Biology subjects

Klingeman, D. M.

Publications and source records attributed to Klingeman, D. M..

10 recordsLinked to original sources

bioRxiv2026

Parallel tracking by sequencing reveals the impact of restriction modification systems on transfer of an integrative and conjugative element

Horizontal gene transfer (HGT) is a promising avenue for microbiome engineering that enables DNA delivery to microbes in their native environment. Integrative and conjugative elements (ICEs), a class of genomically-integrated mobile elements, are ideal vectors for this purpose. Developing targeted ICE transfer for controlled microbiome engineering requires a better understanding of ICE mobility in complex communities. However, current methods for tracking horizontal gene transfer are not high throughput. To improve upon current methods, we developed a sequencing-based strategy to track and quantify ICE transfer in complex mixtures that we refer to as ICE-seq. This method was able to assess ICE transfer in a synthetic community of 150 recipients simultaneously and in combination with phenotypic assays demonstrated that restriction-modification (RM) systems in the donor and recipient alter ICE transfer rate with subspecies resolution by up to 10,000-fold. We propose that manipulating RM systems can be a strategy to engineer targeted ICE transfer for directed microbiome engineering. ImportanceTypical approaches to engineer microbiomes involve adding beneficial strains to existing microbiomes, for example the addition of probiotics to the human gut microbiome or soil amendments to improve plant health or for bioremediation. However, these beneficial bacteria often do not survive for extended periods of time, which limits their overall effectiveness. To circumvent this issue, we can transfer beneficial genes by native mechanisms into existing bacteria that are already in the target environment where they can then perform the beneficial function encoded by these genes. In this work, we developed a technique to track gene transfer into multiple recipients simultaneously. Furthermore, bacteria have innate defense systems to protect against invading DNA that can reduce this gene transfer. Our technique allowed us to identify the impact of these defense systems and their potential function for targeting gene transfer to specific recipients.

microbiology↗

bioRxiv2025

Populus trichocarpa associated microbiomes vary across a natural temperature, elevation and rainfall gradient

Abiotic stresses associated with warming and drought are increasing in frequency and severity due to climate change. However, microbial associations can partially mitigate their adverse effects on plant physiology. We examined the bacterial/archaeal and fungal communities with 16S rRNA and ITS2 gene region amplicon sequencing of bulk soil, rhizosphere soil, and root endosphere samples of Populus trichocarpa trees from 11 sites along a 200 km transect with highly variable precipitation, temperature, and soil conditions over the Cascade Mountains in Washington, USA. Foliar stress response was examined with qRT-PCR and field physiological measurements. Soil conditions were examined with physiochemical sample analysis and site climate conditions were approximated using DAYMET. For each niche sampled, bacterial/archaeal and fungal communities clustered into three distinct micro-climate groups: a high-precipitation, low-stress community on the western side of the range, and two stressed climate communities: a high-elevation community nearer the crest of the pass, and a warmer and drier community along the eastern slope. The differences between these micro-climate communities, though still distinct, were notably diminished within the P. trichocarpa root endosphere, indicating that P. trichocarpa likely has a homogenizing effect on the microbiome under stress, but climate plays a major role in shaping these microbial communities.

ecology↗

bioRxiv2025

Genome shuffling enables quantitative trait locus mapping in Bacillus subtilis

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 SummaryBacterial genome shuffling mimics sexual recombination and enables genetic mapping of complex phenotypes to causal alleles.

microbiology↗

bioRxiv2025

Combinatorial pathway engineering using multiplex Serine recombinase-Assisted Genome Engineering (mSAGE)

Low efficiency of DNA integration into the chromosome limits multiplexed bacterial genetic engineering. We address this by establishing multiplexed Serine recombinase-Assisted Genome Engineering (mSAGE), enabling simultaneous, site-specific insertion of three DNAs into the chromosome of Pseudomonas putida KT2440. We construct a combinatorial library of isophthalate catabolism and transport genes through a single, pooled transformation of three gene libraries, rapidly identifying the best combination of genes for biodegradation of this plastic comonomer.

microbiology↗

bioRxiv2025

When good guides go bad: empirical evaluation of all unique Cas9 targets in E. coli reveal widespread functionality and rules for gRNA biological design

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 ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/651106v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1038ddcorg.highwire.dtl.DTLVardef@566e37org.highwire.dtl.DTLVardef@5df6b2org.highwire.dtl.DTLVardef@ef453c_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

BONCAT-Live for isolation and cultivation of active environmental bacteria

In diverse environments, microbes drive a myriad of processes, from geochemical and nutrient cycling to interspecies interactions, including in association with plants and animals. Their physiological state is dynamic and impacted by abiotic and biotic conditions, responding to environmental fluctuations by changes in cellular metabolism, according to their genetic potential. Molecular, cellular and genomic approaches can identify and measure microbial responses and adaptation to environmental changes in native communities. However, isolating the individual microbes that respond to specific changes has been difficult. To address that, we implemented BONCAT-Live, by integrating bio-orthogonal non-canonical amino acid tagging (BONCAT) in diverse native communities, with isolation and cultivation of cells responding to specific stimuli, at different time scales. In frozen Arctic permafrost samples, we identified and isolated dormant bacteria that become active after thawing under native or nutrient enriched conditions. From a Populus tree rhizosphere, we isolated strains that thrive under high concentrations of root exudates that act as defense compounds and nutrients. In the human oral microbiome, we identified and isolated bacteria that rapidly proliferated when exposed to metabolites provided by the host or other co-occurring microbes. Further characterization of isolated bacterial strains will provide opportunities for in depth determination of how these microbes adapt to changes in their environments, individually and as part of model communities. ImportanceDynamic microbial activity transforms environments and impacts health and disease in associations with plants and animals, including humans. Identifying the contribution of individual microbes to those processes in real time has not been generally compatible with their selective cultivation. BONCAT-Live tracks which microbes in environmental samples are translationally active and couples it with single cell isolation and cultivation. By studying the response of individual community member to specific natural or induced physical or chemical changes in the environment and culturing those organisms, BONCAT-Live enables further insights into microbial metabolic strategies, community dynamics and environmental adaptations.

microbiology↗

bioRxiv2025

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression

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. NoticeThis 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 Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/651296v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1cb97a4org.highwire.dtl.DTLVardef@150bf11org.highwire.dtl.DTLVardef@2ffefforg.highwire.dtl.DTLVardef@10e24c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Elevated temperature alters microbial communities, but not decomposition rates, during three years of in-situ peat decomposition

Peatlands store approximately one-third of the global terrestrial carbon and are historically considered carbon sinks due to primary production outpacing microbial decomposition of organic matter. Climate change has the potential to alter the rate at which peatlands store or release carbon, and results from the Spruce and Peatland Responses Under Changing Environments (SPRUCE) experiment have shown net losses of organic matter and increased greenhouse gas production from a boreal peatland in response to whole-ecosystem warming. In this study, we utilized the SPRUCE sites to investigate how warming and elevated CO2 impact peat microbial communities and peat soil decomposition. We deployed peat soil decomposition ladders across warming and CO2 treatment enclosures for three years, after which we characterized bacterial, archaeal, and fungal communities through amplicon sequencing and measured peat mass and compositional changes across four depth increments. Microbial diversity and community composition were significantly impacted by soil depth, temperature, and CO2 treatment. Bacterial/archaeal -diversity increased significantly with increasing temperature, and fungal -diversity was significantly lower under elevated CO2 treatment. Trans-domain microbial networks showed higher complexity (nodes, edges, degree, betweenness centrality) of microbial communities in decomposition ladders from warmed enclosures, and the number of highly connected, hub taxa within the networks was positively correlated with temperature. Methanogenic hubs were identified in the networks constructed from the warmest enclosures, indicating increased importance of methanogenesis in response to warming. Microbial community responses were not however reflected in measures of peat soil decomposition, as warming and elevated CO2 had no significant short-term effects on soil mass loss or composition. Regardless of treatment, on average only 4.5% of the original soil mass was lost after three years and variation between replicates was high, potentially masking treatment effects. Many previous studies from the SPRUCE experiment have shown that warming is accelerating organic-matter decomposition and CO2 and CH4 production, and our results suggest that these changes may be driven by warming-induced shifts in microbial communities.

ecology↗

bioRxiv2020

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Computational analyses suggested that this bias is due to the action of restriction-modification systems whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that the intensity of recombination is uniform across the genome without concentration into hotspots. Unexpectedly, our results revealed that large species-level genetic distance did not affect key recombination parameters. Our study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

microbiology↗

bioRxiv2019

One-time Nitrogen Fertilization Shifts Switchgrass Soil Microbiomes within a Context of Larger Spatial and Temporal Variation

Soil microbiome responses to short-term nitrogen (N) inputs within the context of existing spatio-temporal variability remain uncertain. Here, we examined soil bacterial and fungal communities pre/post-N fertilization in an 8 year-old switchgrass field, in which twenty-four plots received N fertilization at three levels (0, 100, and 200 kg N ha-1 as NH4NO3) for the first time since planting. Soils were collected at two depths, 0-5 and 5-15 cm, for DNA extraction and amplicon sequencing of 16S rRNA genes and ITS regions, and soil metagenomic analysis. Baseline assessment prior to fertilization revealed no pre-existing differences in either bacterial or fungal communities across plots. The one-time N fertilization increased switchgrass yields and tissue N content, and the added N was nearly completely removed from the soil of fertilized plots by the end of the growing season. Both bacterial/archaeal and fungal communities showed large spatial (by depth) and temporal variation (by season) within each plot, accounting for 17 and 12-22 % of the variation in bacterial/archaeal and fungal community composition, respectively. While N fertilization effects accounted for only ~4% of overall variation, some specific microbial groups, including the bacterial genus Pseudonocardia and the fungal genus Archaeorhizomyces, were notably repressed by fertilization at 200 kg N ha-1. Bacterial groups varied with both depth in the soil profile and time of sampling, while temporal variability shaped the fungal community more significantly than vertical heterogeneity in the soil. Thus, variability within the field might override the changes induced by N addition. Continued analyses of these trends over time with fertilization and management are needed to understand whether these transient effects change over time.

ecology↗