Biology subjects

Lynd, L. R.

Publications and source records attributed to Lynd, L. R..

3 recordsLinked to original sources

bioRxiv2025

Characterization of new thermophilic antibiotic resistance markers

The genetic engineering of thermophilic bacteria is constrained by limited availability of thermostable antibiotic resistance markers for selection. Clostridium thermocellum, a promising candidate for consolidated bioprocessing of lignocellulosic biomass, requires reliable selection systems that function at elevated temperatures. Here, we systematically evaluated antibiotic susceptibility profiles and identified novel resistance markers for this thermophile through bioinformatic screening and experimental validation. We screened 823 thermophilic genomes against the Comprehensive Antibiotic Resistance Database, identifying 1,115 antibiotic resistance genes. From these, we selected candidates with highest homology to resistance determinants for rifampicin, tetracycline, erythromycin, thiamphenicol, and neomycin. We identified three novel antibiotic resistance systems that function in this organism: tetracycline/tet(45), erythromycin/cmeC, and rifampicin/rbpA. Of these, the rifampicin/rbpA provided the highest selection range , > 10,000-fold. Our results establish rbpA as an outstanding selectable marker for thermophilic genetic engineering and provide a validated workflow for discovering thermostable resistance determinants in high-temperature microorganisms. ImportanceThermophilic bacteria like Clostridium thermocellum hold tremendous potential for sustainable biofuel production from plant biomass, but their genetic manipulation has been severely limited by the lack of selection markers that work at high temperatures. Many existing antibiotic resistance systems do not function at thermophilic temperatures, and many approaches to genetic manipulation require multiple antibiotic resistance markers. Currently only two markers are available for C. thermocellum, and only one (cat) functions well. The newly-developed rbpA marker functions well in C. thermocellum and is likely to provide dramatic new opportunities for engineering thermophilic host organisms.

microbiology↗

bioRxiv2021

A single nucleotide change in the polC DNA polymerase III in Clostridium thermocellum is sufficient to create a hypermutator phenotype

Clostridium thermocellum is a thermophilic, anaerobic, bacterium that natively ferments cellulose to ethanol, and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here we reintroduce this mutation using recently-developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation appears to function by interfering with metal ion coordination in the PHP domain responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. ImportanceCellulosic biofuels are a promising approach to decarbonize the heavy duty transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of the material to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production, however they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially-relevant organism, which can reduce the time needed for adaptive evolution experiments.

bioengineering↗

bioRxiv2017

Hydrogen Isotope Composition Of Thermoanaerobacterium saccharolyticum Lipids: Comparing Wild Type To A nfn- Transhydrogenase Mutant

AbstractThe 2H/1H ratio in microbial fatty acids can record information about the energy metabolism of microbes and about the isotopic composition of environmental water. However, the mechanisms involved in the fractionation of hydrogen isotopes between water and lipid are not fully resolved. We provide data aimed at understanding this fractionation in the Gram-positive obligately thermophilic anaerobe, Thermoanaerobacterium saccharolyticum, by comparing a wild-type strain to a deletion mutant in which the nfnAB genes encoding electron-bifurcating transhydrogenase have been removed. The wild-type strain showed faster growth rates and larger overall fractionation [Formula] than the mutant strain [Formula]. The overall trend in growth rate and fractionation, along with the isotopic ordering of individual lipids, is consistent with results reported for the Gram-negative sulfate reducer, Desulfovibrio alaskensis G20.

microbiology↗