Biology subjects

Waite, D.

Publications and source records attributed to Waite, D..

4 recordsLinked to original sources

bioRxiv2026

De novo mutation of an RNA virus is increased in the presence of engineered synonymous mutations that disrupt RNA structural elements

Using a combination of methods including selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), a complete RNA structure map of the cucumber mosaic virus (CMV) RNA 3 segment was mapped (Watters et al. (2018) Nucleic Acids Research 46, 2573-2584). To explore the effect of structural perturbations on genomic stability, infectious mutants were engineered to contain changes in one of four open reading frame (ORF) stem-loop (SL) structures SL1362, SL1439, SL1745 and SL1816, or in a highly conserved structure SL2073 in the 3 untranslated region. The mutations occurred either in the loop or stem, thereby either maintaining or disrupting the predicted SL structure. The mutants were inoculated and then passaged on plants of Nicotiana tabacum. After the eighth passage, the full-length (8 kb) sequence of the virus population was sequenced by an amplicon tiling approach using the Illumina Nextera platform. All virus passages were sequenced as technical duplicates and compared against virus wild-type passages. Across the whole genome, a significant increase in de novo SNPs (synonymous and non-synonymous) occurred in some mutants (vs. wt) as minor ([≥]10%) and dominant populations, suggestive of compensatory genome-wide epistatic changes induced by changes in SLs. ImportanceThis work further supports and expands on the novel observation that minor synonymous nucleotide changes in virus RNA structural elements can significantly increase de novo nucleotide changes across the whole virus genome, inducing, in some instances, amino acid substitutions. This phenomenon has broad implications for the interpretation of natural sequence variation, and more specifically for antiviral strategies.

evolutionary biology↗

bioRxiv2020

A draft genome of grass pea (Lathyrus sativus), a resilient diploid legume

We have sequenced the genome of grass pea (Lathyrus sativus), a resilient diploid (2n=14) legume closely related to pea (Pisum sativum). We determined the genome size of the sequenced European accession (LS007) as 6.3 Gbp. We generated two assemblies of this genome, i) EIv1 using Illumina PCR-free paired-end sequencing and assembly followed by long-mate-pair scaffolding and ii) Rbp using Oxford Nanopore Technologies long-read sequencing and assembly followed by polishing with Illumina paired-end data. EIv1 has a total length of 8.12 Gbp (including 1.9 billion Ns) and scaffold N50 59,7 kbp. Annotation has identified 33,819 high confidence genes in the assembly. Rbp has a total length of 6.2 Gbp (with no Ns) and a contig N50 of 155.7 kbp. Gene space assessment using the eukaryote BUSCO database showed completeness scores of 82.8 % and 89.8%, respectively.

genomics↗

bioRxiv2019

Broad spectrum antibiotic-degrading metallo-β-lactamases are phylogenetically diverse and widespread in the environment.

Antibiotic resistance has emerged as a major global health threat. The Zn2+-dependent metallo-{beta}-lactamases (MBLs) are of particular concern as they act on the most widely prescribed class of antibiotics, the {beta}-lactams, and are largely unaffected by commonly used {beta}-lactamase antagonists such as clavulanic acid. MBLs are subdivided into three groups (B1 to B3); despite low overall sequence similarity, their catalytic centers are conserved with two closely spaced Zn2+ binding sites ( and {beta} site). We recovered almost 1500 B3 MBLs from >100,000 public microbial genomes representing a wide range of habitats including pristine sites not impacted by human activity. Although homologs were predominantly identified in members of the bacterial phylum Proteobacteria, the recovered B3 MBLs represent a much broader phylogenetic diversity than is currently appreciated based on the study of model pathogens. This includes three active site variants inferred to have arisen from the ancestral B3 enzyme. One of these variants, B3-RQK, is noteworthy for being broadly sensitive to clavulanic acid. Through targeted mutations we demonstrate that the presence of a lysine residue (Lys263) in the {beta} site of the catalytic center of this variant confers sensitivity to this compound. Replacing this lysine with the canonical histidine (His263) found in all other MBLs restored resistance. Crystallographic and computational data reveal that clavulanic acid inhibits B3-RQK MBLs by displacing the Zn2+ ion in the {beta} site. Therefore, modifying clavulanic acid to effectively interact with His263 may increase the therapeutic range of this widely used antibiotic resistance drug.\n\nSignificanceThis study surveys the environmental and phylogenetic diversity of the B3 subgroup of antibiotic-degrading metallo-{beta}-lactamases (MBLs). B3-like MBLs are more widespread in the environment than previously appreciated suggesting multiple unrecognized reservoirs of antibiotic resistance. Three variants of the canonical active site were identified, including B3-RQK, which amongst the B3 MBLs is uniquely inhibited by the antibiotic resistance drug clavulanic acid. We demonstrate that the mode of inhibition involves the displacement of a catalytically essential Zn2+ ion from the active site. It may thus be possible to modify clavulanic acid so that it can compete with the Zn2+ ions in other MBLs as well, increasing the therapeutic range of this compound.

biochemistry↗

bioRxiv2018

Characterisation of gut microbiota of farmed Chinook salmon using metabarcoding

With the growing importance of aquaculture worldwide, characterisation of the microbial flora of high-value aquaculture species and identification of gut flora shifts induced by changes in fish physiology or nutrition is of special interest.\n\nHere we report the first metabarcoding survey of the intestinal bacteria of Chinook salmon (Oncorhynchus tshawytscha), an economically important aquacultured species. The microbiota of 30 farmed Chinook salmon from a single cohort was surveyed using metabarcode profiling of the V3-V4 hypervariable region of the bacterial 16S rRNA gene. Seawater, feed and intestinal samples, and controls were sequenced in quadruplicate to assess both biological and technical variation in the microbial profiles.\n\nOver 1,000 operational taxonomic units (OTUs) were identified within the cohort, providing a first glimpse into the gut microbiota of farmed Chinook salmon. The taxonomic distribution of the salmon microbiota was reasonably stable, with around two thirds of individuals dominated by members of the family Vibrionaceae.\n\nThis survey was performed amid a summer heat wave, during which the fish exhibited reduced feeding. Although the sampled fish appeared healthy, they had minimal intestinal content, and the observed intestinal flora may represent the microbiota of fasting and stressed fish. Limited comparison between Mycoplasma and Vibrio sequences from the Chinook salmon gut and published microbial sequences from the intestines of a variety of fish species (including Atlantic salmon) indicated that despite the starvation and temperature variations, the replacement of Vibrio with Mycoplasma is occurring within expected ecological parameters and does not necessarily reflect colonisation by atypical microbes.\n\nDATA SUMMARYRaw sequences from Chinook salmon intestinal microbiome 16S survey generated on the Illumina platform are publically available through NCBI Sequence Read Archive (SRA) database:\n\nBioproject PRJNA421844\n\nSRA study SRP134829\n\nhttps://www.ncbi.nlm.nih.gov/bioproject/PRJNA421844\n\nIMPACT STATEMENTAlthough 16S metabarcoding surveys are becoming routine, little is still known about the microbiota of fish. This is the first survey of the intestinal microbiota of Chinook salmon, a species native to the Pacific Northwest which is farmed in New Zealand and Chile. While most intestinal microbiota studies are performed on faecal material, we directly sampled the intestine epithelium and content.\n\nDuring the time of sampling, the farmed fish population was experiencing stress from a summer heat wave and had little intestinal content. Over 1,000 operational taxonomic units (OTUs) were identified within the intestines of the cohort, providing a first glimpse into the gut microbiota of farmed Chinook salmon.\n\nWe believe this survey will be of interest not only to those interested in fish biology and aquaculture, but also as an addition to the ongoing debate in the literature on sampling and DNA extraction methods for challenging samples.

microbiology↗