Biology subjects

Crofts, T. S.

Publications and source records attributed to Crofts, T. S..

4 recordsLinked to original sources

bioRxiv2026

Abundance-weighted analysis and deep functional metagenomic selection reveals unrecognized diversity of soil-derived streptothricin acetyltransferases

Streptothricins (STCs) are broad-spectrum antibiotics under investigation for their therapeutic potential against antibiotic-resistant Gram-negative pathogens. However, the extent and diversity of genes that confer resistance to STCs, or their environmental resistome, remains largely unknown and is essentially limited to a handful of STC acetyltransferases. Here, we used a deep functional metagenomic approach to uncover previously unrecognized STC resistance determinants. A functional metagenomic library containing ca. 700 Gigabases of soil metagenomic DNA was selected for resistance to nourseothricin (NTC), a mixture of STC congeners, resulting in collection and sequencing of nearly 30,000 resistant clones. An abundance-weighted sequence analysis identified 265 acetyltransferase-like candidates spanning broad phylogenetic, taxonomic, and genomic diversity. Re-cloning and experimental testing of 18 candidates chosen to cover acetyltransferase sequence space confirmed that 10 conferred significant NTC resistance in Escherichia coli. Phylogenetic analyses identified three previously uncharacterized STC acetyltransferase lineages, designated as SatC, SatD, and SatE. Despite substantial sequence divergence from characterized streptothricin acetyltransferases (maximum amino acid identities of approximately 31% for SatC and SatD and up to 47.5% for SatE), these enzymes largely retained key residues associated with streptothricin binding and catalysis. In addition to strongly increasing NTC resistance, expression of the SatC construct in E. coli also significantly decreased susceptibility to the aminoglycoside antibiotics amikacin and kanamycin. Together, these findings reveal a substantially broader than previously known environmental repertoire of STC acetyltransferases and demonstrate how deep functional metagenomic selections, combined with abundance-weighted hit calling, phylogenetic analysis, and structural prediction, can uncover novel resistance functions.

microbiology↗

bioRxiv2025

A functionally selected Acinetobacter sp. phosphoethanolamine transferase gene from the goose fecal microbiome confers colistin resistance in E. coli

Polymyxins are last-resort antibiotics for infections caused by multidrug resistant Gram-negative bacteria such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. This makes the rise of bacteria exhibiting polymyxin E (colistin) resistance, largely through modification of lipid A moieties, concerning and suggests that it is important to document potential sources of the corresponding resistance genes. This study searched for potential emerging colistin-resistance genes from the environment by investigating a previously performed functional metagenomic selection for colistin resistance of a goose fecal microbiome. We found that the selection captured Acinetobacter sp. DNA fragments which all contained eptA genes. We confirmed their ability to confer significant colistin resistance in E. coli via modification of lipid A in the outer membrane. Furthermore, we found evidence for mobilization of closely related eptA genes in Acinetobacter strains, marking them as potential mcr genes or their precursors. This study highlights the goose fecal microbiome as a potential source for colistin resistance in the environment.

microbiology↗

bioRxiv2024

Functional metagenomic discovery of novel tetracycline and acarbose resistance genes from low biomass samples using METa assembly

A significant challenge in the field of microbiology is the functional annotation of sequence novel genes from microbiomes. The increasing pace of sequencing technology development has made solving this challenge in a high-throughput manner even more important. Functional metagenomics offer a sequence-naive and cultivation-independent solution. Unfortunately, most methods for constructing functional metagenomic libraries require large input masses of metagenomic DNA, putting many sample types out of reach. Here, we show that our functional metagenomic library preparation method, METa assembly, can be used to prepare useful libraries from much lower input DNA quantities. Standard methods of functional metagenomic library preparation generally call for 5 g to 60 g of input metagenomic DNA. We demonstrate that the threshold for input DNA mass can be lowered at least to 30.5 ng, a three-log decrease from prior art. We prepared functional metagenomic libraries using between 30.5 ng and 100 ng of metagenomic DNA, and found that, despite their limited input mass, they were sufficient to link MFS transporters lacking substrate-specific annotations to tetracycline resistance and to capture a gene encoding a sequence novel GNAT family acetyltransferase that represents a new streptothricin acetyltransferase, satB. Our preparation of functional metagenomic libraries from aquatic samples and a human stool swab demonstrate that METa assembly can be used to prepare functional metagenomic libraries from microbiomes that were previously incompatible with this approach.

microbiology↗

bioRxiv2021

Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic librarie

Functional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naive and cultivation-independent discovery of novel genes from microbial communities. Preparation of these libraries is limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, METa assembly, for extremely efficient functional metagenomic library preparation. We apply tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergizes with homology-based assembly cloning to result in a 300-fold increase in library size compared to traditional blunt cloning based protocols. Compared to published libraries prepared by state-of-the-art protocols we show that METa assembly is on average 23- to 270-fold more efficient and can be effectively used to prepare gigabase-sized libraries with as little as 200 ng of input DNA. We demonstrate the utility of METa assembly to capture novel genes based on their function by discovering novel aminoglycoside (26% amino acid identity) and colistin (36% amino acid identity) resistance genes in soil and goose gut microbiomes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low biomass or scarce microbiomes. IMPORTANCEMedically and industrially important genes can be recovered from microbial communities by high-throughput sequencing but are limited to previously sequenced genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library is a sequence-naive and cultivation-independent method to discover novel genes. This directly connects genes to functions, but the process of preparing these libraries is DNA greedy and inefficient. Here we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and from a goose fecal microbiome and allowed us to discover novel antibiotic resistance genes. This new library preparation method will potentially allow for the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such biomedical swabs or exotic samples.

microbiology↗