Dawn M. Klingeman

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion in Bacillus subtilis to generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes, including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size (~15 kb), and these associations were validated through targeted genetic swaps. This B. subtilis QTL population can easily be used to map additional phenotypes and the general approach for QTL mapping is applicable to a wide range of Gram-positive and Gram-negative bacteria using diverse methods for genome-wide recombination.

The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease with which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption persists within the E. coli community that inactive gRNAs are rare. To resolve these contradictions, we evaluated the activity of 463 000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional. These nonfunctional gRNAs exhibit strong spacer self-interaction, which can either be excluded using a simple design rule or “repaired” during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million empirically evaluated E. coli gRNAs as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. Lastly, we have produced a webapp to allow users to easily browse and extract gRNA sequences from the E. coli genome, which can be accessed at https://grna.ornl.gov.

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

Abiotic stresses like heat and drought pose major threats to forest ecosystems and are anticipated to increase due to anthropogenic climate change. One mechanism by which plants cope with stress conditions is tailoring their microbiomes to aid in stress tolerance. However, experimentally imposing abiotic stresses to examine impacts on tree microbiomes over long timescales is impractical. Naturally occurring climate gradients provide an opportunity to observe the longer-term effects of abiotic stress on soil and tree-associated microbiomes, potentially informing projections on how these relationships will respond to climate change. Populus trichocarpa is an ecologically important tree species, a major source of wood products, a potential bioenergy feedstock, and has emerged as a model species for both biological and ecological processes. To understand how belowground plant-microbe interactions changed across climatic gradients, we collected bulk soil, rhizosphere soil, and root endosphere samples from P. trichocarpa trees found along a 200 km transect over the Cascade Mountains in Washington, USA within the Cowlitz River Basin on the wetter, western side and along the Tieton River basin on the drier, eastern side. Precipitation rates and temperatures varied considerably over the ~1300-meter elevation span. We performed 16S and ITS2 amplicon sequencing to examine the bacterial/archaeal and fungal communities, as well as soil chemical and physical characteristics, stand characteristics, and host tree physiology. We hypothesized that the soil and P. trichocarpa -associated rhizosphere and root endosphere microbiomes would vary in response to the naturally occurring climate gradient along the transect. We found that for each niche sampled, bacterial/archaeal and fungal communities clustered into three distinct micro-climate groups: a wetter community common across the Cowlitz River sites, a drier community of the lower altitude Tieton River sites, and a high elevation community at the crest of the pass and along the upper Tieton River. The community differences between these micro-climate groups were diminished within the P. trichocarpa root endosphere, though still evident, indicating that while P. trichocarpa may have a selective and homogenizing effect on the microbiome, abiotic factors play a major role in the overall shaping of these microbial communities.

Monolignol-derived dimers containing β–β linkages are synthesized by vascular plants and can be released during lignin depolymerization. In this work, we isolated a bacterium, Novosphingobium rhizosphaerae LY, that grows with the β–β lignan (+)-pinoresinol as a sole growth substrate. Sequence analysis suggested that this strain encodes a broad range of pathways for assimilation of aromatic monomers as well as one enzyme implicated in pinoresinol catabolism but lacks other known pathways for aromatic dimer catabolism. We constructed a genome-wide barcoded transposon library and identified genes required for pinoresinol catabolism. Using feeding studies, compound isolation, targeted synthesis, and analysis of purified enzymes, we elucidated the biochemical intermediates and reaction pathway involved in pinoresinol catabolism. We demonstrated that the first enzymatic reaction is the reductive cleavage of a furan ring in (±)-pinoresinol with retention of configuration to yield lariciresinol. We additionally confirmed that the final pathway enzyme, PinU, is related to lignostilbene dioxygenases and oxidatively cleaves a diguaiacylbutadiene intermediate to yield vanillin and coniferaldehyde. Finally, based on the enzyme characterization, we demonstrated that the strain can grow with a second β–β lignan, (–)-syringaresinol, as a sole growth substrate. In combination, these results demonstrate a new biocatalytic route for transforming a widely occurring group of plant phenylpropanoid natural products. IMPORTANCE: Plants synthesize a variety of aromatic phenylpropanoid compounds containing β–β linkages, including lignin, a major structural polymeric component of the vascular plant cell wall, and lignans, biochemically related secondary metabolites with a wide range of bioactivities. Although microbial catabolic pathways have been described for dimeric phenylpropanoids featuring other interunit linkages, relatively little is known about pathways for catabolism of β–β-linked compounds. In this work, we isolated a Novosphingobium strain capable of degrading the β–β-linked lignan (+)-pinoresinol and elucidated the catabolic pathway. Understanding how bacteria catabolize β–β-linked compounds provides a basis for new biocatalytic transformations of lignans and oligolignols and has the potential to improve bacterial lignin valorization.

Bacteria are recognized for their diverse metabolic capabilities, yet the impact of microbe-microbe interactions on multispecies community structure and dynamics is poorly understood. Cell-to-cell signaling in the form of quorum sensing (QS) often regulates secondary metabolite production and microbial interactions. Here, we examine how acylhomoserine lactone (AHL)-mediated QS impacts microbial community structure in a 10-member synthetic community of isolates from Populus deltoides . To explore the role of QS in microbial community structure and dynamics, we disrupted AHL signaling by exogenous addition of AiiA-lactonase, an enzyme that cleaves the lactone ring. Microbial community structure resulting from signal inactivation, as measured by 16S rRNA amplicon sequencing and secondary metabolite production, was assessed after successive passaging of the community. Further, we investigated the impact of quorum quenching on specific microbe-microbe interactions using pairwise inhibition assays. Our results indicate that AHL inactivation alters the relative abundance of dominant community members at later passages but does not impact the overall membership in the community. Quorum quenching significantly alters the metabolic profile in lactonase-treated communities. This metabolic alteration impacts microbe-microbe interactions through decreased inhibition of other community members. Together, these results indicate that QS impacts microbial community structure through the regulation of secondary metabolites in dominant members and that the membership of microbial communities can be relatively stable despite changes in metabolic profiles. IMPORTANCE: In terrestrial ecosystems, bacteria exist as multispecies consortia and provide diverse ecosystem services. Interactions among microbes contribute to determining their abundance and population structure and are often mediated by cell-to-cell communication. However, the role of microbial communication in community assembly is poorly understood. In this study, we investigated the disruption of AHL-based quorum sensing on bacterial community structure using a synthetic microbial community derived from a plant host. We found that disrupting AHL signaling did not change the membership but shifted the relative abundance of the dominant community members. Metabolic profiles of disrupted communities reveal alterations in key secondary metabolites that likely reduce antagonistic behavior. Investigating the driving mechanisms underlying microbial community assembly is fundamental to understanding microbial ecosystem ecology and can be broadly applied toward understanding sustainable systems and facilitating agricultural applications where plant-associated microbes are of growing importance.

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion in Bacillus subtilis to generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size (∼10 kb), and these associations were validated through targeted genetic swaps. This B. subtilis QTL population can easily be used to map additional phenotypes, and the general approach for QTL mapping is applicable in a wide range of bacteria. One-Sentence Summary: Bacterial genome shuffling mimics sexual recombination and enables genetic mapping of complex phenotypes to causal alleles.

The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease in which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption that inactive gRNAs are rare persists in the E. coli community. To resolve these contradictions, we evaluated the activity of nearly 500,000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional.These nonfunctional gRNAs exhibit strong spacer self-interaction, which can be either excluded using a simple design rule or “repaired” during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million E. coli gRNAs that have been empirically evaluated in vivo as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. GRAPHICAL

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We show that the most highly repressing RNAT, hsp17rep , significantly reduces population skew with a library of characterized guide RNAs (gRNAs). This information should be applicable to all Cas9-based methods and technologies. Notice: This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( https://www.energy.gov/doe-public-access-plan ). Graphical

Cancer therapy-induced oral mucositis is a frequent major oncological problem, secondary to cytotoxicity of chemo-radiation treatment. Oral mucositis commonly occurs 7–10 days after initiation of therapy; it is a dose-limiting side effect causing significant pain, eating difficulty, need for parenteral nutrition and a rise of infections. The pathobiology derives from complex interactions between the epithelial component, inflammation, and the oral microbiome. Our longitudinal study analysed the dynamics of the oral microbiome (bacteria and fungi) in nineteen patients undergoing chemo-radiation therapy for oral and oropharyngeal squamous cell carcinoma as compared to healthy volunteers. The microbiome was characterized in multiple oral sample types using rRNA and ITS sequence amplicons and followed the treatment regimens. Microbial taxonomic diversity and relative abundance may be correlated with disease state, type of treatment and responses. Identification of microbial-host interactions could lead to further therapeutic interventions of mucositis to re-establish normal flora and promote patients’ health. Data presented here could enhance, complement and diversify other studies that link microbiomes to oral disease, prophylactics, treatments, and outcome.