Biology subjects

Overholt, W. A.

Publications and source records attributed to Overholt, W. A..

9 recordsLinked to original sources

bioRxiv2026

Epidemiology of Legionella: Genome-bAsed Typing (el_gato) - a new bioinformatic tool for identifying sequence-based types of Legionella pneumophila from whole genome sequencing data

Sequence-based typing (SBT) via Sanger sequencing has been the standard for describing Legionella pneumophila relationships for two decades. SBT involves sequencing seven loci, identifying alleles using the United Kingdom Health Security Agency (UKHSA) database, and inferring the corresponding sequence type (ST). While similar SBT approaches for other organisms can be easily adapted to whole genome sequencing (WGS), L. pneumophila presents two known challenges for this adaptation: multiple copies of one locus (mompS) and extensive heterogeneity in a second locus (neuA/neuAh). Although several computational methods have been proposed to address these issues, a WGS-based replacement with equal resolution to traditional SBT has been elusive. To address this gap, we developed el_gato (Epidemiology of Legionella: Genome-bAsed Typing; https://github.com/CDCgov/el_gato), which offers several advantages over existing methods: (1) a novel approach for resolving multiple mompS alleles identified in the same isolate, (2) the ability to capture diverse neuA/neuAh alleles, (3) fast runtime with an average of 27.7 seconds per sample, (4) easy installation via Bioconda or Docker and (5) an updated database as of March 2025. el_gato works with either paired-end short reads or genome assemblies, performing more accurately with paired-end short reads at least 250 base pairs (bp) in length. We compared el_gato against two other in silico SBT tools ("mompS", hereafter referred to as mompS tool and "legsta") using a dataset of 441 isolates with sequence types (STs) previously determined by Sanger-based sequencing. el_gato correctly identified the ST for 98.9% of the test isolates, compared to 95.2% for the mompS tool and 42.2% for legsta, demonstrating a significant improvement compared to the mompS tool (adjusted p = 1.06e-3) and legsta (adjusted p = 4.24e-55) in ST identification. Furthermore, el_gatos determination of ST was not significantly different from Sanger sequencing (adjusted p = 0.442). In summary, el_gato significantly improves in silico SBT and given its growing adoption, is poised to support the public health community.

bioinformatics↗

bioRxiv2024

Genetic potential for aerobic respiration and denitrification in globally distributed respiratory endosymbionts

The endosymbiont Candidatus Azoamicus ciliaticola generates ATP for its eukaryotic host, an anaerobic ciliate of the Plagiopylea class, fulfilling a function analogous to mitochondria in other eukaryotic cells. The discovery of this respiratory endosymbiosis has major implications for both evolutionary history and ecology of microbial eukaryotes. However, with only a single species described, knowledge of its environmental distribution and diversity is limited. Here we report four new complete, circular metagenome assembled genomes (cMAGs) representing respiratory endosymbionts inhabiting groundwater in California, Ohio, and Germany. These cMAGs form two lineages comprising a monophyletic clade within the uncharacterized gammaproteobacterial order UBA6186, enabling evolutionary analysis of their key protein complexes. Strikingly, all four novel cMAGs encode a cytochrome cbb3 oxidase, which indicates the capacity for both aerobic and anaerobic respiration. Accordingly, we detect these respiratory endosymbionts in diverse habitats worldwide, thus significantly expanding the ecological scope of this respiratory symbiosis.

microbiology↗

bioRxiv2023

Genome streamlining in CPR bacteria transitioning from soil to groundwater

To better understand the influence of habitat on the genetic content of Candidate Phyla Radiation (CPR) bacteria, we studied the effects of transitioning from soil to groundwater on genomic divergences of these organisms. Bacterial metagenome-assembled genomes (318 total, 35 of CPR bacteria) were generated from seepage waters and compared directly to groundwater counterparts. Seepage water CPR bacteria exhibited 1.24-fold greater mean genome size, while their inferred mean replication rate was 21.1% lower than groundwater lineages. While exploring gene loss and adaptive gains in closely related lineages in groundwater, we identified a zinc transporter, a surface protein, and a lipogylcopeptide resistance gene unique to a seepage Parcubacterium. A nitrite reductase gene unique to the groundwater Parcubacterium, likely acquired from pelagic microbes via horizontal gene transfer, was also identified. Groundwater Parcubacteria harbored nearly double the fraction (9.4%) of pseudogenes than their seepage kin (4.9%), suggesting further genome streamlining.

microbiology↗

bioRxiv2021

Rates of primary production in groundwater rival those in oligotrophic marine systems

The terrestrial subsurface contains nearly all of Earths freshwater reserves1 and harbors upwards of 60% of our planets total prokaryotic biomass2,3. While genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the translocation of photosynthetically derived organic carbon4-6, corroborating measurements of carbon fixation in the subsurface are absent. Using a novel ultra-low level 14C-labeling technique, we show that in situ carbon fixation rates in a carbonate aquifer reached 10% of the median rates measured in oligotrophic marine surface waters, and were up to six-fold greater than those observed in lower euphotic zone waters where deep chlorophyll levels peak. Empirical carbon fixation rates were substantiated by both nitrification and anammox rate data. Metagenomic analyses revealed a remarkable abundance of putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria - the first representatives of this class expected to fix carbon via the Wood-Ljungdahl pathway. Based on these fixation rates, we extrapolate global primary production in carbonate groundwaters to be 0.11 Pg of carbon per year.

microbiology↗

bioRxiv2021

The economical lifestyle of CPR bacteria in groundwater allows little preference for environmental drivers

The highly diverse Cand. Patescibacteria are predicted to have minimal biosynthetic and metabolic pathways, which hinders understanding of how their populations differentiate to environmental drivers or host organisms. Their metabolic traits to cope with oxidative stress are largely unknown. Here, we utilized genome-resolved metagenomics to investigate the adaptive genome repertoire of Patescibacteria in oxic and anoxic groundwaters, and to infer putative host ranges. Within six groundwater wells, Cand. Patescibacteria was the most dominant (up to 79%) super-phylum across 32 metagenomes obtained from sequential 0.2 and 0.1 {micro}m filtration. Of the reconstructed 1275 metagenome-assembled genomes (MAGs), 291 high-quality MAGs were classified as Cand. Patescibacteria. Cand. Paceibacteria and Cand. Microgenomates were enriched exclusively in the 0.1 {micro}m fractions, whereas candidate division ABY1 and Cand. Gracilibacteria were enriched in the 0.2 {micro}m fractions. Patescibacteria enriched in the smaller 0.1 {micro}m filter fractions had 22% smaller genomes, 13.4% lower replication measures, higher fraction of rod-shape determining proteins, and genomic features suggesting type IV pili mediated cell-cell attachments. Near-surface wells harbored Patescibacteria with higher replication rates than anoxic downstream wells characterized by longer water residence time. Except prevalence of superoxide dismutase genes in Patescibacteria MAGs enriched in oxic groundwaters (83%), no major metabolic or phylogenetic differences were observed based on oxygen concentrations. The most abundant Patescibacteria MAG in oxic groundwater encoded a nitrate transporter, nitrite reductase, and F-type ATPase, suggesting an alternative energy conservation mechanism. Patescibacteria consistently co-occurred with one another or with members of phyla Nanoarchaeota, Bacteroidota, Nitrospirota, and Omnitrophota. However, only 8% of MAGs showed highly significant one-to-one association, mostly with Omnitrophota. Genes coding for motility and transport functions in certain Patescibacteria were highly similar to genes from other phyla (Omnitrophota, Proteobacteria and Nanoarchaeota). Other than genes to cope with oxidative stress, we found little genomic evidence for niche adaptation of Patescibacteria to oxic or anoxic groundwaters. Given that we could detect specific host preference only for a few MAGs, we propose that the majority of Patescibacteria can attach to multiple hosts just long enough to loot or exchange supplies with an economic lifestyle of little preference for geochemical conditions.

microbiology↗

bioRxiv2021

Sulfur-fueled chemolithoautotrophs replenish organic carbon inventory in groundwater

The current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2 fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically-fixed carbon, the flux of CO2-derived carbon through various subpopulations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here, we implement novel stable isotope cluster analysis to render a time-resolved and quantitative evaluation of 13CO-derived carbon flow through a groundwater microbiome stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not obligate chemolithoautotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the recycling of organic carbon and, when chance afforded, supplemented their carbon requirements via chemolithoautotrophy and uptake of available organic compounds. Mixotrophic activity facilitated the replacement of 43 and 80% of total microbial carbon stores with 13C in just 21 and 70 days, respectively. This opportunistic "utilize whatever pathways net the greatest advantage in fitness" strategy may explain the great abundances of mixotrophs in other oligotrophic habitats, like the upper ocean and boreal lakes.

microbiology↗

bioRxiv2020

Biology and rearing of the cogongrass gall midge, Orseolia javanica Kieffer & Docters van Leeuwen-Reijnvaan (Diptera: Cecidomyiidae)

Imperata cylindrica (L.) Beauv. (Poaceae) is one of the most harmful weeds in the world because of its ability to spread and form high density, monospecific stands that exclude other vegetation. The cogongrass gall midge, Orseolia javanica Kieffer & Docters van Leeuwen-Reijnvaan (Diptera: Cecidomyiidae), is a stem galling insect that is only known to develop in cogongrass and has only been found on the island of Java in Indonesia. The midge attacks very young shoots, which stimulates abnormal growth, resulting in the formation of a purplish, elongate stem gall tappered to a point at the apical end. The aim of the current research was to describe the biology of the midge and develop a rearing method. Orseolia javanica completed its life cycle in 12-38 days with average egg, larval, and pupal periodes of 4.0 {+/-} 0.0, 13.5 {+/-} 3.8, and 8.6 {+/-} 6.6 days (mean {+/-} SD), respectively. Mated female, unmated female, and male longevities were 1.7 {+/-} 0.47, 1.2 {+/-} 0.41, and 1.0 {+/-} 0.00 days (mean {+/-} SD). Galls began to appear 29 days after larval infestation, and stem death coincided with emergence of the adult midge. The midge may have potential for biological control of cogongrass if future studies confirm a restricted host range.

ecology↗

bioRxiv2019

Inclusion of Oxford Nanopore long reads improves all microbial and phage metagenome-assembled genomes from a complex aquifer system

Assembling microbial and phage genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. In order to compare the recovery of genomes from microorganisms and their phages from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from Illumina-only assemblies and a hybrid assembly approach. Strikingly, the hybrid approach more than doubled the number of mid to high-quality MAGs (> 50% completion, < 10% redundancy), generated nearly four-fold more phage genomes, and improved all associated genome metrics relative to the Illumina only method. The hybrid assemblies yielded MAGs that were on average 7.8% more complete, with 133 fewer contigs and a 14 kbp greater N50. Furthermore, the longer contigs from the hybrid approach generated microbial MAGs that had a higher proportion of rRNA genes. We demonstrate this usefulness by linking microbial MAGs containing 16S rRNA genes with extensive amplicon dataset. This work provides quantitative data to inform a cost-benefit analysis on the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.

microbiology↗

bioRxiv2018

Defining the seafloor microbiome of the Gulf of Mexico and its response to oil perturbation

The microbial ecology of oligotrophic deep ocean sediments is understudied relative to their shallow counterparts, and this lack of understanding hampers our ability to predict responses to current and future perturbations. The Gulf of Mexico has experienced two of the largest accidental marine oil spills, the 1979 Ixtoc-1 blowout and the 2010 Deepwater Horizon (DWH) discharge. Here, microbial communities were characterized for 29 sites across multiple years in >700 samples. The composition of the seafloor microbiome was broadly consistent across the region and was well approximated by the overlying water depth and depth within the sediment column, while geographic distance played a limited role. Biogeographical distributions were employed to generate predictive models for over 4000 OTU that leverage easy-to-obtain geospatial variables which are linked to measured sedimentary oxygen profiles. Depth stratification and putative niche diversification are evidenced by the distribution of taxa that mediate the microbial nitrogen cycle. Further, these results demonstrate that sediments impacted by the DWH spill had returned to near baseline conditions after two years. The distributions of benthic microorganisms in the Gulf can be constrained, and moreover deviations from these predictions may pinpoint impacted sites and aid in future response efforts or long-term stability studies.

ecology↗