Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2.

Azoxy compounds are a distinctive group of bioactive secondary metabolites characterized by a unique RN═N+(O-)R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here, we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S. albidoflavus J1074 and S. coelicolor M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knockout using CRISPR-Cas9 base editing in the native producer, S. mirabilis P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to an azo compound followed by further oxygenation. Enzymes involved in modifications of the precursor could be postulated based on their predicted function and the intermediates identified in the knockout strains. Moreover, the distribution of the azoxy biosynthetic gene clusters across Streptomyces spp. genomes is explored, highlighting the presence of these clusters in over 20% of the Streptomyces spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A-like clusters are widely distributed across the phylogenetic tree.

Complete Genome Sequences of the Two Strains Methylorubrum extorquens NBC_00036 and NBC_00404.

Here, we report the complete genome sequences of Methylorubrum extorquens NBC_00036 and Methylorubrum extorquens NBC_00404. The genomes were sequenced using the Oxford Nanopore Technologies MinION and Illumina NovaSeq systems. Both genomes are circular, with sizes of 5,661,342 bp and 5,869,086 bp, respectively.

Identification and heterologous expression of the globomycin biosynthetic gene cluster.

Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens. Its mode of action is based on the competitive inhibition of the lipoprotein signal peptidase II (LspA), which is absent in eukaryotes and considered an attractive target for the development of new antibiotics. Despite its interesting biological properties, the gene cluster encoding its biosynthesis has not yet been identified. In this study we employed a genome-mining approach in the globomycin-producing Streptomyces sp. CA-278952 to identify a candidate gene cluster responsible for its biosynthesis. A null mutant was constructed using CRISPR base editing where production was abolished, strongly suggesting its involvement in the biosynthesis. The putative gene cluster was then cloned and heterologously expressed in Streptomyces albus J1074 and Streptomyces coelicolor M1146, therefore unambiguously linking globomycin and its biosynthetic gene cluster. Our work paves the way for the biosynthesis of new globomycin derivatives with improved pharmacological properties.

Treatment of Diabetic Foot Ulcers With Inforatio Technique to Promote Wound Healing: A Feasibility Trial.

Chronic foot ulcers have extensive consequences for diabetic patients' quality of life and increase risks of amputation and death. The aim of this trial was to assess the feasibility of conducting a larger clinical trial to evaluate the clinical effect of inforatio technique on healing of diabetic foot ulcers (DFUs). Inforatio technique is a novel minimal invasive procedure where small cuts are made on wound beds with punch biopsy tools. This study was a feasibility trial conducted at an outpatient wound care clinic at Zealand University Hospital. Twelve patients with DFUs were included. During a 90-day follow-up, participants visited the clinic 5 times and received inforatio technique twice. Feasibility was assessed with regard to recruitment, acceptability, burden, benefits, protocol adherence, and adverse events. The recruitment rate was 1 patient per eighth day (95% confidence interval [CI] = [4th-13th]), and the retention rate was 100% (95% CI = [74-100]). During follow-up, healing was observed for 4 ulcers (33%, 95% CI = [10-65]) with a mean time for healing of 59 days (range, 22-89) (95% CI = [5-113]). Five ulcers had a reduction of wound area and 3 ulcers had an increase in area from baseline to 90-day follow-up. No temporal relationship was found between inforatio application and wound area increase. There were no patient-reported harmful effects and no adverse events with probable relation to inforatio technique. Patient acceptability and participant adherence were promising. Thus, a larger clinical trial for evaluating the clinical effect of inforatio technique is considered feasible to conduct.

Identification of the Biosynthetic Gene Cluster for Pyracrimycin A, an Antibiotic Produced by Streptomyces sp.

Actinomycetes make a wealth of complex, structurally diverse natural products, and a key challenge is to link them to their biosynthetic gene clusters and delineate the reactions catalyzed by each of the enzymes. Here, we report the biosynthetic gene cluster for pyracrimycin A, a set of nine genes that includes a core nonribosomal peptide synthase (pymB) that utilizes serine and proline as precursors and a monooxygenase (pymC) that catalyzes Baeyer-Villiger oxidation. The cluster is similar to the one for brabantamide A; however, pyracrimycin A biosynthesis differs in that feeding experiments with isotope-labeled serine and proline suggest that a ring opening reaction takes place and a carbon is lost from serine downstream of the oxidation reaction. Based on these data, we propose a full biosynthesis pathway for pyracrimycin A.

Complete Genome Sequence of the Collinolactone Producer Streptomyces sp. Strain Gö40/10.

The actinomycete Streptomyces sp. strain Gö40/10 has the potential to produce a range of secondary metabolites, one of which is collinolactone, a compound with neuroprotective properties and potential for pharmaceutical applications. The genome was sequenced with Oxford Nanopore Technologies MinION and Illumina MiSeq systems and consists of a single 9,635,564-nucleotide linear chromosome.

A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing.

CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.

Complete Genome Sequence of Amycolatopsis sp. CA-230715, Encoding a 35-Module Type I Polyketide Synthase.

We report the sequencing, assembly, and annotation of the genome of Amycolatopsis sp. CA-230715, a potentially interesting producer of natural products. The genome of CA-230715 was sequenced using PacBio, Illumina, and Nanopore technologies. It consists of a circular 10,363,158-nucleotide (nt) chromosome and a circular 12,080-nt plasmid.

Characterization and engineering of Streptomyces griseofuscus DSM 40191 as a potential host for heterologous expression of biosynthetic gene clusters.

Streptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including the production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by the full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5.19% genome reduction. DEL2 can be characterized by faster growth and inability to produce three main native metabolites: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to observe a formation of a blue halo, indicating a potential production of actinorhodin by both DEL2 and a wild type.

Complete Genome Sequence of the Rare Actinobacterium Kutzneria sp. Strain CA-103260.

Here, we report the sequencing, assembly, and annotation of the genome of the rare actinobacterium Kutzneria sp. strain CA-103260. The genome of CA-103260 was sequenced using PacBio and Illumina technologies and it consists of a circular 11,609,901-bp chromosome.

Complete Genome Sequence of Streptomyces sp. Strain CA-256286.

Here, we report the sequencing, assembly, and annotation of the genome of Streptomyces sp. strain CA-256286. The genome consists of a linear 7,726,360-nucleotide chromosome and a linear 466,817-nucleotide putative plasmid. This strain is predicted to produce a range of novel secondary metabolites.

High-Quality Sequencing, Assembly, and Annotation of the Streptomyces griseofuscus DSM 40191 Genome.

Here, we report the sequencing, assembly, and annotation of the genome of Streptomyces griseofuscus DSM 40191. The genome of S. griseofuscus was sequenced using PacBio and Illumina technologies. It consists of a linear 8,721,740-bp chromosome and three plasmids, pSGRIFU1 (220 kb), pSGRIFU2 (88 kb), and pSGRIFU3 (86 kb).

CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes.

Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.

Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST.

Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.

Expanding the biodiversity of Oenococcus oeni through comparative genomics of apple cider and kombucha strains.

BACKGROUND: Oenococcus oeni is a lactic acid bacteria species adapted to the low pH, ethanol-rich environments of wine and cider fermentation, where it performs the crucial role of malolactic fermentation. It has a small genome and has lost the mutS-mutL DNA mismatch repair genes, making it a hypermutable and highly specialized species. Two main lineages of strains, named groups A and B, have been described to date, as well as other subgroups correlated to different types of wines or regions. A third group "C" has also been hypothesized based on sequence analysis, but it remains controversial. In this study we have elucidated the species population structure by sequencing 14 genomes of new strains isolated from cider and kombucha and performing comparative genomics analyses. RESULTS: Sequence-based phylogenetic trees confirmed a population structure of 4 clades: The previously identified A and B, a third group "C" consisting of the new cider strains and a small subgroup of wine strains previously attributed to group B, and a fourth group "D" exclusively represented by kombucha strains. A pair of complete genomes from group C and D were compared to the circularized O. oeni PSU-1 strain reference genome and no genomic rearrangements were found. Phylogenetic trees, K-means clustering and pangenome gene clusters evidenced the existence of smaller, specialized subgroups of strains. Using the pangenome, genomic differences in stress resistance and biosynthetic pathways were found to uniquely distinguish the C and D clades. CONCLUSIONS: The obtained results, including the additional cider and kombucha strains, firmly established the O. oeni population structure. Group C does not appear as fully domesticated as group A to wine, but showed several unique patterns which may be due to ongoing specialization to the cider environment. Group D was shown to be the most divergent member of O. oeni to date, appearing as the closest to a pre-domestication state of the species.

Gelatinous plankton is important in the diet of European eel (Anguilla anguilla) larvae in the Sargasso Sea.

Limited insight into eel larvae feeding and diet prevents a holistic overview of the life-cycle of catadromous eels and an understanding of the ecological position of their early stages in marine waters. The present study evaluated the diet of larval European eel, Anguilla anguilla - a critically endangered species. Next-generation 18S rRNA gene sequencing data of Sargasso Sea eel larvae gut contents and marine snow aggregates was compared with a reference plankton database to assess the trophic relations of eel larvae. Gut contents of A. anguilla larvae were not well explained by the eukaryotic composition of marine snow aggregates; gut contents being dominated by gene sequences of Hydrozoa taxa (phylum Cnidaria), while snow aggregates were dominated by Crustacea taxa. Pronounced differences between gut contents and marine snow aggregates were also seen in the prokaryotic 16S rRNA gene composition. The findings, in concert with significant abundances of Hydrozoa in the study area, suggest that Hydrozoa plankton are important in the diet of A. anguilla larvae, and that consideration of these organisms would further our understanding of A. anguilla feeding strategies in the oligotrophic Sargasso Sea, which may be important for potential future rearing of A. anguilla larvae in captivity.

Probenecid Inhibits α-Adrenergic Receptor-Mediated Vasoconstriction in the Human Leg Vasculature.

Coordination of vascular smooth muscle cell tone in resistance arteries plays an essential role in the regulation of peripheral resistance and overall blood pressure. Recent observations in animals have provided evidence for a coupling between adrenoceptors and Panx1 (pannexin-1) channels in the regulation of sympathetic nervous control of peripheral vascular resistance and blood pressure; however, evidence for a functional coupling in humans is lacking. We determined Panx1 expression and effects of treatment with the pharmacological Panx1 channel inhibitor probenecid on the vasoconstrictor response to α1- and α2-adrenergic receptor stimulation in the human forearm and leg vasculature of young healthy male subjects (23±3 years). By use of immunolabeling and confocal microscopy, Panx1 channels were found to be expressed in vascular smooth muscle cells of arterioles in human leg skeletal muscle. Probenecid treatment increased (P<0.05) leg vascular conductance at baseline by ≈15% and attenuated (P<0.05) the leg vasoconstrictor response to arterial infusion of tyramine (α1- and α2-adrenergic receptor stimulation) by ≈15%, whereas the response to the α1-agonist phenylephrine was unchanged. Inhibition of α1-adrenoceptors prevented the probenecid-induced increase in baseline leg vascular conductance, but did not alter the effect of probenecid on the vascular response to tyramine. No differences with probenecid treatment were detected in the forearm. These observations provide the first line of evidence in humans for a functional role of Panx1 channels in setting resting tone via α1-adrenoceptors and in the constrictive effect of noradrenaline via α2-adrenoceptors, thereby contributing to the regulation of peripheral vascular resistance and blood pressure in humans.

Metagenomic Comparison of Antibiotic Resistance Genes Associated with Liquid and Dewatered Biosolids.

Municipal biosolids (MBs) that are land-applied in North America are known to possess an active microbial population that can include human pathogens. Activated sludge is a hotspot for the accumulation of antibiotics and has been shown to be a selective environment for microorganisms that contain antibiotic resistance genes (ARGs); however, the prevalence of ARGs in MBs is not well characterized. In this study, we enriched the plasmid metagenome from raw sewage sludge and two CP2 MBs, a mesophilic anaerobic digestate and a dewatered digestate, to evaluate the presence of ARGs in mobile genetic elements. The CP2-class biosolids are similar to Class B biosolids in the United States. The CP2 biosolids must meet a microbiological cut off of 2 × 10 colony-forming units (CFU) per dry gram or 100 mL of biosolids. The enriched plasmid DNA was sequenced (Illumina MiSeq). Sequence matching against databases, including the Comprehensive Antibiotic Resistance Database (CARD), MG-RAST, and INTEGRALL, identified potential genes of interest related to ARGs and their ability to transfer. The presence and abundance of different ARGs varied between treatments with heterogeneity observed among the same sample types. The MBs plasmid-enriched metagenomes contained ARGs associated with resistance to a variety of antibiotics, including ß-lactams, rifampicin, quinolone, and tetracycline as well as the detection of extended spectrum ß-lactamase genes. Cultured bacteria from CP2 MBs possessed antibiotic resistances consistent with the MBs metagenome data including multiantibiotic-resistant isolates. The results from this study provide a better understanding of the ARG and MGE profile of the plasmid-enriched metagenome of CP2 MBs.

Extrachromosomal circular DNA is common in yeast.

Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥ 15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution.

Current strategies for mobilome research.

Mobile genetic elements (MGEs) are pivotal for bacterial evolution and adaptation, allowing shuffling of genes even between distantly related bacterial species. The study of these elements is biologically interesting as the mode of genetic propagation is kaleidoscopic and important, as MGEs are the main vehicles of the increasing bacterial antibiotic resistance that causes thousands of human deaths each year. The study of MGEs has previously focused on plasmids from individual isolates, but the revolution in sequencing technology has allowed the study of mobile genomic elements of entire communities using metagenomic approaches. The problem in using metagenomic sequencing for the study of MGEs is that plasmids and other mobile elements only comprise a small fraction of the total genetic content that are difficult to separate from chromosomal DNA based on sequence alone. The distinction between plasmid and chromosome is important as the mobility and regulation of genes largely depend on their genetic context. Several different approaches have been proposed that specifically enrich plasmid DNA from community samples. Here, we review recent approaches used to study entire plasmid pools from complex environments, and point out possible future developments for and pitfalls of these approaches. Further, we discuss the use of the PacBio long-read sequencing technology for MGE discovery.