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Mycoparasite secondary metabolites control fungal infections or diseases in agriculture and human health. Among genes involved in synthesizing secondary metabolites, cytochrome P450 monooxygenases (CYPs/P450s) play a key role in synthesizing and attributing diversity to the secondary metabolites. Despite the importance of P450s, a comparative analysis of P450s in mycoparasites has yet to be reported. This study is aimed at addressing this research gap. Genome-wide analysis of P450s in 43 fungi representing six fungal phyla and three distinct lifestyles, such as mycoparasitic (24 species), saprophytic (5 species), and ectomycorrhizal (14 species), revealed the expansion of P450s in Pezizomycete mycoparasites for the synthesis of secondary metabolites. The number of P450s and their families and subfamilies, the number of secondary-metabolite biosynthetic gene clusters (SMBGCs), and the number of P450s that are part of these SMBGCs were found to be highest in Pezizomycete mycoparasites compared to their counterparts of saprophytes and ectomycorrhiza, indicating P450s also play a key role in mycoparasitism. An analysis of P450 location as part of SMBGCs and the available literature on Pezizomycete P450s revealed that P450s play a key role in the synthesis of anti-fungal secondary metabolites such as trichothecene sesquiterpene, harzianum A, heptelidic acid, and gliotoxin. The mycoparasite Trichoderma virens Tv29.8 P450 CYP68Q3 is found to be a bifunctional enzyme with epoxidation and oxidation capability, and CYP5117A3 performs a Baeyer–Villiger oxidation reaction with regioselectivity. This study serves as a reference for future annotation of P450s in mycoparasites.

Prochlorococcus, a cyanobacteria genus of the smallest and most abundant oceanic phototrophs, encompasses ecotype strains adapted to high-light (HL) and low-light (LL) niches. To elucidate the adaptive evolution of this genus, we analyzed 40 Prochlorococcus marinus ORFeomes, including two cornerstone strains, MED4 and NATL1A. Employing deep learning with robust statistical methods, we detected new protein family distributions in the strains and identified key genes differentiating the HL and LL strains. The HL strains harbor genes (ABC-2 transporters) related to stress resistance, such as DNA repair and RNA processing, while the LL strains exhibit unique chlorophyll adaptations (ion transport proteins, HEAT repeats). Additionally, we report the finding of variable, depth-dependent endogenous viral elements in the 40 strains. To generate biological resources to experimentally study the HL and LL adaptations, we constructed the ORFeomes of two representative strains, MED4 and NATL1A synthetically, covering 99% of the annotated protein-coding sequences of the two species, totaling 3976 cloned, sequence-verified open reading frames (ORFs). These comparative genomic analyses, paired with MED4 and NATL1A ORFeomes, will facilitate future genotype-to-phenotype mappings and the systems biology exploration of Prochlorococcus ecology.

Abstract Horse chestnut (Aesculus chinensis) is an important medicinal tree that contains various bioactive compounds, such as aescin, barrigenol-type triterpenoid saponins (BAT), and aesculin, a glycosylated coumarin. Herein, we report a 470.02 Mb genome assembly and characterize an Aesculus-specific whole-genome duplication event, which leads to the formation and duplication of two triterpenoid biosynthesis-related gene clusters (BGCs). We also show that AcOCS6, AcCYP716A278, AcCYP716A275, and AcCSL1 genes within these two BGCs along with a seed-specific expressed AcBAHD6 are responsible for the formation of aescin. Furthermore, we identify seven Aesculus-originated coumarin glycoside biosynthetic genes and achieve the de novo synthesis of aesculin in E. coli. Collinearity analysis shows that the collinear BGC segments can be traced back to early-diverging angiosperms, and the essential gene-encoding enzymes necessary for BAT biosynthesis are recruited before the splitting of Aesculus, Acer, and Xanthoceras. These findings provide insight on the evolution of gene clusters associated with medicinal tree metabolites.

Abstract Larval eastern oysters (Crassostrea virginica) grown in shellfish hatcheries are susceptible to bacterial diseases, particularly vibriosis. Probiotics are microbes that confer health benefits to the host and have been identified as promising tools to manage diseases in aquaculture. The marine bacterium Phaeobacter inhibens S4 (S4) protects larval eastern oysters against challenge with the bacterial pathogen Vibrio coralliilyticus RE22 (RE22). A concentrated liquid formulation of probiont S4 that maintained high cell viability after long‐term storage was developed for commercial use in shellfish hatcheries. The safety and efficacy of the formulation were tested in six different trials in two hatcheries. The S4 formulation was added to C. virginica larvae culture tanks daily at 104 colony forming units (CFU)/mL from Day 1 post fertilisation until Day 6, 12 or 14, depending on the trial. Treatment of larvae in the hatchery with the S4 formulation did not significantly affect the survival and growth of the larvae. Formulated probiont S4 treatment in the hatchery led to a significant increase in relative percent survival (RPS) when larvae were subsequently challenged with the pathogen RE22 (105 CFU/mL) for 24 h in a laboratory challenge as compared to probiotic‐untreated RE22‐challenged larvae (RPS increase of 46%–74%, p

Abstract Recent work has demonstrated that many bee species have specific cytochrome P450 enzymes (P450s) that can efficiently detoxify certain insecticides. The presence of these P450s, belonging or closely related to the CYP9Q subfamily (CYP9Q‐related), is generally well conserved across the diversity of bees. However, the alfalfa leafcutter bee, Megachile rotundata, lacks CYP9Q‐related P450s and is 170–2500 times more sensitive to certain insecticides than bee pollinators with these P450s. The extent to which these findings apply to other Megachilidae bee species remains uncertain. To address this knowledge gap, we sequenced the transcriptomes of four Megachile species and leveraged the data obtained, in combination with publicly available genomic data, to investigate the evolution and function of P450s in the Megachilidae. Our analyses reveal that several Megachilidae species, belonging to the Lithurgini, Megachilini and Anthidini tribes, including all species of the Megachile genus investigated, lack CYP9Q‐related genes. In place of these genes Megachile species have evolved phylogenetically distinct CYP9 genes, the CYP9DM lineage. Functional expression of these P450s from M. rotundata reveal they lack the capacity to metabolize the neonicotinoid insecticides thiacloprid and imidacloprid. In contrast, species from the Osmiini and Dioxyini tribes of Megachilidae have CYP9Q‐related P450s belonging to the CYP9BU subfamily that are able to detoxify thiacloprid. These findings provide new insight into the evolution of P450s that act as key determinants of insecticide sensitivity in bees and have important applied implications for pesticide risk assessment.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1’s overall activity were identified and highlighted this enzyme’s rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug–P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure–function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Abstract A central goal in evolutionary biology is to determine the predictability of adaptive genetic changes. Despite many documented cases of convergent evolution at individual loci, little is known about the repeatability of gene family expansions and contractions. To address this void, we examined gene family evolution in the redheaded pine sawfly Neodiprion lecontei, a noneusocial hymenopteran and exemplar of a pine‐specialized lineage evolved from angiosperm‐feeding ancestors. After assembling and annotating a draft genome, we manually annotated multiple gene families with chemosensory, detoxification, or immunity functions before characterizing their genomic distributions and molecular evolution. We find evidence of recent expansions of bitter gustatory receptor, clan 3 cytochrome P450, olfactory receptor, and antimicrobial peptide subfamilies, with strong evidence of positive selection among paralogs in a clade of gustatory receptors possibly involved in the detection of bitter compounds. In contrast, these gene families had little evidence of recent contraction via pseudogenization. Overall, our results are consistent with the hypothesis that in response to novel selection pressures, gene families that mediate ecological interactions may expand and contract predictably. Testing this hypothesis will require the comparative analysis of high‐quality annotation data from phylogenetically and ecologically diverse insect species and functionally diverse gene families. To this end, increasing sampling in under‐sampled hymenopteran lineages and environmentally responsive gene families and standardizing manual annotation methods should be prioritized.

Abstract Despite the availability of efficacious direct-acting antiviral (DAA) therapy, the number of people infected with hepatitis C virus (HCV) continues to rise, and HCV remains a leading cause of liver-related morbidity, liver transplantation, and mortality. We developed and validated machine learning (ML) algorithms to predict DAA treatment failure. Using the HCV-TARGET registry of adults who initiated all-oral DAA treatment, we developed elastic net (EN), random forest (RF), gradient boosting machine (GBM), and feedforward neural network (FNN) ML algorithms. Model performances were compared with multivariable logistic regression (MLR) by assessing C statistics and other prediction evaluation metrics. Among 6525 HCV-infected adults, 308 patients (4.7%) experienced DAA treatment failure. ML models performed similarly in predicting DAA treatment failure (C statistic [95% CI]: EN, 0.74 [0.69–0.79]; RF, 0.74 [0.69–0.80]; GBM, 0.72 [0.67–0.78]; FNN, 0.75 [0.70–0.80]), and all 4 outperformed MLR (C statistic [95% CI]: 0.51 [0.46–0.57]), and EN used the fewest predictors (n = 27). With Youden index, the EN had 58.4% sensitivity and 77.8% specificity, and nine patients were needed to evaluate to identify 1 DAA treatment failure. Over 60% treatment failure were classified in top three risk decile subgroups. EN-identified predictors included male sex, treatment 1.2 g/dL, advanced liver disease, and use of tobacco, alcohol, or vitamins. Addressing modifiable factors of DAA treatment failure may reduce the burden of retreatment. Machine learning algorithms have the potential to inform public health policies regarding curative treatment of HCV.

Although ginkgo terpenoids have been studied extensively for their pharmaceutical properties, knowledge on their biosynthesis remains limited. Here, the authors identify five multifunctional cytochrome P450s that catalyze the generation of the tert-butyl group and one of the lactone rings towards the biosynthesis of ginkgolides.

Cytochrome P450 monooxygenases (CYPs; P450s) are a superfamily of heme-containing enzymes that are recognized for their vast substrate range and oxidative multifunctionality. CYP107 family members perform hydroxylation and epoxidation processes, producing a variety of biotechnologically useful secondary metabolites. Despite their biotechnological importance, a thorough examination of CYP107 protein structures regarding active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 44 CYP107 crystal structures were investigated in this study. We demonstrate that the CYP107 active site cavity is very flexible, with ligand binding reducing the volume of the active site in some situations and increasing volume size in other instances. Polar interactions between the substrate and active site residues result in crucial salt bridges and the formation of proton shuttling pathways. Hydrophobic interactions, however, anchor the substrate within the active site. The amino acid residues within the binding pocket influence substrate orientation and anchoring, determining the position of the hydroxylation site and hence direct CYP107’s catalytic activity. Additionally, the amino acid dynamics within and around the binding pocket determine CYP107’s multifunctionality. This study serves as a reference for understanding the structure–function analysis of CYP107 family members precisely and the structure–function analysis of P450 enzymes in general. Finally, this work will aid in the genetic engineering of CYP107 enzymes to produce novel molecules of biotechnological interest.

Lichens are unique organisms that exhibit a permanent symbiosis between fungi and algae or fungi and photosynthetic bacteria. Lichens have been found to produce biotechnologically valuable secondary metabolites. A handful of studies showed that tailoring enzymes such as cytochrome P450 monooxygenases (CYPs/P450s) play a key role in synthesizing these metabolites. Despite the critical role of P450s in the biosynthesis of secondary metabolites, the systematic analysis of P450s in lichens has yet to be reported. This study is aimed to address this research gap. A genome-wide analysis of P450s in five lichens from the fungal class Lecanoromycetes revealed the presence of 434 P450s that are grouped into 178 P450 families and 345 P450 subfamilies. The study indicated that none of the P450 families bloomed, and 15 P450 families were conserved in all five Lecanoromycetes. Lecanoromycetes have more P450s and higher P450 family diversity compared to Pezizomycetes. A total of 73 P450s were found to be part of secondary metabolite gene clusters, indicating their potential involvement in the biosynthesis of secondary metabolites. Annotation of P450s revealed that CYP682BG1 and CYP682BG2 from Cladonia grayi and Pseudevernia furfuracea (physodic acid chemotype) are involved in the synthesis of grayanic acid and physodic acid, CYP65FQ2 from Stereocaulon alpinum is involved in the synthesis of atranorin, and CYP6309A2 from Cladonia uncialis is involved in the synthesis of usnic acid. This study serves as a reference for future annotation of P450s in lichens.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme proteins that play a role in organisms’ primary and secondary metabolism. P450s play an important role in organism adaptation since lifestyle influences P450 composition in their genome. This phenomenon is well-documented in bacteria but less so in fungi. This study observed this phenomenon where diverse P450 complements were identified in saprophytic and ectomycorrhizal Pezizomycetes. Genome-wide data mining, annotation, and phylogenetic analysis of P450s in 19 Pezizomycetes revealed 668 P450s that can be grouped into 153 P450 families and 245 P450 subfamilies. Only four P450 families, namely, CYP51, CYP61, CYP5093, and CYP6001, are conserved across 19 Pezizomycetes, indicating their important role in these species. A total of 5 saprophyte Pezizomycetes have 103 P450 families, whereas 14 ectomycorrhizal Pezizomycetes have 89 P450 families. Only 39 P450 families were common, and 50 and 64 P450 families, respectively, were unique to ectomycorrhizal and saprophytic Pezizomycetes. These findings suggest that the switch from a saprophytic to an ectomycorrhizal lifestyle led to both the development of diverse P450 families as well as the loss of P450s, which led to the lowest P450 family diversity, despite the emergence of novel P450 families in ectomycorrhizal Pezizomycetes.

Ferredoxins, iron-sulfur (Fe-S) cluster proteins, play a key role in oxidoreduction reactions. To date, evolutionary analysis of these proteins across the domains of life have been confined to observing the abundance of Fe-S cluster types (2Fe-2S, 3Fe-4S, 4Fe-4S, 7Fe-8S (3Fe-4s and 4Fe-4S) and 2[4Fe-4S]) and the diversity of ferredoxins within these cluster types was not studied. To address this research gap, here we propose a subtype classification and nomenclature for ferredoxins based on the characteristic spacing between the cysteine amino acids of the Fe-S binding motif as a subtype signature to assess the diversity of ferredoxins across the living organisms. To test this hypothesis, comparative analysis of ferredoxins between bacterial groups, Alphaproteobacteria and Firmicutes and ferredoxins collected from species of different domains of life that are reported in the literature has been carried out. Ferredoxins were found to be highly diverse within their types. Large numbers of alphaproteobacterial species ferredoxin subtypes were found in Firmicutes species and the same ferredoxin subtypes across the species of Bacteria, Archaea, and Eukarya, suggesting shared common ancestral origin of ferredoxins between Archaea and Bacteria and lateral gene transfer of ferredoxins from prokaryotes (Archaea/Bacteria) to eukaryotes. This study opened new vistas for further analysis of diversity of ferredoxins in living organisms.

Abstract The cytochrome P450 (CYP) superfamily is a diverse and important enzyme family, playing a central role in chemical defense and in synthesis and metabolism of major biological signaling molecules. The CYPomes of four cnidarian genomes (Hydra vulgaris, Acropora digitifera, Aurelia aurita, Nematostella vectensis) were annotated; phylogenetic analyses determined the evolutionary relationships amongst the sequences and with existing metazoan CYPs. 155 functional CYPs were identified and 90 fragments. Genes were from 24 new CYP families and several new subfamilies; genes were in 9 of the 12 established metazoan CYP clans. All species had large expansions of clan 2 diversity, with H. vulgaris having reduced diversity for both clan 3 and mitochondrial clan. We identified potential candidates for xenobiotic metabolism and steroidogenesis. That each genome contained multiple, novel CYP families may reflect the large evolutionary distance within the cnidarians, unique physiology in the cnidarian classes, and/or different ecology of the individual species.

Abstract Background The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. Results This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. Conclusions The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Medicaid prior authorization (PA) policies for treatment of hepatitis C virus (HCV) with direct‐acting antiviral (DAA) therapy are changing. We aimed to evaluate effects of changes in PA requirements on treatment uptake and to determine the factors associated with DAA treatment among Florida Medicaid beneficiaries with HCV. This is a retrospective cohort analysis of Florida’s Medicaid administrative claims and electronic medical records (2013‐2018). A total of 14,063 newly diagnosed patients with HCV were grouped based on human immunodeficiency virus (HIV) co‐infection and/or a substance use disorder (SUD) (7,735 HCV mono‐infected with a SUD, 5,180 HCV mono‐infected without a SUD, 564 HCV/HIV co‐infected with a SUD, and 584 HCV/HIV co‐infected without a SUD). Although the treatment rate increased three‐fold after June 1, 2016, when a fibrosis‐stage restriction was eliminated, only 8% received DAAs. Compared to HCV mono‐infected without a SUD, HCV mono‐infected with a SUD and HCV/HIV co‐infected with a SUD were 47% (adjusted hazard ratio, 0.53; 95% confidence interval, 0.47‐0.60) and 59% (adjusted hazard ratio, 0.41; 95% confidence interval, 0.28‐0.61) less likely to initiate DAAs. Those with HCV/HIV/SUD did not experience a DAA initiation increase after a fibrosis‐stage restriction was eliminated. Compared with Whites, Blacks were less likely to receive DAAs but were more likely to complete treatment. Use of medication‐assisted therapy was low, despite those on medication‐assisted therapy being 60% more likely to initiate DAA therapy and no more likely to discontinue therapy. Conclusion: Despite changes in Florida’s Medicaid PA requirements for DAA treatment, only 8% received treatment. Disparities in treatment access were found among patients with HIV and a SUD, and who were Black.

Cytochrome P450 monooxygenases (CYPs/P450s) and their redox partners, ferredoxins, are ubiquitous in organisms. P450s have been studied in biology for over six decades owing to their distinct catalytic activities, including their role in drug metabolism. Ferredoxins are ancient proteins involved in oxidation-reduction reactions, such as transferring electrons to P450s. The evolution and diversification of P450s in various organisms have received little attention and no information is available for archaea. This study is aimed at addressing this research gap. Genome-wide analysis revealed 1204 P450s belonging to 34 P450 families and 112 P450 subfamilies, where some families and subfamilies are expanded in archaea. We also identified 353 ferredoxins belonging to the four types 2Fe-2S, 3Fe-4S, 7Fe-4S and 2[4Fe-4S] in 40 archaeal species. We found that bacteria and archaea shared the CYP109, CYP147 and CYP197 families, as well as several ferredoxin subtypes, and that these genes are co-present on archaeal plasmids and chromosomes, implying the plasmid-mediated lateral transfer of these genes from bacteria to archaea. The absence of ferredoxins and ferredoxin reductases in the P450 operons suggests that the lateral transfer of these genes is independent. We present different scenarios for the evolution and diversification of P450s and ferredoxins in archaea. Based on the phylogenetic analysis and high affinity to diverged P450s, we propose that archaeal P450s could have diverged from CYP109, CYP147 and CYP197. Based on this study’s results, we propose that all archaeal P450s are bacterial in origin and that the original archaea had no P450s.

Abstract Background Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species’ feeding and habitat traits, defining potential targets for pest management strategies. Results Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys’ capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. Conclusions Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Abstract Background Diatoms, which can accumulate large amounts of carotenoids, are a major group of microalgae and the dominant primary producer in marine environments. Phaeodactylum tricornutum, a model diatom species, acquires little silicon for its growth although silicon is known to contribute to gene regulation and play an important role in diatom intracellular metabolism. In this study, we explored the effects of artificial high-silicate medium (i.e. 3.0 mM sodium metasilicate) and LED illumination conditions on the growth rate and pigment accumulation in P. tricornutum, which is the only known species so far that can grow without silicate. It’s well known that light-emitting diodes (LEDs) as novel illuminants are emerging to be superior monochromatic light sources for algal cultivation with defined and efficient red and blue lights. Results Firstly, we cultivated P. tricornutum in a synthetic medium supplemented with either 0.3 mM or 3.0 mM silicate. The morphology and size of diatom cells were examined: the proportion of the oval and triradiate cells decreased while the fusiform cells increased with more silicate addition in high-silicate medium; the average length of fusiform cells also slightly changed from 14.33 µm in 0.3 mM silicate medium to 12.20 µm in 3.0 mM silicate medium. Then we cultivated P. tricornutum under various intensities of red light in combination with the two different levels of silicate in the medium. Higher biomass productivity also achieved in 3.0 mM silicate medium than in 0.3 mM silicate medium under red LED light irradiation at 128 μmol/m2/s or higher light intensity. Increasing silicate reversed the down-regulation of fucoxanthin and chlorophyll a under high red-light illumination (i.e. 255 μmol/m2/s). When doubling the light intensity, fucoxanthin content decreased under red light but increased under combined red and blue (50:50) lights while chlorophyll a content reduced under both conditions. Fucoxanthin accumulation and biomass productivity increased with enhanced red and blue (50:50) lights. Conclusion High-silicate medium and blue light increased biomass and fucoxanthin production in P. tricornutum under high light conditions and this strategy may be beneficial for large-scale production of fucoxanthin in diatoms.

Abstract Background The salmon louse (Lepeophtheirus salmonis) infests farmed and wild salmonid fishes, causing considerable economic damage to the salmon farming industry. Infestations of farmed salmon are controlled using a combination of non-medicinal approaches and veterinary drug treatments. While L. salmonis has developed resistance to most available salmon delousing agents, relatively little is known about the molecular mechanisms involved. Members of the cytochrome P450 (CYP) superfamily are typically monooxygenases, some of which are involved in the biosynthesis and metabolism of endogenous compounds, while others have central roles in the detoxification of xenobiotics. In terrestrial arthropods, insecticide resistance can be based on the enhanced expression of CYPs. The reported research aimed to characterise the CYP superfamily in L. salmonis and assess its potential roles in drug resistance. Methods Lepeophtheirus salmonis CYPs were identified by homology searches of the genome and transcriptome of the parasite. CYP transcript abundance in drug susceptible and multi-resistant L. salmonis was assessed by quantitative reverse transcription PCR, taking into account both constitutive expression and expression in parasites exposed to sublethal levels of salmon delousing agents, ecdysteroids and environmental chemicals. Results The above strategy led to the identification of 25 CYP genes/pseudogenes in L. salmonis, making its CYP superfamily the most compact characterised for any arthropod to date. Lepeophtheirus salmonis possesses homologues of a number of arthropod CYP genes with roles in ecdysteroid metabolism, such as the fruit fly genes disembodied, shadow, shade, spook and Cyp18a1. CYP transcript expression did not differ between one drug susceptible and one multi-resistant strain of L. salmonis. Exposure of L. salmonis to emamectin benzoate or deltamethrin caused the transcriptional upregulation of certain CYPs. In contrast, neither ecdysteroid nor benzo[a]pyrene exposure affected CYP transcription significantly. Conclusions The parasite L. salmonis is demonstrated to possess the most compact CYP superfamily characterised for any arthropod to date. The complement of CYP genes in L. salmonis includes conserved CYP genes involved in ecdysteroid biosynthesis and metabolism, as well as drug-inducible CYP genes. The present study does not provide evidence for a role of CYP genes in the decreased susceptibility of the multiresistant parasite strain studied.

Cytochrome P450 monooxygenases (P450s/CYPs) are ubiquitous enzymes with unique regio- and stereo-selective oxidation activities. Due to these properties, P450s play a key role in the biosynthesis of natural metabolites. Mycobacterial species are well-known producers of complex metabolites that help them survive in diverse ecological niches, including in the host. In this study, a comprehensive analysis of P450s and their role in natural metabolite synthesis in 2666 mycobacterial species was carried out. The study revealed the presence of 62,815 P450s that can be grouped into 182 P450 families and 345 subfamilies. Blooming (the presence of more than one copy of the same gene) and expansion (presence of the same gene in many species) were observed at the family and subfamily levels. CYP135 was the dominant family in mycobacterial species. The mycobacterial species have distinct P450 profiles, indicating that lifestyle impacts P450 content in their genome vis a vis P450s, playing a key role in organisms’ adaptation. Analysis of the P450 profile revealed a gradual loss of P450s from non-pathogenic to pathogenic mycobacteria. Pathogenic mycobacteria have more P450s in biosynthetic gene clusters that produce natural metabolites. This indicates that P450s are recruited for the biosynthesis of unique metabolites, thus helping these pathogens survive in their niches. This study is the first to analyze P450s and their role in natural metabolite synthesis in many mycobacterial species.

Summary: Diatoms are a major group of microalgae that initiate biofouling by surface colonization of human-made underwater structures; however, the involved regulatory pathways remain uncharacterized. Here, we describe a protocol for identifying and validating regulatory genes involved in the morphology shift of the model diatom species Phaeodactylum tricornutum during surface colonization. We also provide a workflow for characterizing biofouling transformants. By using this protocol, gene targets such as GPCR signaling genes could be identified and manipulated to turn off diatom biofouling.For complete information on the generation and use of this protocol, please refer to Fu et al. (2020).

Avicennia marina forests fulfill essential blue carbon and ecosystem services, including halting coastal erosion and supporting fisheries. Genetic studies of A. marina tissues could yield insight into halophyte adaptive strategies, empowering saline agriculture research. We compare transcriptomes from A. marina pneumatophores, stems, leaves, flowers, seeds, and transcriptomes across four widely divergent environments in the Indo-Pacific (Red Sea, Arabian Gulf, Bay of Bengal, and Red River Delta) to decipher the shared and location-, tissue-, and condition-specific functions. On average, 4.8% of transcripts per tissue were uniquely expressed in that tissue, and 12.2% were shared in all five tissues. Flowers’ transcript expression was the most distinct, with domain-centric gene ontology analysis showing high enrichment for stimulus-responsive processes, as well as genes implicated in flowering (hydroxygeraniol dehydrogenase, TPM = 3687) and floral scent biosynthesis (e.g., benzoyl_coenzyme_A, 2497.2 TPM). Pneumatophores highly expressed antioxidant genes, such as glutathione S-transferase (GST, TPM = 4759) and thioredoxin (TRX, TPM = 936.2), as well as proteins in the GO term ‘Hydroquinone:oxygen oxidoreductase activity’ (enrichment Z = 7.69, FDR-corr. p = 0.000785). Tissue-specific metabolic pathway reconstruction revealed unique processes in the five tissues; for example, seeds showed the most complete expression of lipid biosynthetic and degradation pathways. The leaf transcriptome had the lowest functional diversity among the expressed genes in any tissue, but highly expressed a catalase (TPM = 4181) and was enriched for the GO term ‘transmembrane transporter activity’ (GO:0015238; Z = 11.83; FDR-corr. p = 1.58 × 10−9), underscoring the genes for salt exporters. Metallothioneins (MTs) were the highest-expressed genes in all tissues from the cultivars of all locations; the dominant expression of these metal-binding and oxidative-stress control genes indicates they are essential for A. marina in its natural habitats. Our study yields insight into how A. marina tissue-specific gene expression supports halotolerance and other coastal adaptative strategies in this halophytic angiosperm.

Recent studies have suggested a negative impact of hepatocellular carcinoma (HCC) on sustained virologic response (SVR) to hepatitis C virus (HCV) direct acting antivirals (DAAs). We compared the effectiveness of DAAs in patients with cirrhosis, with and without HCC, and in those with HCC partially treated or untreated (PT/UT‐HCC) versus completely treated (CT‐HCC). HCC status was based on imaging 6 months before or 2 months after start of DAA therapy. Absence and presence of enhancing lesions after HCC treatment defined CT‐HCC and PT/UT‐HCC, respectively. Using minimally adjusted logistic regression, the association between the presence of HCC and SVR rates was estimated. Among the 1,457 patients with cirrhosis from HCV‐TARGET with complete virologic data (per‐protocol population) who did not undergo liver transplantation during treatment and followup, 1,300 were without HCC, 91 with CT‐HCC, and 66 with PT/UT‐HCC. Most patients were genotype 1 (81%) and treatment‐experienced (56%), 41% had history of prior decompensation, and the median pretreatment Model for End‐Stage Liver Disease was 9 (range 6‐39). The SVR rates were 91% for patients without HCC, 84% for CT‐HCC, and 80% for PT/UT‐HCC. The presence of HCC (versus not having HCC) was associated with significantly lower odds of achieving SVR (odds ratio [OR] = 0.51, 95% confidence interval [CI]: 0.33‐0.81; P = 0.003). However, among those with HCC, HCC treatment status (PT/UT‐HCC versus CT‐HCC) did not show association with SVR (OR = 0.79, 95% CI: 0.35‐1.79, P = 0.569). Conclusions: The presence of HCC reduces the likelihood of SVR by 50%, but with no evident difference in those with completely treated HCC versus partially treated/untreated HCC.

Introduction and aim: Direct-acting antiviral (DAA) agents are highly effective for treatment of chronic hepatitis C virus (HCV) yet access to treatment remains a serious challenge. The aim of this study was to identify barriers to treatment initiation with DAA-containing regimens in an urban clinic setting. Materials and methods: A retrospective cohort of all chronic HCV patients seen in an urban academic practice in Jacksonville, FL, USA from 1/2014 to 1/2017 was analyzed. Baseline characteristics were recorded and a review of medical records was performed to identify barriers to treatment initiation and overall success rates. Results: Two-hundred and forty patients with chronic HCV were analyzed. Fifty-six percent of patients were African-American and 63% were insured through Medicaid/county programs or uninsured. Sixty-nine percent had barriers to initiating antiviral therapy categorized as psychosocial (n = 112), provider (n = 26), medical (n = 20), and insurance-related factors (n = 7). The most commonly encountered psychosocial barriers included failure to keep appointments (79/240, 33%), active substance abuse (18/240, 8%), and failure to obtain laboratory testing (11/240, 5%). Overall, only 27% of patients evaluated were initiated on DAA-containing regimens with 18% reaching SVR12 within the 36-month study period. Conclusion: In conclusion, only 27% of patients who presented to an urban academic practice with chronic HCV received DAA-containing regimens over a 36-month period. Psychosocial issues were the major barriers to antiviral therapy. These findings illustrate the need for an integrated approach that addresses psychosocial factors as well as comorbidities and adherence to care in order to increase rates of HCV treatment in at risk patients.

Summary: The activities of microalgae support nutrient cycling that helps to sustain aquatic and terrestrial ecosystems. Most microalgal species, especially those from the subtropics, are genomically uncharacterized. Here we report the isolation and genomic characterization of 22 microalgal species from subtropical coastal regions belonging to multiple clades and three from temperate areas. Halotolerant strains including Halamphora, Dunaliella, Nannochloris, and Chloroidium comprised the majority of these isolates. The subtropical-based microalgae contained arrays of methyltransferase, pyridine nucleotide-disulfide oxidoreductase, abhydrolase, cystathionine synthase, and small-molecule transporter domains present at high relative abundance. We found that genes for sulfate transport, sulfotransferase, and glutathione S-transferase activities were especially abundant in subtropical, coastal microalgal species and halophytic species in general. Our metabolomics analyses indicate lineage- and habitat-specific sets of biomolecules implicated in niche-specific biological processes. This work effectively expands the collection of available microalgal genomes by ∼50%, and the generated resources provide perspectives for studying halophyte adaptive traits. : Global Nutrient Cycle; Algology; Genomics; Metabolomics Subject Areas: Global Nutrient Cycle, Algology, Genomics, Metabolomics

For the last six decades, cytochrome P450 monooxygenases (CYPs/P450s), heme thiolate proteins, have been under the spotlight due to their regio- and stereo-selective oxidation activities, which has led to the exploration of their applications in almost all known areas of biology. The availability of many genome sequences allows us to understand the evolution of P450s in different organisms, especially in the Bacteria domain. The phenomenon that “P450s play a key role in organisms’ adaptation vis a vis lifestyle of organisms impacts P450 content in their genome” was proposed based on studies on a handful of individual bacterial groups. To have conclusive evidence, one must analyze P450s and their role in secondary metabolism in species with diverse lifestyles but that belong to the same category. We selected species of the phylum Proteobacteria classes, Alpha, Beta, Gamma, Delta, and Epsilon, to address this research gap due to their diverse lifestyle and ancient nature. The study identified that the lifestyle of alpha-, beta-, gamma-, delta-, and epsilon-proteobacterial species profoundly affected P450 profiles in their genomes. The study determined that irrespective of the species associated with different proteobacterial classes, pathogenic species or species adapted to a simple lifestyle lost or had few P450s in their genomes. On the contrary, species with saprophytic or complex lifestyles had many P450s and secondary metabolite biosynthetic gene clusters. The study findings prove that the phenomenon mentioned above is factual, and there is no link between the number and diversity of P450s and the age of the bacteria.

Species belonging to the bacterial phyla Bacteroidetes and Firmicutes represent over 90% of the gastrointestinal microbiota. Changes in the ratio of these two bacterial groups were found to have contrasting health effects, including obesity and inflammatory diseases. Despite the availability of many bacterial genomes, comparative genomic studies on the gene pools of these two bacterial groups concerning cytochrome P450 monooxygenases (P450s), ferredoxins, and secondary metabolite biosynthetic gene clusters (smBGCs) are not reported. This study is aimed to address this research gap. The study revealed the presence of diverse sets of P450s, ferredoxins, and smBGCs in their genomes. Bacteroidetes species have the highest number of P450 families, ferredoxin cluster-types, and smBGCs compared to Firmicutes species. Only four P450 families, three ferredoxin cluster types, and five smBGCs are commonly shared between these two bacterial groups. Considering the above facts, we propose that the contrasting effects of these two bacterial groups on the host are partly due to the distinct nature of secondary metabolites produced by these organisms. Thus, the cause of the contrasting health effects of these two bacterial groups lies in their gene pools.

Background: Survivors of COVID-19 pneumonia often suffer from chronic critical illness (CCI) and require long-term hospitalization. Long-term acute care (LTAC) hospitals are vital in the care of CCI patients, but their role for patients post COVID-19 infection is not known. Barlow Respiratory Hospital (BRH) is a 105-bed, LTAC hospital network serving ventilator-dependent and medically-complex patients transferred from the ICUs of hospitals in southern California. We report patient characteristics of our first series of COVID-19 survivors admitted to the post-acute venue of an LTAC hospital. Methods: Single-center observational descriptive report of patients recovering from acute infectious complications of COVID-19 pneumonia requiring long-term respiratory support. Results: From 28 April to 7 September 2020, 41 patients were admitted to BRH for continued recovery from COVID-19 pneumonia. Median age: 68 [44-94] years, 25/41 (61%) male, 33/41 (80.5%) with tracheostomy, 21/41 (51.2%) on invasive mechanical ventilation, 9/41 (22%) receiving hemodialysis. All mechanical ventilation and hemodialysis interventions were initiated at the transferring hospital. Conclusions: To our knowledge, this is the first report to characterize CCI and medically complex COVID-19 patients transferred to the post-acute venue of an LTAC hospital. Patients on average spent over six weeks in the transferring hospital mostly in the ICU, are largely elderly, carry the known risk factors for COVID-19 infection, and experienced respiratory failure necessitating prolonged mechanical ventilation via tracheostomy. Our findings suggest that these patients will continue to require considerable medical interventions and treatments, including weaning from mechanical ventilation, owing to the numerous sequelae of the infection and the burden of acute-on-chronic diseases. As ICU survival rates improve, this research further emphasizes the important role of the LTAC hospital in responding to the COVID-19 crisis.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme thiolate proteins present in species across the biological kingdoms. By virtue of their broad substrate promiscuity and regio- and stereo-selectivity, these enzymes enhance or attribute diversity to secondary metabolites. Actinomycetes species are well-known producers of secondary metabolites, especially Salinispora species. Despite the importance of P450s, a comprehensive comparative analysis of P450s and their role in secondary metabolism in Salinispora species is not reported. We therefore analyzed P450s in 126 strains from three different species Salinispora arenicola, S. pacifica, and S. tropica. The study revealed the presence of 2643 P450s that can be grouped into 45 families and 103 subfamilies. CYP107 and CYP125 families are conserved, and CYP105 and CYP107 families are bloomed (a P450 family with many members) across Salinispora species. Analysis of P450s that are part of secondary metabolite biosynthetic gene clusters (smBGCs) revealed Salinispora species have an unprecedented number of P450s (1236 P450s-47%) part of smBGCs compared to other bacterial species belonging to the genera Streptomyces (23%) and Mycobacterium (11%), phyla Cyanobacteria (8%) and Firmicutes (18%) and the classes Alphaproteobacteria (2%) and Gammaproteobacteria (18%). A peculiar characteristic of up to six P450s in smBGCs was observed in Salinispora species. Future characterization Salinispora species P450s and their smBGCs have the potential for discovering novel secondary metabolites.

Summary: Surface colonization allows diatoms, a dominant group of phytoplankton in oceans, to adapt to harsh marine environments while mediating biofoulings to human-made underwater facilities. The regulatory pathways underlying diatom surface colonization, which involves morphotype switching in some species, remain mostly unknown. Here, we describe the identification of 61 signaling genes, including G-protein-coupled receptors (GPCRs) and protein kinases, which are differentially regulated during surface colonization in the model diatom species, Phaeodactylum tricornutum. We show that the transformation of P. tricornutum with constructs expressing individual GPCR genes induces cells to adopt the surface colonization morphology. P. tricornutum cells transformed to express GPCR1A display 30% more resistance to UV light exposure than their non-biofouling wild-type counterparts, consistent with increased silicification of cell walls associated with the oval biofouling morphotype. Our results provide a mechanistic definition of morphological shifts during surface colonization and identify candidate target proteins for the screening of eco-friendly, anti-biofouling molecules.

Introduction: Health research registries have great potential to increase awareness of research opportunities among diverse patient populations and reduce disparities in clinical trial accrual. However, little research has focused on patients’ intentions to participate in clinical trials once they are enrolled in the registry and their intentions to remain in the registry over time. Methods: Patients (N = 312) enrolled in a university-based health research registry (i.e., Consent2Share) in the southeastern region of the US participated in an online survey. Results: Health research registry knowledge, perceived values, self-efficacy, trust, having chronic health concerns, and consent recall were positively correlated with intentions to remain enrolled in the research registry and participate in future clinical trials. Health research registry consent recall had significant positive associations with registry knowledge, perceived values, trust, registry retention, and participating in future trials. Conclusion: The process of consenting patients to the health research registry is important for recruitment, registry retention, and participation in future clinical trials. We identified key points of emphasis to expand participation in research registries as a strategy to increase clinical trial enrollment, such as deploying precision messages and tailored interventions. Keywords: Research registry, Clinical trials, Recruitment, Consent recall, Communication

Introduction: Although registries can rapidly identify clinical study participants, it is unknown which follow up methods for recruiting are most effective. We examined the efficacy of three communication strategies for recruiting and enrolling patients who were identified via a contact registry (i.e., registry linked to a consent to re-contact program) into a clinical study. Methods: Patients who met the study criteria were identified via the contact registry and targeted for recruitment. In condition 1, patients established in the university hepatology specialty clinics were contacted one time via phone call by the study coordinator and asked to participate (C1). In condition 2, non-established specialty clinic patients were mailed an IRB-approved letter with study information and instructions for calling the study coordinator to participate (C2). Condition 2A included patients who called within two weeks of receiving the letter (C2A); condition 2B included patients who did not call after receiving the letter but were subsequently contacted via phone call. Results: A registry identified 1060 patients, of which 661 were eligible and targeted for recruiting. All 37 patients were reached in C1 and 17 (45.9%) were recruited. Nineteen of the 624 patients in C2A were reached and 10 were recruited whereas 120 of the 605 patients in C2B were reached and 53 (8.7%) were recruited. Seventy patients enrolled with C2B being the most effective (total, cost) recruitment strategy (n = 50) (p

Two-dimensional elastic membranes show great potential as functional materials, but how to engineer them with tunable mechanical properties remains challenging. Here, Yllanes et al. propose a method to induce the onset of crumpling transition in graphene-like sheets by simply altering their geometries.

Abstract Background Vibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum. Results Seven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout. Conclusions The data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain.

Cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins, are well-known players in the generation of chemicals valuable to humans and as a drug target against pathogens. Understanding the evolution of P450s in a bacterial population is gaining momentum. In this study, we report comprehensive analysis of P450s in the ancient group of the bacterial class Alphaproteobacteria. Genome data mining and annotation of P450s in 599 alphaproteobacterial species belonging to 164 genera revealed the presence of P450s in only 241 species belonging to 82 genera that are grouped into 143 P450 families and 214 P450 subfamilies, including 77 new P450 families. Alphaproteobacterial species have the highest average number of P450s compared to Firmicutes species and cyanobacterial species. The lowest percentage of alphaproteobacterial species P450s (2.4%) was found to be part of secondary metabolite biosynthetic gene clusters (BGCs), compared other bacterial species, indicating that during evolution large numbers of P450s became part of BGCs in other bacterial species. Our study identified that some of the P450 families found in alphaproteobacterial species were passed to other bacterial species. This is the first study to report on the identification of CYP125 P450, cholesterol and cholest-4-en-3-one hydroxylase in alphaproteobacterial species (Phenylobacterium zucineum) and to predict cholesterol side-chain oxidation capability (based on homolog proteins) by P. zucineum.

Abstract Background The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach. Results Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation. Conclusions The carotenogenic yeast X. dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the carotenogenic yeast X. dendrorhous and their potential biotechnological applications.

Cytochrome P450 family enzymes have an essential role in the creation of triterpenoid diversity in plants. Here, the authors describe triterpenoid synthesis as mediated by CYP716 enzymes in medicinal plant species, and perform phylogenetic analysis to describe CYP716 molecular evolution in plants.

Larval oysters in hatcheries are susceptible to diseases caused by bacterial pathogens, including Vibrio spp. Previous studies have shown that daily addition of the probiotic Bacillus pumilus RI06-95 to water in rearing tanks increases larval survival when challenged with the pathogen Vibrio coralliilyticus. We propose that the presence of probiotics causes shifts in bacterial community structure in rearing tanks, leading to a net decrease in the relative abundance of potential pathogens. During three trials spanning the 2012–2015 hatchery seasons, larvae, tank biofilm, and rearing water samples were collected from control and probiotic-treated tanks in an oyster hatchery over a 12-day period after spawning. Samples were analyzed by 16S rRNA sequencing of the V4 or V6 regions followed by taxonomic classification, in order to determine bacterial community structures. There were significant differences in bacterial composition over time and between sample types, but no major effect of probiotics on the structure and diversity of bacterial communities (phylum level, Bray–Curtis k = 2, 95% confidence). Probiotic treatment, however, led to a higher relative percent abundance of Oceanospirillales and Bacillus spp. in water and oyster larvae. In the water, an increase in Vibrio spp. diversity in the absence of a net increase in relative read abundance suggests a likely decrease in the abundance of specific pathogenic Vibrio spp., and therefore lower chances of a disease outbreak. Co-occurrence network analysis also suggests that probiotic treatment had a systemic effect on targeted members of the bacterial community, leading to a net decrease in potentially pathogenic species.

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.

The bed bug, Cimex lectularius, is a ubiquitous human ectoparasite with global distribution. Here, the authors sequence the genome of the bed bug and identify reductions in chemosensory genes, expansion of genes associated with blood digestion and genes linked to pesticide resistance.

Ticks transmit a large number of pathogens that cause human diseases. Here, the authors sequence the genome of the tick Ixodes scapularisand uncover expansion of genes associated with parasitic processes unique to ticks and tick-host interactions.

Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms’ primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing Mycobacterium tuberculosis H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different in silico approaches to understand CYP128 P450 family’s distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 CYP128 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between M. tuberculosis complex (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with CYP124A1, or together with CYP124A1 and CYP121A1. The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in M. tuberculosis.

Unraveling the role of cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins present in living and non-living entities, in secondary metabolite synthesis is gaining momentum. In this direction, in this study, we analyzed the genomes of 203 Streptomyces species for P450s and unraveled their association with secondary metabolism. Our analyses revealed the presence of 5460 P450s, grouped into 253 families and 698 subfamilies. The CYP107 family was found to be conserved and highly populated in Streptomyces and Bacillus species, indicating its key role in the synthesis of secondary metabolites. Streptomyces species had a higher number of P450s than Bacillus and cyanobacterial species. The average number of secondary metabolite biosynthetic gene clusters (BGCs) and the number of P450s located in BGCs were higher in Streptomyces species than in Bacillus, mycobacterial, and cyanobacterial species, corroborating the superior capacity of Streptomyces species for generating diverse secondary metabolites. Functional analysis via data mining confirmed that many Streptomyces P450s are involved in the biosynthesis of secondary metabolites. This study was the first of its kind to conduct a comparative analysis of P450s in such a large number (203) of Streptomyces species, revealing the P450s’ association with secondary metabolite synthesis in Streptomyces species. Future studies should include the selection of Streptomyces species with a higher number of P450s and BGCs and explore the biotechnological value of secondary metabolites they produce.

The prokaryotic phylum Cyanobacteria are some of the oldest known photosynthetic organisms responsible for the oxygenation of the earth. Cyanobacterial species have been recognised as a prosperous source of bioactive secondary metabolites with antibacterial, antiviral, antifungal and/or anticancer activities. Cytochrome P450 monooxygenases (CYPs/P450s) contribute to the production and diversity of various secondary metabolites. To better understand the metabolic potential of cyanobacterial species, we have carried out comprehensive analyses of P450s, predicted secondary metabolite biosynthetic gene clusters (BGCs), and P450s located in secondary metabolite BGCs. Analysis of the genomes of 114 cyanobacterial species identified 341 P450s in 88 species, belonging to 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Only 8% of P450s were found to be part of BGCs. Comparative analyses with other bacteria Bacillus, Streptomyces and mycobacterial species have revealed a lower number of P450s and BGCs and a percentage of P450s forming part of BGCs in cyanobacterial species. A mathematical formula presented in this study revealed that cyanobacterial species have the highest gene-cluster diversity percentage compared to Bacillus and mycobacterial species, indicating that these diverse gene clusters are destined to produce different types of secondary metabolites. The study provides fundamental knowledge of P450s and those associated with secondary metabolism in cyanobacterial species, which may illuminate their value for the pharmaceutical and cosmetics industries.

Despite the importance of fluid flow for transporting and organizing populations, few laboratory systems exist to systematically investigate the impact of advection on their spatial evolutionary dynamics. To address this problem, we study the morphology and genetic spatial structure of microbial colonies growing on the surface of a nutrient-laden fluid 10^{4} to 10^{5} times more viscous than water in Petri dishes; the extreme but finite viscosity inhibits undesired thermal convection and allows populations to effectively live at the air-liquid interface due to capillary forces. We discover that S. cerevisiae (baker’s yeast) growing on a viscous liquid behave like “active matter”: They metabolically generate fluid flows many times larger than their unperturbed colony expansion speed, and that flow, in turn, can dramatically impact their colony morphology and spatial population genetics. We show that yeast cells generate fluid flows by consuming surrounding nutrients and decreasing the local substrate density, leading to misaligned fluid pressure and density contours, which ultimately generates vorticity via a thresholdless baroclinic instability. Numerical simulations with experimentally measured parameters demonstrate that an intense vortex ring is produced below the colony’s edge. As the viscosity of the substrate is lowered and the self-induced flow intensifies, we observe three distinct morphologies: At the highest viscosity, cell proliferation and movement produces compact circular colonies with, however, a stretched regime of exponential expansion; intermediate viscosities give rise to compact colonies with “fingers” that are usually monoclonal and then break into smaller cell clusters; at the lowest viscosity, the expanding colony fractures into many genetically diverse, mutually repelling, islandlike fragments that can colonize an entire 94-mm-diameter Petri dish within 36 hours. We propose a simple phenomenological model that predicts the early colony dynamics. Our results provide rich opportunities to study the interplay between fluid flow and spatial population genetics for future investigations.

Tremellomycetes, a fungal class in the subphylum Agaricomycotina, contain well-known opportunistic and emerging human pathogens. The azole drug fluconazole, used in the treatment of diseases caused by some species of Tremellomycetes, inhibits cytochrome P450 monooxygenase CYP51, an enzyme that converts lanosterol into an essential component of the fungal cell membrane ergosterol. Studies indicate that mutations and over-expression of CYP51 in species of Tremellomycetes are one of the reasons for fluconazole resistance. Moreover, the novel drug, VT-1129, that is in the pipeline is reported to exert its effect by binding and inhibiting CYP51. Despite the importance of CYPs, the CYP repertoire in species of Tremellomycetes has not been reported to date. This study intends to address this research gap. Comprehensive genome-wide CYP analysis revealed the presence of 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of Tremellomycetes that can be grouped into 38 CYP families and 72 CYP subfamilies. Twenty-three CYP families are new and three CYP families (CYP5139, CYP51 and CYP61) were conserved across 23 species of Tremellomycetes. Pathogenic cryptococcal species have 50% fewer CYP genes than non-pathogenic species. The results of this study will serve as reference for future annotation and characterization of CYPs in species of Tremellomycetes.

Tuberculosis (TB) is one of the top infectious diseases causing numerous human deaths in the world. Despite enormous efforts, the physiology of the causative agent, Mycobacterium tuberculosis, is poorly understood. To contribute to better understanding the physiological capacity of these microbes, we have carried out extensive in silico analyses of the 1111 mycobacterial species genomes focusing on revealing the role of the orphan cytochrome P450 monooxygenase (CYP) CYP139 family. We have found that CYP139 members are present in 894 species belonging to three mycobacterial groups: M. tuberculosis complex (850-species), Mycobacterium avium complex (34-species), and non-tuberculosis mycobacteria (10-species), with all CYP139 members belonging to the subfamily “A”. CYP139 members have unique amino acid patterns at the CXG motif. Amino acid conservation analysis placed this family in the 8th among CYP families belonging to different biological domains and kingdoms. Biosynthetic gene cluster analyses have revealed that 92% of CYP139As might be associated with producing different secondary metabolites. Such enhanced secondary metabolic potentials with the involvement of CYP139A members might have provided mycobacterial species with advantageous traits in diverse niches competing with other microbial or viral agents, and might help these microbes infect hosts by interfering with the hosts’ metabolism and immune system.