Specialized Metabolites from Ribosome Engineered Strains of Streptomyces clavuligerus

Bacterial specialized metabolites are of immense importance because of their medicinal, industrial, and agricultural applications. Streptomyces clavuligerus is a known producer of such compounds; however, much of its metabolic potential remains unknown, as many associated biosynthetic gene clusters are silent or expressed at low levels. The overexpression of ribosome recycling factor (frr) and ribosome engineering (induced rpsL mutations) in other Streptomyces spp. has been reported to increase the production of known specialized metabolites. Therefore, we used an overexpression strategy in combination with untargeted metabolomics, molecular networking, and in silico analysis to annotate 28 metabolites in the current study, which have not been reported previously in S. clavuligerus. Many of the newly described metabolites are commonly found in plants, further alluding to the ability of S. clavuligerus to produce such compounds under specific conditions. In addition, the manipulation of frr and rpsL led to different metabolite production profiles in most cases. Known and putative gene clusters associated with the production of the observed compounds are also discussed. This work suggests that the combination of traditional strain engineering and recently developed metabolomics technologies together can provide rapid and cost-effective strategies to further speed up the discovery of novel natural products.

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Genome analysis suggests the bacterial family Acetobacteraceae is a source of undiscovered specialized metabolites

Antonie van Leeuwenhoek ◽

10.1007/s10482-021-01676-7 ◽

2021 ◽

Author(s):

Juan Guzman ◽

Andreas Vilcinskas

Keyword(s):

Hot Springs ◽

In Silico Analysis ◽

Gene Clusters ◽

Acetic Acid Bacteria ◽

Specialized Metabolites ◽

The Family ◽

Genes Encoding ◽

Peptide Biosynthesis ◽

Taxonomic Groups ◽

Type 3

AbstractAcetobacteraceae is an economically important family of bacteria that is used for industrial fermentation in the food/feed sector and for the preparation of sorbose and bacterial cellulose. It comprises two major groups: acetous species (acetic acid bacteria) associated with flowers, fruits and insects, and acidophilic species, a phylogenetically basal and physiologically heterogeneous group inhabiting acid or hot springs, sludge, sewage and freshwater environments. Despite the biotechnological importance of the family Acetobacteraceae, the literature does not provide any information about its ability to produce specialized metabolites. We therefore constructed a phylogenomic tree based on concatenated protein sequences from 141 type strains of the family and predicted the presence of small-molecule biosynthetic gene clusters (BGCs) using the antiSMASH tool. This dual approach allowed us to associate certain biosynthetic pathways with particular taxonomic groups. We found that acidophilic and acetous species contain on average ~ 6.3 and ~ 3.4 BGCs per genome, respectively. All the Acetobacteraceae strains encoded proteins involved in hopanoid biosynthesis, with many also featuring genes encoding type-1 and type-3 polyketide and non-ribosomal peptide synthases, and enzymes for aryl polyene, lactone and ribosomal peptide biosynthesis. Our in silico analysis indicated that the family Acetobacteraceae is a potential source of many undiscovered bacterial metabolites and deserves more detailed experimental exploration.

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Production of Plant-Associated Volatiles by Select Model and Industrially Important Streptomyces spp.

Microorganisms ◽

10.3390/microorganisms8111767 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1767 ◽

Cited By ~ 1

Author(s):

Zhenlong Cheng ◽

Sean McCann ◽

Nicoletta Faraone ◽

Jody-Ann Clarke ◽

E. Abbie Hudson ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Biological Activities ◽

Gene Clusters ◽

Gas Chromatography Mass Spectrometry ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Specialized Metabolites ◽

Streptomyces Spp ◽

Volatile Organic

The Streptomyces produce a great diversity of specialized metabolites, including highly volatile compounds with potential biological activities. Volatile organic compounds (VOCs) produced by nine Streptomyces spp., some of which are of industrial importance, were collected and identified using gas chromatography–mass spectrometry (GC-MS). Biosynthetic gene clusters (BGCs) present in the genomes of the respective Streptomyces spp. were also predicted to match them with the VOCs detected. Overall, 33 specific VOCs were identified, of which the production of 16 has not been previously reported in the Streptomyces. Among chemical classes, the most abundant VOCs were terpenes, which is consistent with predicted biosynthetic capabilities. In addition, 27 of the identified VOCs were plant-associated, demonstrating that some Streptomyces spp. can also produce such molecules. It is possible that some of the VOCs detected in the current study have roles in the interaction of Streptomyces with plants and other higher organisms, which might provide opportunities for their application in agriculture or industry.

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A global co-expression network approach for connecting genes to specialized metabolic pathways in plants

10.1101/093914 ◽

2016 ◽

Cited By ~ 3

Author(s):

Jennifer H. Wisecaver ◽

Alexander T. Borowsky ◽

Vered Tzin ◽

Georg Jander ◽

Daniel J. Kliebenstein ◽

...

Keyword(s):

Genetic Basis ◽

Meta Analysis ◽

Null Distribution ◽

Gene Clusters ◽

Cytochrome P450s ◽

Model Organisms ◽

Biosynthetic Gene Clusters ◽

Terpene Synthases ◽

Specialized Metabolites ◽

Data Source

AbstractPlants produce a tremendous diversity of specialized metabolites (SMs) to interact with and manage their environment. A major challenge hindering efforts to tap this seemingly boundless source of pharmacopeia is the identification of SM pathways and their constituent genes. Given the well-established observation that the genes comprising a SM pathway are co-regulated in response to specific environmental conditions, we hypothesized that genes from a given SM pathway would form tight associations (modules) with each other in gene co-expression networks, facilitating their identification. To evaluate this hypothesis, we used 10 global co-expression datasets—each a meta-analysis of hundreds to thousands of expression experiments—across eight plant model organisms to identify hundreds of modules of co-expressed genes for each species. In support of our hypothesis, 15.3-52.6% of modules contained two or more known SM biosynthetic genes (e.g., cytochrome P450s, terpene synthases, and chalcone synthases), and module genes were enriched in SM functions (e.g., glucoside and flavonoid biosynthesis). Moreover, modules recovered many experimentally validated SM pathways in these plants, including all six known to form biosynthetic gene clusters (BGCs). In contrast, genes predicted based on physical proximity on a chromosome to form plant BGCs were no more co-expressed than the null distribution for neighboring genes. These results not only suggest that most predicted plant BGCs do not represent genuine SM pathways but also argue that BGCs are unlikely to be a hallmark of plant specialized metabolism. We submit that global gene co-expression is a rich, but largely untapped, data source for discovering the genetic basis and architecture of plant natural products, which can be applied even without knowledge of the genome sequence.

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Elucidating the Regulatory Elements for Transcription Termination and Posttranscriptional Processing in the Streptomyces clavuligerus Genome

mSystems ◽

10.1128/msystems.01013-20 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Soonkyu Hwang ◽

Namil Lee ◽

Donghui Choe ◽

Yongjae Lee ◽

Woori Kim ◽

...

Keyword(s):

Secondary Metabolite ◽

Transcription Termination ◽

Gene Clusters ◽

Streptomyces Clavuligerus ◽

Regulatory Elements ◽

Regulation Of Transcription ◽

Content Type ◽

Transcriptional Regulatory Elements ◽

Transcriptional Regulatory ◽

Posttranscriptional Processing

ABSTRACT Identification of transcriptional regulatory elements in the GC-rich Streptomyces genome is essential for the production of novel biochemicals from secondary metabolite biosynthetic gene clusters (smBGCs). Despite many efforts to understand the regulation of transcription initiation in smBGCs, information on the regulation of transcription termination and posttranscriptional processing remains scarce. In this study, we identified the transcriptional regulatory elements in β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27064 by determining a total of 1,427 transcript 3′-end positions (TEPs) using the term-seq method. Termination of transcription was governed by three classes of TEPs, of which each displayed unique sequence features. The data integration with transcription start sites and transcriptome data generated 1,648 transcription units (TUs) and 610 transcription unit clusters (TUCs). TU architecture showed that the transcript abundance in TU isoforms of a TUC was potentially affected by the sequence context of their TEPs, suggesting that the regulatory elements of TEPs could control the transcription level in additional layers. We also identified TU features of a xenobiotic response element (XRE) family regulator and DUF397 domain-containing protein, particularly showing the abundance of bidirectional TEPs. Finally, we found that 189 noncoding TUs contained potential cis- and trans-regulatory elements that played a major role in regulating the 5′ and 3′ UTR. These findings highlight the role of transcriptional regulatory elements in transcription termination and posttranscriptional processing in Streptomyces sp. IMPORTANCE Streptomyces sp. is a great source of bioactive secondary metabolites, including antibiotics, antifungal agents, antiparasitic agents, immunosuppressant compounds, and other drugs. Secondary metabolites are synthesized via multistep conversions of the precursor molecules from primary metabolism, governed by multicomplex enzymes from secondary metabolite biosynthetic gene clusters. As their production is closely related with the growth phase and dynamic cellular status in response to various intra- and extracellular signals, complex regulatory systems tightly control the gene expressions related to secondary metabolism. In this study, we determined genome-wide transcript 3′-end positions and transcription units in the β-lactam antibiotic producer Streptomyces clavuligerus ATCC 27064 to elucidate the transcriptional regulatory elements in transcription termination and posttranscriptional processing by integration of multiomics data. These unique features, such as transcript 3′-end sequence, potential riboregulators, and potential 3′-untranslated region (UTR) cis-regulatory elements, can be potentially used to design engineering tools that can regulate the transcript abundance of genes for enhancing secondary metabolite production.

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Bacterial secondary metabolite biosynthetic potential in soil varies with phylum, depth, and vegetation type

10.1101/818815 ◽

2019 ◽

Cited By ~ 4

Author(s):

Allison M. Sharrar ◽

Alexander Crits-Christoph ◽

Raphaël Méheust ◽

Spencer Diamond ◽

Evan P. Starr ◽

...

Keyword(s):

Secondary Metabolite ◽

Vegetation Type ◽

Bacterial Species ◽

Gene Clusters ◽

Metagenomic Sequencing ◽

Soil Bacterial Diversity ◽

Community Members ◽

Clinical Value ◽

Specialized Metabolites ◽

Shallow Soils

AbstractBacteria isolated from soils are major sources of specialized metabolites, including antibiotics and other compounds with clinical value that likely shape interactions among microbial community members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies across the phylogenetic diversity of bacterial species in soils, and whether the genetic potential for production of these metabolites differs with soil type. We sampled soils and saprolite from three sites in a northern California Critical Zone Observatory with varying vegetation and bedrock characteristics and used metagenomic sequencing and assembly to reconstruct 1,334 microbial genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite production. We obtained genomes for prolific producers of secondary metabolites, including novel groups within the Actinobacteria, Chloroflexi and candidate phylum Dormibactereota. Surprisingly, one genome of a Candidate Phyla Radiation bacterium encoded for a ribosomally synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by taxonomy.

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Three unlinked gene clusters are involved in clavam metabolite biosynthesis in Streptomyces clavuligerus

Canadian Journal of Microbiology ◽

10.1139/w04-070 ◽

2004 ◽

Vol 50 (10) ◽

pp. 803-810 ◽

Cited By ~ 24

Author(s):

Kapil Tahlan ◽

Hyeon Ung Park ◽

Susan E Jensen

Keyword(s):

Clavulanic Acid ◽

Gel Electrophoresis ◽

Genomic Dna ◽

Biosynthetic Pathway ◽

Gene Clusters ◽

Streptomyces Clavuligerus ◽

Pulsed Field Gel Electrophoresis ◽

Single Family ◽

Chromosome Walking ◽

Restriction Fragments

In Streptomyces clavuligerus, three groups of genes are known to be involved in the biosynthesis of the clavam metabolites. Since antibiotic biosynthetic genes are invariably clustered on the chromosome in prokaryotes, chromosome walking was undertaken in an attempt to show that the three groups of clavam genes would resolve into a single super-cluster when analyzed at larger scale. However, no evidence of linkage between the three groups was obtained. Furthermore, Southern analysis of macro-restriction fragments of genomic DNA separated by pulsed-field gel electrophoresis also indicated that the three groups of genes are not linked. Despite the structural and biosynthetic relatedness of the clavam metabolites, our results suggest that the genes involved in their production lie in three unlinked gene clusters. We believe that this represents the first instance in bacteria of genes involved in the biosynthesis of a single family of antibiotics sharing a common biosynthetic pathway and yet residing in three separate locations on the chromosome.Key words: Streptomyces, clavulanic acid, clavams, paralogues, gene clusters.

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PCR-RFLP Detection and Genogroup Identification of Piscirickettsia salmonis in Field Samples

Pathogens ◽

10.3390/pathogens9050358 ◽

2020 ◽

Vol 9 (5) ◽

pp. 358

Author(s):

Pamela Aravena ◽

Rodrigo Pulgar ◽

Javiera Ortiz-Severín ◽

Felipe Maza ◽

Alexis Gaete ◽

...

Keyword(s):

High Throughput Sequencing ◽

In Silico Analysis ◽

Cost Effective ◽

Rrna Gene ◽

Bacterial Composition ◽

Bacterial Dna ◽

Phenotypic Differences ◽

Field Samples ◽

Pcr Rflp ◽

Digestion Pattern

Piscirickettsia salmons, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the P. salmonis genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the P. salmonis genogroups. Using an in silico analysis of the P. salmonis 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 P. salmonis strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the P. salmonis genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation.

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Potential Rhodopsin- and Bacteriochlorophyll-Based Dual Phototrophy in a High Arctic Glacier

mBio ◽

10.1128/mbio.02641-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Yonghui Zeng ◽

Xihan Chen ◽

Anne Mette Madsen ◽

Athanasios Zervas ◽

Tue Kjærgaard Nielsen ◽

...

Keyword(s):

Solar Energy ◽

Energy Production ◽

Gene Clusters ◽

High Arctic ◽

Proton Pumping ◽

The Other ◽

Life Strategy ◽

Metabolic Potential ◽

Expression Control ◽

Arctic Glacier

ABSTRACT Conserving additional energy from sunlight through bacteriochlorophyll (BChl)-based reaction center or proton-pumping rhodopsin is a highly successful life strategy in environmental bacteria. BChl and rhodopsin-based systems display contrasting characteristics in the size of coding operon, cost of biosynthesis, ease of expression control, and efficiency of energy production. This raises an intriguing question of whether a single bacterium has evolved the ability to perform these two types of phototrophy complementarily according to energy needs and environmental conditions. Here, we report four Tardiphaga sp. strains (Alphaproteobacteria) of monophyletic origin isolated from a high Arctic glacier in northeast Greenland (81.566° N, 16.363° W) that are at different evolutionary stages concerning phototrophy. Their >99.8% identical genomes contain footprints of horizontal operon transfer (HOT) of the complete gene clusters encoding BChl- and xanthorhodopsin (XR)-based dual phototrophy. Two strains possess only a complete XR operon, while the other two strains have both a photosynthesis gene cluster and an XR operon in their genomes. All XR operons are heavily surrounded by mobile genetic elements and are located close to a tRNA gene, strongly signaling that a HOT event of the XR operon has occurred recently. Mining public genome databases and our high Arctic glacial and soil metagenomes revealed that phylogenetically diverse bacteria have the metabolic potential of performing BChl- and rhodopsin-based dual phototrophy. Our data provide new insights on how bacteria cope with the harsh and energy-deficient environment in surface glacier, possibly by maximizing the capability of exploiting solar energy. IMPORTANCE Over the course of evolution for billions of years, bacteria that are capable of light-driven energy production have occupied every corner of surface Earth where sunlight can reach. Only two general biological systems have evolved in bacteria to be capable of net energy conservation via light harvesting: one is based on the pigment of (bacterio-)chlorophyll and the other is based on proton-pumping rhodopsin. There is emerging genomic evidence that these two rather different systems can coexist in a single bacterium to take advantage of their contrasting characteristics in the number of genes involved, biosynthesis cost, ease of expression control, and efficiency of energy production and thus enhance the capability of exploiting solar energy. Our data provide the first clear-cut evidence that such dual phototrophy potentially exists in glacial bacteria. Further public genome mining suggests this understudied dual phototrophic mechanism is possibly more common than our data alone suggested.

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Molecular Mechanisms Underpinning Aggregation in Acidiphilium sp. C61 Isolated from Iron-Rich Pelagic Aggregates

Microorganisms ◽

10.3390/microorganisms8030314 ◽

2020 ◽

Vol 8 (3) ◽

pp. 314

Author(s):

Qianqian Li ◽

Rebecca E. Cooper ◽

Carl-Eric Wegner ◽

Kirsten Küsel

Keyword(s):

Hot Spots ◽

Biofilm Formation ◽

Extracellular Polymeric Substances ◽

Molecular Mechanisms ◽

Core Genome ◽

Gene Clusters ◽

Microbial Interactions ◽

Aggregate Formation ◽

Rna Seq ◽

Metabolic Potential

Iron-rich pelagic aggregates (iron snow) are hot spots for microbial interactions. Using iron snow isolates, we previously demonstrated that the iron-oxidizer Acidithrix sp. C25 triggers Acidiphilium sp. C61 aggregation by producing the infochemical 2-phenethylamine (PEA). Here, we showed slightly enhanced aggregate formation in the presence of PEA on different Acidiphilium spp. but not other iron-snow microorganisms, including Acidocella sp. C78 and Ferrovum sp. PN-J47. Next, we sequenced the Acidiphilium sp. C61 genome to reconstruct its metabolic potential. Pangenome analyses of Acidiphilium spp. genomes revealed the core genome contained 65 gene clusters associated with aggregation, including autoaggregation, motility, and biofilm formation. Screening the Acidiphilium sp. C61 genome revealed the presence of autotransporter, flagellar, and extracellular polymeric substances (EPS) production genes. RNA-seq analyses of Acidiphilium sp. C61 incubations (+/− 10 µM PEA) indicated genes involved in energy production, respiration, and genetic processing were the most upregulated differentially expressed genes in the presence of PEA. Additionally, genes involved in flagellar basal body synthesis were highly upregulated, whereas the expression pattern of biofilm formation-related genes was inconclusive. Our data shows aggregation is a common trait among Acidiphilium spp. and PEA stimulates the central cellular metabolism, potentially advantageous in aggregates rapidly falling through the water column.

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