A candidate gene cluster for the bioactive natural product gyrophoric acid in lichen-forming fungi

Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Yet they a have limited implementation in industry as for most of the natural products, the corresponding genes remain unknown. Here we implement a long-read sequencing and bioinformatic approach to identify the biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus Umbilicaria, we identify the most likely GA cluster and investigate cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria with only three genes that are conserved across species, including the PKS gene. In addition, our results suggest that the same cluster codes for different but structurally similar NPs, i.e., GA, umbilicaric acid and hiascic acid, bringing new evidence that lichen metabolite diversity is also generated through regulatory mechanisms at the molecular level. Ours is the first study to identify the most likely GA cluster. This information is essential for opening up avenues for biotechnological approaches to producing and modifying GA, and possibly other lichen compounds. We show that bioinformatics approaches are useful in linking genes and potentially associated natural products. Genome analyses help unlocking the pharmaceutical potential of organisms such as lichens, which are biosynthetically diverse, but slow growing, and usually uncultivable due to their symbiotic nature.

Insight into the symbiotic lifestyle of DPANN archaea revealed by cultivation and genome analyses

Decades of culture-independent analyses have resulted in proposals of many tentative archaeal phyla with no cultivable representative. Members of DPANN (an acronym of the names of the first included phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota), an archaeal superphylum composed of at least 10 of these tentative phyla, are generally considered obligate symbionts dependent on other microorganisms. While many draft/complete genome sequences of DPANN archaea are available and their biological functions have been considerably predicted, only a few examples of their successful laboratory cultivation have been reported, limiting our knowledge of their symbiotic lifestyles. Here, we investigated physiology, morphology, and host specificity of an archaeon of the phylum “Candidatus Micrarchaeota” (ARM-1) belonging to the DPANN superphylum by cultivation. We constructed a stable coculture system composed of ARM-1 and its original host Metallosphaera sp. AS-7 belonging to the order Sulfolobales. Further host-switching experiments confirmed that ARM-1 grew on five different archaeal species from three genera—Metallosphaera, Acidianus, and Saccharolobus—originating from geologically distinct hot, acidic environments. The results suggested the existence of DPANN archaea that can grow by relying on a range of hosts. Genomic analyses showed inferred metabolic capabilities, common/unique genetic contents of ARM-1 among cultivated micrarchaeal representatives, and the possibility of horizontal gene transfer between ARM-1 and members of the order Sulfolobales. Our report sheds light on the symbiotic lifestyles of DPANN archaea and will contribute to the elucidation of their biological/ecological functions.

CONGA: Copy number variation genotyping in ancient genomes and low-coverage sequencing data

To date, ancient genome analyses have been largely confined to the study of single nucleotide polymorphisms (SNPs). Copy number variants (CNVs) are a major contributor of disease and of evolutionary adaptation, but identifying CNVs in ancient shotgun-sequenced genomes is hampered by (a) most published genomes being <1x coverage, (ii) ancient DNA fragments being typically <80 bps. These characteristics preclude state-of-the-art CNV detection software to be effectively applied to ancient genomes. Here we present CONGA, an algorithm tailored for genotyping deletion and duplication events in genomes with low depths of coverage. Simulations show that CONGA can genotype deletions and duplications >1 Kbps with F-scores >0.77 and >0.82, respectively at >=0.5x. Further, down-sampling experiments using published ancient BAM files reveal that >1 Kbps deletions could be genotyped at F-score >0.75 at >=1x coverage. Using CONGA, we analyse deletion events at 10,018 loci in 56 ancient human genomes spanning the last 50,000 years, with coverages 0.4x-26x. We find inter-individual genetic diversity measured using deletions and SNPs to be highly correlated, suggesting that deletion frequencies broadly reflect demographic history. We also identify signatures of purifying selection on deletions, such as an excess of singletons compared to those in SNPs. CONGA paves the way for systematic studies of drift, mutation load, and adaptation in ancient and modern-day gene pools through the lens of CNVs.

Comparative Chloroplast Genome Analyses of the Winter-Blooming Eastern Asian Endemic Genus Chimonanthus (Calycanthaceae) With Implications For Its Phylogeny and Diversification

Chimonanthus of Calycanthaceae is a small endemic genus in China, with unusual winter-blooming sweet flowers widely cultivated for ornamentals and medicinal uses. The evolution of Chimonanthus plastomes and its phylogenetic relationships remain unresolved due to limited availability of genetic resources. Here, we report fully assembled and annotated chloroplast genomes of five Chimonanthus species. The chloroplast genomes of the genus (size range 153,010 – 153,299 bp) reveal high similarities in gene content, gene order, GC content, codon usage, amino acid frequency, simple sequence repeats, oligonucleotide repeats, synonymous and non-synonymous substitutions, and transition and transversion substitutions. Signatures of positive selection are detected in atpF and rpoB genes in C. campanulatus. The correlations among substitutions, InDels, and oligonucleotide repeats reveal weak to strong correlations in distantly related species at the intergeneric levels, and very weak to weak correlations among closely related Chimonanthus species. Chloroplast genomes are used to reconstruct a well-resolved phylogenetic tree, which supports the monophyly of Chimonanthus. Within Chimonanthus, C. praecox and C. campanulatus form one clade, while C. grammatus, C. salicifolius, C. zhejiangensis, and C. nitens constitute another clade. Chimonanthus nitens appears paraphyletic and is closely related to C. salicifolius and C. zhejiangensis, suggesting the need to reevaluate the species delimitation of C. nitens. Chimonanthus and Calycanthus diverged in mid-Oligocene; the radiation of extant Chimonanthus species was dated to the mid-Miocene, while C. grammatus diverged from other Chimonanthus species in the late Miocene. C. salicifolius, C. nitens(a), and C. zhejiangensis are inferred to have diverged in the Pleistocene of the Quaternary period, suggesting recent speciation of a relict lineage in the subtropical forest regions in eastern China. This study provides important insights into the chloroplast genome features and evolutionary history of Chimonanthus and family Calycanthaceae.

Isolation of a Bacillus safensis from mine tailings in Peru, genomic characterization and characterization of its cyanide- degrading enzyme CynD

Cyanide is widely used in industry as a potent lixiviant due to its capacity to tightly bind metals. This property imparts cyanide enormous toxicity to all known organisms. Thus, industries that utilize this compound must reduce its concentration in recycled or waste waters. Physical, chemical, and biological treatments have been used for cyanide remediation; however, none of them meet all the desired characteristics: efficiency, low cost and low environmental impact. A better understanding of metabolic pathways and biochemistry of enzymes involved in cyanide degradation is a necessary step to improve cyanide bioremediation efficacy to satisfy the industry requirements. Here, we used several approaches to explore this topic. We have isolated three cyanide-degrading Bacillus strains from water in contact with mine tailings from Lima, Peru, and classified them as Bacillus safensis PER-URP-08, Bacillus licheniformis PER-URP-12, and Bacillus subtilis PER-URP-17 based on 16S rRNA gene sequencing and core genome analyses. Additionally, core genome analyses of 132 publicly available genomes of Bacillus pumilus group including B. safensis and B. altitudinis allowed us to reclassify some strains and identify two strains that did not match with any known species of the Bacillus pumilus group. We searched for possible routes of cyanide-degradation in the genomes of these three strains and identified putative B. licheniformis PER-URP-12 and B. subtilis PER-URP-17 rhodaneses and B. safensis PER-URP-08 cyanide dihydratase (CynD) sequences possibly involved cyanide degradation. We identified characteristic C-terminal residues that differentiate CynD from B. pumilus and B. safensis, and showed that, differently from CynD from B. pumilus C1, recombinant CynD from the Bacillus safensis PER-URP-08 strain remains active up to pH 9 and presents a distinct oligomerization pattern at pH 8 and 9. Moreover, transcripts of B. safensis PER-URP-08 CynD (CynDPER-URP-08) are strongly induced in the presence of cyanide. Our results warrant further investigation of B. safensis PER-URP-08 and CynDPER-URP-08 as potential tools for cyanide-bioremediation.

Genomic Insights into Denitrifying Methane-Oxidizing Bacteria Gemmobacter fulva sp. Nov., Isolated from an Anabaena Culture

The genus Gemmobacter grows phototrophically, aerobically, or anaerobically, and utilizes methylated amine. Here, we present two high-quality complete genomes of the strains con4 and con5T isolated from a culture of Anabaena. The strains possess sMMO (soluble methane monooxygenase)-oxidizing alkanes to carbon dioxide. Functional genes for methane-oxidation (prmAC, mimBD, adh, gfa, fdh) were identified. The genome of strain con5T contains nirB, nirK, nirQ, norB, norC, and norG genes involved in dissimilatory nitrate reduction. The presence of nitrite reductase gene (nirK) and the nitric-oxide reductase gene (norB) indicates that it could potentially use nitrite as an electron acceptor in anoxic environments. Taxonomic investigations were also performed on two strains through polyphasic methods, proposing two isolates as a novel species of the genus Gemmobacter. The findings obtained through the whole genome analyses provide genome-based evidence of complete oxidation of methane to carbon dioxide. This study provides a genetic blueprint of Gemmobacter fulva con5T and its biochemical characteristics, which help us to understand the evolutionary biology of the genus Gemmobacter.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p.pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Sanguibacter suaedae sp. nov., isolated from the root of Suaeda aralocaspica in north-west PR China

A bacterial strain, designated YZGR15T, was isolated from the root of an annual halophyte Suaeda aralocaspica, collected from the southern edge of the Gurbantunggut desert, north-west PR China. Cells of the isolate were Gram-stain-positive, facultatively anaerobic, irregular rods. Growth occurred at 4–42 °C (optimum, 30–37 °C), at pH 6.0–9.0 (optimum, pH 7.0–7.5) and in the presence of 0–9 % (w/v) NaCl (optimum, 2–5 %). Phylogenetic analysis using 16S rRNA gene sequences indicated that strain YZGR15T showed the highest sequence similarity to Sanguibacter keddieii (98.27 %), Sanguibacter antarcticus (98.20 %) and Sanguibacter inulinus (98.06 %). Results of genome analyses of strain YZGR15T indicated that the genome size was 3.16 Mb, with a genomic DNA G+C content of 71.9 mol%. Average nucleotide identity and digital DNA–DNA hybridization values between strain YZGR15Tand three type strains were in the range of 76.5–77.8 % and 20.0–22.2 %, respectively. Analysis of the cellular component of strain YZGR15T revealed that the primary fatty acids were anteiso-C15 : 0, C16 : 0, C14 : 0 and iso-C16 : 0 and the polar lipids included diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids and two unidentified glycolipids. The cell-wall characteristic amino acids were glutamic acid, alanine and an unknown amino acid. The whole-cell sugars for the strain were mannose, ribose, rhamnose, glucose and an unidentified sugar. The predominant respiratory quinone was MK-9(H4). Based on the results of genomic, phylogenetic, phenotypic and chemotaxonomic analyses, strain YZGR15T represents a novel species of the genus Sanguibacter , for which the name Sanguibacter suaedae sp. nov. is proposed. The type strain is YZGR15T (=CGMCC 1.18691T=KCTC 49659T)