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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of secondary metabolites that have garnered scientific attention due to their complex scaffolds with potential roles in medicine, agriculture, and chemical ecology. RiPPs derive from the cleavage of ribosomally synthesized proteins and additional modifications, catalyzed by various enzymes to alter the peptide backbone or side chains. Of these enzymes, cytochromes P450 (P450s) are a superfamily of heme-thiolate proteins involved in many metabolic pathways, including RiPP biosyntheses. In this review, we focus our discussion on P450 involved in RiPP pathways and the unique chemical transformations they mediate. Previous studies have revealed a wealth of P450s distributed across all domains of life. While the number of characterized P450s involved in RiPP biosyntheses is relatively small, they catalyze various enzymatic reactions such as C-C or C-N bond formation. Formation of some RiPPs is catalyzed by more than one P450, enabling structural diversity. With the continuous improvement of the bioinformatic tools for RiPP prediction and advancement in synthetic biology techniques, it is expected that further cytochrome P450-mediated RiPP biosynthetic pathways will be discovered.SummaryThe presence of genes encoding P450s in gene clusters for ribosomally synthesized and post-translationally modified peptides expand structural and functional diversity of these secondary metabolites, and here, we review the current state of this knowledge.

The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%-50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences.

Cells of most bacterial species are around 2 micrometers in length, with some of the largest specimens reaching 750 micrometers. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Candidatus (Ca.) Thiomargarita magnifica, a bacterium that has an average cell length greater than 9000 micrometers and is visible to the naked eye. These cells grow orders of magnitude over theoretical limits for bacterial cell size, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes into daughter cells. These features, along with compartmentalization of genomic material and ribosomes in translationally active organelles bound by bioenergetic membranes, indicate gain of complexity in the Thiomargarita lineage and challenge traditional concepts of bacterial cells.

Streptomycetes are highly metabolically gifted bacteria with the abilities to produce bioproducts that have profound economic and societal importance. These bioproducts are produced by metabolic pathways including those for the biosynthesis of secondary metabolites and catabolism of plant biomass constituents. Advancements in genome sequencing technologies have revealed a wealth of untapped metabolic potential from Streptomyces genomes. Here, we report the largest Streptomyces pangenome generated by using 205 complete genomes. Metabolic potentials of the pangenome and individual genomes were analyzed, revealing degrees of conservation of individual metabolic pathways and strains potentially suitable for metabolic engineering. Of them, Streptomyces bingchenggensis was identified as a potent degrader of plant biomass. Polyketide, non-ribosomal peptide, and gamma-butyrolactone biosynthetic enzymes are primarily strain specific while ectoine and some terpene biosynthetic pathways are highly conserved. A large number of transcription factors associated with secondary metabolism are strain-specific while those controlling basic biological processes are highly conserved. Although the majority of genes involved in morphological development are highly conserved, there are strain-specific varieties which may contribute to fine tuning the timing of cellular differentiation. Overall, these results provide insights into the metabolic potential, regulation and physiology of streptomycetes, which will facilitate further exploitation of these important bacteria.

Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcription factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is laborious and not scalable. Here, we demonstrate regulon identification by in vitro transcription-sequencing (RIViT-seq) that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViT-seq data expands the transcriptional regulatory network in this bacterium, discovering regulatory cascades and crosstalk between sigma factors. Implementation of RIViT-seq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology.

The National Microbiome Data Collaborative (NMDC) Data Portal (https://data.microbiomedata.org) supports microbiome multi-omics data exploration and access through an integrated, distributed data framework aligned with the FAIR (Findable, Accessible, Interoperable and Reusable) data principles (1). The NMDC Data Portal currently hosts 10.2 terabytes of multi-omics microbiome data, spanning five data types (metagenomes, metatranscriptomes, metaproteomes, metabolomes, and natural organic matter characterizations), generated at two Department of Energy User Facilities, the Joint Genome Institute (JGI) at Lawrence Berkeley National Laboratory (LBNL) and the Environmental Molecular Systems Laboratory (EMSL) at Pacific Northwest National Laboratory (PNNL). A flexible data schema (https://github.com/microbiomedata/nmdc-schema) leveraging community-driven standards underpins how data is managed and integrated. Annotated multi-omic data products are produced by the NMDC workflows and linked through common biosamples to enable search capabilities based on environmental context, instrumentation, and functional attributes. As a pilot system, the NMDC Data Portal offers download capabilities and several search components, including interactive geographic visualization of samples; environmental classification distribution visualized through an interactive Sankey diagram; time-series slider to select longitudinal samples of interest; and an upset plot displaying the number of multi-omics data generated from the same biosample within a study.

Phenazines (Phzs), a family of chemicals with a phenazine backbone, are secondary metabolites with diverse properties such as antibacterial, anti-fungal, or anticancer activity. The core derivatives of phenazine, phenazine-1-carboxylic acid (PCA) and phenazine-1,6-dicarboxylic acid (PDC), are themselves precursors for various other derivatives. Recent advances in genome mining tools have enabled researchers to identify many biosynthetic gene clusters (BGCs) that might produce novel Phzs. To characterize the function of these BGCs efficiently, we performed modular construct assembly and subsequent multi-chassis heterologous expression using chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE allowed rapid integration of a PCA BGC into 23 diverse γ-proteobacteria species and allowed us to identify top PCA producers. We then used the top five chassis hosts to express four partially refactored PDC BGCs. A few of these platforms produced high levels of PDC. Specifically, Xenorhabdus doucetiae and Pseudomonas simiae produced PDC at a titer of 293 mg/L and 373 mg/L, respectively, in minimal media. These titers are significantly higher than those previously reported. Furthermore, selectivity toward PDC production over PCA production was improved by up to 9-fold. The results show that these strains are promising chassis for production of PCA, PDC, and their derivatives, as well as for function characterization of Phz BGCs identified via bioinformatics mining.

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth's continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.

In the version of this article initially published, four people were missing from the alphabetical list of IMG/M Data Consortium members: Lauren V. Alteio of the Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria; Jeffrey L. Blanchard of the Biology Department, University of Massachusetts Amherst, Amherst, MA, USA; Kristen M. DeAngelis of the Department of Microbiology, University of Massachusetts Amherst, Amherst, MA, USA; and William Rodriguez-Reillo of the Research Computing Division, Harvard Medical School, Boston, MA, USA. The error has been corrected in the PDF and HTML versions of the article.

This paper was originally published under standard Springer Nature copyright (© The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license. The error has been corrected in the print, HTML and PDF versions of the article.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery.

Microbial secondary metabolism is a reservoir of bioactive compounds of immense biotechnological and biomedical potential. The biosynthetic machinery responsible for the production of these secondary metabolites (SMs) (also called natural products) is often encoded by collocated groups of genes called biosynthetic gene clusters (BGCs). High-throughput genome sequencing of both isolates and metagenomic samples combined with the development of specialized computational workflows is enabling systematic identification of BGCs and the discovery of novel SMs. In order to advance exploration of microbial secondary metabolism and its diversity, we developed the largest publicly available database of predicted BGCs combined with experimentally verified BGCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc-public). Here we describe the first major content update of the IMG-ABC knowledgebase, since its initial release in 2015, refreshing the BGC prediction pipeline with the latest version of antiSMASH (v5) as well as presenting the data in the context of underlying environmental metadata sourced from GOLD (https://gold.jgi.doe.gov/). This update has greatly improved the quality and expanded the types of predicted BGCs compared to the previous version.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

Natural products are a large family of diverse and complex chemical molecules that have roles in both primary and secondary metabolism, and over 210,000 natural products have been described. Secondary metabolite natural products are of high commercial and societal value with therapeutic uses as antibiotics, antifungals, antitumor and antiparasitic products and in agriculture as products for crop protection and animal health. There is a resurgence of activity in exploring natural products for a wide range of applications, due to not only increasing antibiotic resistance, but the advent of next-generation genome sequencing and new technologies to interrogate and investigate natural product biosynthesis. Genome mining has revealed a previously undiscovered richness of biosynthetic potential in novel biosynthetic gene clusters for natural products. Complementing these computational processes are new experimental platforms that are being developed and deployed to access new natural products.

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

The mechanism behind the regulation of dimethyl sulfoxide (DMSO) reductase genes of the phototroph Rhodobacter sphaeroides by both DMSO and oxygen was studied by investigating the transcriptional regulation of the dorC, dorR and dorS promoters. The dorC::lacZ expression was minimized by up to one-third when the cells were grown photosynthetically with DMSO and with medium to high lighting. The induction also depends on the DorR and DorS proteins.

Rhodobacter sphaeroides expresses different cytochrome oxidases depending on the availability of oxygen. Cytochrome oxidase aa3 is expressed with high oxygen tension while cbb3 is expressed under microaerophilic conditions. The genetic control of cbb3 expression is investigated in a fused transcriptional system using the promoter of cbb3, ccoN and the lacZ gene. Results indicate that oxygen concentration influences cbb3 expression and the protein FnrL is a positive regulator of cbb3 expression.

Research was conducted to describe the genes encoding the dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T. Recombinant DNA methods were utilized for the construction of mutants while automated DNA sequencing was carried out with an ABI 373A automatic DNA sequencer. Results indicated that the level of DorA protein expressed from the plasmids was sufficient to restore growth to the mutants. They also showed that dorS and dorR gene products are important for the production of DorA protein expression.

Research was conducted to study the functions of the fnrL gene in Rhodobacter capsulatus. R. capsulatus was prepared in a Sistrom's succinate minimal medium while DNA manipulations and enyzymatic treatments were determined according to standard protocols. Based on the identity of the amino acid sequences of the gene products, it is suggested that the fnrL gene identified in the study was the R. capsulatus homology of R. sphaeroides fnrL.

The molybdenum-dependent regulation of transcription of the operons of Azotobacter vinelandii, modEABC and modG, is mediated by the modE, the first gene in the modEABC operon. However, modE was not observed to have any control on the Mo-dependent expression of alternative nitrogenase structural genes. Mo accumulation of cells in the presence of molybdate were substantially reduced in a modE mutant, contrary to expectations of maximal rates of Mo transport with the constitutive expression of the high-affinity Mo-transport system.

An open reading frame, modG, that may encode a protein with a tandem repeat sequence similar to the molybdopterin-binding protein Mop was found upstream of the modEABC operon in Azotobacter vinelandii. Mutations in these mod genes, involved in the molybdenum transport system, appeared to reveal separate high and low affinity molybdenum transport systems. The modE and modG genes may have a role in molybdate processing, which may be important for nitrogenase cofactor biosynthesis.

The mutants of strain N44-1 in which the gene encoding the membrane-bound glucose dehydrogenase was inactivated either alone or together with the gene encoding the cytoplasmic glucose dehydrogenase to understand the low growth yield of glucose metabolism in the cytoplasm. The conversion of glucose to gluconate and ketogluconates in the cytoplasm was found to have strong negative impact on the growth of Gluconobacter oxydans N44-1.

Research was conducted to examine the effects of redox-dependent gene regulation in Rhodobacter sphaeroides 2.4.1(super T) on dimethyl sulfoxide (DMSO) reductase (dor) gene expression. Expression of dor::lacZ transcriptional fusions in the CcoP mutant provided evidence that the effects of the ccoP mutation occurred at the transcriptional level. Results demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in R sphaeroides.

Provides information on a study which examined the role of redox potentiation in the regulation of dimethyl sulfoxide reductase (DOR) expression. Materials and methods; Results of the study; Conclusion.