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The National Microbiome Data Collaborative (NMDC) is a multi-organizational effort to integrate microbiome data across diverse areas in environmental science. Data provided by the NMDC can then undergo advanced analysis and provide new insights into metagenomics, metatranscriptomics, metaproteomics, and metabolomics. To address these challenges, we have developed our schema using the Linked data Modeling Language (LinkML). This allows us to easily map data to existing standards and ontologies.

The nascent field of microbiome science is transitioning from a descriptive approach of cataloging taxa and functions present in an environment to applying multi-omics methods to investigate microbiome dynamics and function. A large number of new tools and algorithms have been designed and used for very specific purposes on samples collected by individual investigators or groups. While these developments have been quite instructive, the ability to compare microbiome data generated by many groups of researchers is impeded by the lack of standardized application of bioinformatics methods. Additionally, there are few examples of broad bioinformatics workflows that can process metagenome, metatranscriptome, metaproteome and metabolomic data at scale, and no central hub that allows processing, or provides varied omics data that are findable, accessible, interoperable and reusable (FAIR). Here, we review some of the challenges that exist in analyzing omics data within the microbiome research sphere, and provide context on how the National Microbiome Data Collaborative has adopted a standardized and open access approach to address such challenges., Competing Interests: Author DW is employed by Polyneme LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hu, Canon, Eloe-Fadrosh, Anubhav, Babinski, Corilo, Davenport, Duncan, Fagnan, Flynn, Foster, Hays, Huntemann, Jackson, Kelliher, Li, Lo, Mans, McCue, Mouncey, Mungall, Piehowski, Purvine, Smith, Varghese, Winston, Xu and Chain.)

Microbiome samples are inherently defined by the environment in which they are found. Therefore, data that provide context and enable interpretation of measurements produced from biological samples, often referred to as metadata, are critical. Important contributions have been made in the development of community-driven metadata standards; however, these standards have not been uniformly embraced by the microbiome research community. To understand how these standards are being adopted, or the barriers to adoption, across research domains, institutions, and funding agencies, the National Microbiome Data Collaborative (NMDC) hosted a workshop in October 2019. This report provides a summary of discussions that took place throughout the workshop, as well as outcomes of the working groups initiated at the workshop.

We present redirection of electron flow to more efficient proton pumping branches within respiratory chains as a generally applicable metabolic engineering strategy, which tailors microbial metabolism to the specific requirements of high cell density processes by improving product and biomass yields. For the example of riboflavin production by Bacillus subtilis, we reduced the rate of maintenance metabolism by about 40% in a cytochrome bd oxidase knockout mutant. Since the putative Yth and the caa(3) oxidases were of minor importance, the most likely explanation for this improvement is translocation of two protons per transported electron via the remaining cytochrome aa(3) oxidase, instead of only one proton via the bd oxidase. The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and B. subtilis biomass in fed-batch cultures.

Rhodobacter sphaeroides 2.4.1(T) respires aerobically via a branched respiratory chain consisting of both cytochrome c oxidases and quinol oxidases. Here, genes from chromosome II encoding two distinct quinol oxidases have been characterized. The qoxBA genes encode a putative heme-copper quinol oxidase, whereas the qxtAB genes encode a quinol oxidase homologous to the cyanide-insensitive oxidase of Pseudomonas aeruginosa. No phenotype was observed for mutations in either oxidase in the wild-type background. A strain containing a qxtA mutation in a cytochrome bc(1) complex mutant background was unable to grow aerobically. No role was found for the Qox oxidase, nor was a qoxB::lacZ transcriptional fusion expressed under a variety of conditions. These are the first molecular studies to characterize the quinol oxidases of R. sphaeroides 2.4.1(T).

The ability of Rhodobacter sphaeroides 2.4.1(T) to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the dor locus. Previously, we have demonstrated that dor expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium. Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY. To further examine the role of redox potentiation in the regulation of dor expression, we measured DMSO reductase synthesis and beta-galactosidase activity from dor::lacZ fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression. Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO. When cells were grown photoheterotrophically, dorC::lacZ expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions. Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the ccoP mutation. By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered dor expression in CcoP mutants. These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in R. sphaeroides and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling dor expression.

Under anaerobic-dark growth conditions, in the presence of the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), Rhodobacter sphaeroides 2.4.1(T) respires anaerobically using the molybdoenzyme DMSO reductase (DMSOR). Genes encoding DMSOR and associated proteins are encoded by genes of the dor locus. Previously, we demonstrated that the expression of DMSOR is regulated by both the oxygen status of the cell via the FnrL protein and by the presence of DMSO or TMAO, presumably through the DorS-DorR two-component sensor-regulator system. Here we further investigate expression of the dor genes through the use of transcriptional lacZ fusions to the dorS, dorR, and dorC promoters. The expression of dorC::lacZ was strongly induced by the absence of oxygen and presence of DMSO. In accordance with our previous findings of DMSOR activity, dorC::lacZ expression was reduced by up to one-third when cells were grown photosynthetically in the presence of DMSO with medium or high light, compared to the expression observed after anaerobic-dark growth. The induction of dorC::lacZ expression in the presence of DMSO was dependent on the DorS and DorR proteins. Expression of the dorS and dorR genes was also induced in the absence of oxygen. In an FnrL mutant, dorS::lacZ expression was not induced when oxygen tensions in the media were lowered, in contrast to what occurred in the wild-type strain. The expression of dorS::lacZ and dorR::lacZ was dependent on the DorS and DorR proteins themselves, suggesting the importance of autoregulation. These results demonstrate a cascade regulation of dor gene expression, where the expression of the regulatory proteins DorS and DorR governs the downstream regulation of the dorCBA operon encoding the structural proteins of DMSOR.

The ccoNOQP gene cluster of Rhodobacter sphaeroides 2.4.1T encodes a cbb3 cytochrome oxidase which is utilized in oxygen-limited conditions for aerobic respiration. The beta-galactosidase activity of a ccoN::lacZ transcriptional fusion was low under high (30%)-oxygen and anaerobic growth conditions. Maximal ccoN::lacZ expression was observed when the oxygen concentration was lowered to 2%. In an FnrL mutant, ccoN::lacZ expression was significantly lower than in the wild-type strain, suggesting that FnrL is a positive regulator of genes encoding the cbb3 oxidase.