Multigene Family

Conserved biosynthetic gene clusters (BGCs) are often tied to the production of natural products that perform critical functions in an organism's physiology and ecological interactions. Here, by phylogenetic analysis across the bacterial genus, we report the obligate conservation of a BGC in genomes of cosmopolitan marine Microbulbifer bacteria. This genus is a common member of marine microbiomes, and this BGC was conserved in Microbulbifer genomes regardless of phylogenetic or geographical dispersal. The post-translationally modified peptidic product encoded by this BGC─which was accessed via heterologous production and its structure elucidated using a combination of mass spectrometry and NMR spectroscopy─was found to be a copper chelator. Similar BGCs were then found in genomes of other marine bacterial genera coinhabiting the microbiomes of sponges and corals. The phylogenomic workflows described herein were implemented in a pedagogic setting at the Georgia Institute of Technology to provide hands-on instruction to undergraduate students in bacterial phylogeny, genome mining, and natural product chemistry.

Withanolides are steroidal lactones from nightshade (Solanaceae) plants with untapped drug potential due to limited availability of minor representatives caused by lack of biosynthetic pathway knowledge. Here, we combine phylogenomics with metabolic engineering to overcome this limitation. By sequencing the genome of the medicinal plant ashwagandha (Withania somnifera) and comparing it with nine Solanaceae species, we discover a conserved withanolide biosynthesis gene cluster, consisting of two sub gene clusters with differing expression patterns. We establish metabolic engineering platforms in yeast (Saccharomyces cerevisiae) and the model plant Nicotiana benthamiana to reconstitute the first five oxidations of withanolide biosynthesis, catalysed by the cytochrome P450 monooxygenases CYP87G1, CYP88C7, and CYP749B2 and a short-chain dehydrogenase/reductase, producing the aglycone of withanoside V. Enzyme functions are conserved within both sub gene clusters in W. somnifera and between W. somnifera and Physalis pruinosa. Our work sets the basis for biotechnological withanolide production to unlock their pharmaceutical potential.

Plant-microbe symbioses such as the legume-rhizobium mutualism are vital in the web of ecological relationships within both natural and managed ecosystems, influencing primary productivity, crop yield, and ecosystem services. The outcome of these interactions for plant hosts varies quantitatively and can range from highly beneficial to even detrimental depending on natural genetic variation in microbial symbionts. Here, we take a systems genetics approach, harnessing the genetic diversity present in wild rhizobial populations to predict genes and molecular pathways crucial in determining partner quality, i.e., the benefits of symbiosis for legume hosts. We combine traits, dual-RNAseq of both partners from active nodules, pangenomics/pantranscriptomics, and Weighted Gene Co-expression Network Analysis (WGCNA) for a panel of 20 Sinorhizobium meliloti strains that vary in symbiotic partner quality. We find that genetic variation in the nodule transcriptome predicts host plant biomass, and WGCNA reveals networks of genes in plants and rhizobia that are coexpressed and associated with high-quality symbiosis. Presence-absence variation of gene clusters on the symbiosis plasmid (pSymA), validated in planta, is associated with high or low-quality symbiosis and is found within important coexpression modules. Functionally our results point to management of oxidative stress, amino acid and carbohydrate transport, and NCR peptide signaling mechanisms in driving symbiotic outcomes. Our integrative approach highlights the complex genetic architecture of microbial partner quality and raises hypotheses about the genetic mechanisms and evolutionary dynamics of symbiosis.

A surge in sequenced myxobacteria catalyzed by advancements in long-read genome and metagenome sequencing has provided sufficient data to scrutinize the conserved biosynthetic gene clusters (BGCs) within the phylum Myxococcota. Provided the utility of myxobacteria in environmental nutrient cycles and discovery of novel therapeutic leads, we sought to determine any conserved specialized metabolism in the phylum. Using a pan-genome approach to analyze 11 genera and 195 sequenced genomes, including 10 newly reported myxobacterial isolates, we observed five conserved BGCs. All five clusters encode for characterized metabolites with established ecological roles for four of the metabolites, and none of the metabolites are known toxins. Validation of our approach was done by analyzing Myxococcota genera without sufficient sequenced representatives for pan-genome analysis to observe the presence/absence of these five clusters. This approach enabled observation of genus-level conservation of BGCs with varying degrees of confidence due to the diversity of sequenced species within each genus. The indigoidine BGC typically found in Streptomyces spp. was notably conserved in Melittangium; heterologous expression of the core biosynthetic gene bspA in Escherichia coli and subsequent detection of indigoidine confirmed the identity of the indigoidine cluster. Conserved BGCs in myxobacteria reveal maintenance of biosynthetic pathways and cognate metabolites with ecological roles as chemical signals and stress response; these observations suggest competitive specialization of secondary metabolism and toxin production in myxobacteria.ImportanceCritical contributions to soil nutrient cycles by predatory bacteria, including the Myxococcota, and utility as a resource for the discovery of novel enzymology and metabolism motive continued isolation and characterization of myxobacteria from the environment. Each of these motivating factors involves specialized metabolites produced by myxobacteria and the biosynthetic gene clusters (BGCs) responsible for their assembly. Primarily associated with the predatory lifestyles of myxobacteria, myxobacterial specialized metabolites have been pursued as therapeutic leads for novel antibacterials, antifungals, anthelmintics, and cancer therapies. Despite these efforts and the observation that nearly all genera within the Myxococcota have an extraordinary number of BGCs, there is no consensus view of the conserved BGCs in the phylum. Our study revealed the core BGCs consistently present throughout the phylum. By reporting these core specialized metabolites and their ecological roles, we hope to streamline the discovery and investigation of specialized metabolism in myxobacteria.