiotics, chemotherapeutics, immunosuppressants, and antiparasitic agents (1). They are also well known for their complex, multicellular developmental cycle (2). The Streptomyces life cycle can be broadly divided into two stages: vegetative growth and reproductive development. Unlike most bacteria, Streptomyces organisms are filamentous, and during the vegetative phase of their life cycle, they grow by hyphal tip extension and branching, ultimately forming an interwoven network of cells known as the vegetative mycelium (3). Under certain as yet poorly defined stress conditions, vegetative growth ceases, and reproductive growth ensues. Here, unbranched filamentous cells, termed aerial hyphae, grow away from the vegetative mycelium and extend into the air. These cells then undergo a synchronous round of chromosome segregation, cell division, and cell wall modification to yield chains of dormant exospores (4). These spores are resistant to a wide variety of environmental insults and can be widely dispersed.A dormant cell state is not unique to the streptomycetes, and indeed many well-studied bacteria (e...

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“…1A). Analysis of data from previously published RNA-sequencing (RNA-seq) experiments (32) suggested that each of rnj, dapA, and thyX was expressed from a distinct promoter but also shared low-level readthrough transcription, particularly between dapA and rnj (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…1B). The investigation of available RNA-seq data (32) suggested that while these three genes do indeed appear to be cotranscribed in S. venezuelae, there exists an additional encoding genes (thyX, dapA, and rnj) is shown a graph of relative transcript levels throughout this region, as determined using RNA-seq data from RNA samples representing all stages of S. venezuelae growth (RNA was harvested at three distinct growth stages, vegetative, fragmentation, and sporulation, and was combined prior to sequencing). (B) Genetic organization of the RNase III-encoding rnc region.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were harvested either at time points corresponding to vegetative growth (6 to 8 h) for rRNA analysis and hpf expression analysis or 23 h after ethanol shock for analysis of jadomycin B biosynthetic cluster transcripts. RNA was extracted as described previously (31), using a modified guanidium thiocyanate protocol (32). Cells were lysed by vortexing for 2 min with glass beads in a solution containing 4 M guanidium thiocyanate, 25 mM trisodium citrate dihydrate, 0.5% (wt/vol) sodium N-lauroylsarcosinate, and 0.8% ␤-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

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Development, Antibiotic Production, and Ribosome Assembly in Streptomyces venezuelae Are Impacted by RNase J and RNase III Deletion

2014

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b RNA metabolism is a critical but frequently overlooked control element affecting virtually every cellular process in bacteria. RNA processing and degradation is mediated by a suite of ribonucleases having distinct cleavage and substrate specificity. Here, we probe the role of two ribonucleases (RNase III and RNase J) in the emerging model system Streptomyces venezuelae. We show that each enzyme makes a unique contribution to the growth and development of S. venezuelae and further affects the secondary metabolism and antibiotic production of this bacterium. We demonstrate a connection between the action of these ribonucleases and translation, with both enzymes being required for the formation of functional ribosomes. RNase III mutants in particular fail to properly process 23S rRNA, form fewer 70S ribosomes, and show reduced translational processivity. The loss of either RNase III or RNase J additionally led to the appearance of a new ribosomal species (the 100S ribosome dimer) during exponential growth and dramatically sensitized these mutants to a range of antibiotics. R ibonucleases (RNases) are enzymes that process and degrade RNA molecules; consequently, they are critical for RNA maturation, RNA stability, and posttranscriptional regulation (1). In bacterial cells, the finely tuned balance between RNA synthesis and RNA degradation allows for rapid adaptation to changing environments, proper processing of noncoding RNAs, and efficient recycling of ribonucleotides (2).Our understanding of RNA metabolism to date is based largely on studies of Escherichia coli and Bacillus subtilis, which employ distinct enzymes for RNA processing and degradation. In E. coli, RNase E is the central component of the RNA degradosome; as such, it is responsible for much of the RNA decay in this organism. B. subtilis lacks this enzyme, and acting in its place are three other nucleases: RNase J1/J2 and RNase Y (3, 4). Virtually all bacteria contain at least one of RNase E (or its paralog, RNase G), RNase J, or RNase Y (5). These RNases are unrelated in primary sequence and mechanism of catalysis but have similar substrate specificity: they all have single-strand-specific endonuclease activity and preferentially cleave AU-rich regions (4, 6, 7). Unlike RNase E and Y, however, RNase J has the capacity to act both as an endonuclease and as a 5= exonuclease (8, 9). The analysis of available genome sequences suggests that more than half of all bacteria, and over two-thirds of Archaea, possess an RNase J homologue (6,8). In addition to these diverse single-strand-specific RNases, most bacteria also encode the double-strand-specific RNase III (10). This enzyme is highly conserved in bacteria and eukaryotes and has a critical role in posttranscriptional regulation, where it cleaves double-stranded substrates, such as those resulting from the base pairing of an mRNA and noncoding RNA (11), or those associated with highly structured RNAs, such as ribosomal RNAs (rRNAs) (12, 13). There can be considerable functional interplay between RNases, ...

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Comparative analysis of non-coding RNAs in the antibiotic-producing Streptomyces bacteria

Cited by 56 publications

References 97 publications

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

Resuscitation-Promoting Factors Are Cell Wall-Lytic Enzymes with Important Roles in the Germination and Growth of Streptomyces coelicolor

Development, Antibiotic Production, and Ribosome Assembly in Streptomyces venezuelae Are Impacted by RNase J and RNase III Deletion

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