Biology subjects

Pearson, A.

Publications and source records attributed to Pearson, A..

26 records · Page 2Linked to original sources

bioRxiv2020

Recombination in enteroviruses is a ubiquitous event independent of sequence homology and RNA structure

Genetic recombination in RNA viruses is an important evolutionary mechanism. It contributes to population diversity, host/tissue adaptation and compromises vaccine efficacy. Both the molecular mechanism and initial products of recombination are relatively poorly understood. We used an established poliovirus-based in vitro recombination assay to investigate the roles of sequence identity and RNA structure, both implicated or inferred from analysis of circulating recombinant viruses, in the process. In addition, we used next generation sequencing to investigate the early products of recombination after cellular co-infection with different poliovirus serotypes. In independent studies we find no evidence for a role for RNA identity or structure in determining recombination junctions location. Instead, genome function and fitness are of greater importance in determining the identity of recombinant progeny. These studies provide further insights into this important evolutionary mechanism and emphasise the critical nature of the selection process on a mixed virus population.

microbiology↗

bioRxiv2020

Functional analysis of the fatty acid and alcohol metabolism of Pseudomonas putida using RB-TnSeq

ABSTRACTWith its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.Competing Interest StatementJ.D.K. has financial interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, and Apertor Labs. C.B.E has a financial interest in Perlumi Chemicals.View Full Text

microbiology↗

bioRxiv2020

Multiple environmental parameters impact core lipid cyclization in Sulfolobus acidocaldarius

Environmental reconstructions based on microbial lipids require understanding the coupling between environmental conditions and membrane physiology. The paleotemperature proxy TEX86 is built on the observation that archaea alter the number of five- and six-membered rings in the hydrophobic core of their glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids when growing at different temperatures. However, recent work with these archaea also highlights a role for other factors, such as pH or energy availability in determining the degree of core lipid cyclization. To better understand the role of these variables we cultivated a model Crenarchaeon, Sulfolobus acidocaldarius, over a range in temperature, pH, oxygen flux, or agitation speed, and quantified the changes in growth rate, biomass yield, and core lipid compositions. The average degree of cyclization in core lipids correlated with growth rate under most conditions. When considered alongside other experimental findings from both the thermoacidophilic and mesoneutrophilic archaea, the results suggest the cyclization of archaeal lipids records a universal response to energy availability at the cellular level. Although we isolated the effects of individual parameters, there remains a need for multi-factor experiments (e.g., pH + temperature + redox) to establish a robust framework to interpret biomarker records of environmental change.

microbiology↗

bioRxiv2020

On Contamination of Symbolic Datasets

Data taking values on discrete sample spaces are the embodiment of modern biological research. "Omics" experiments produce millions of symbolic outcomes in the form of reads (i.e., DNA sequences of a few dozens to a few hundred nucleotides). Unfortunately, these intrinsically non-numerical datasets are often highly contaminated, and the possible sources of contamination are usually poorly characterized. This contrasts with numerical datasets where Gaussian-type noise is often well-justified. To overcome this hurdle, we introduce the notion of latent weight, which measures the largest expected fraction of samples from a contaminated probabilistic source that conform to a model in a well-structured class of desired models. We examine various properties of latent weights, which we specialize to the class of exchangeable probability distributions. As proof of concept, we analyze DNA methylation data from the 22 human autosome pairs. Contrary to what it is usually assumed, we provide strong evidence that highly specific methylation patterns are overrepresented at some genomic locations when contamination is taken into account.

genomics↗

bioRxiv2019

The genetic origins of Saint Helena’s liberated Africans

Between the early 16th and late 19th centuries, an estimated 12 million Africans were transported to the Americas as part of the transatlantic slave trade. Following Britains abolition of slave trade in 1807, the Royal Navy patrolled the Atlantic and intercepted slave ships that continued to operate. During this period, the island of St Helena in the middle of the South Atlantic served as a depot for \"liberated\" Africans. Between 1840 and 1867, approximately 27,000 Africans were disembarked on the island. To investigate their origins, we generated genome-wide ancient DNA data for 20 individuals recovered from St Helena. The genetic data indicate that they came from West Central Africa, possibly the area of present-day Gabon and Angola. The data further suggest that they did not belong to a single population, confirming historical reports of cultural heterogeneity among the islands African community. Our results shed new light on the origins of enslaved Africans during the final stages of the slave trade and illustrate how genetic data can be used to complement and validate existing historical sources.

genomics↗

bioRxiv2019

Energy flux controls tetraether lipid cyclization in Sulfolobus acidocaldarius

Microorganisms regulate the composition of their membranes in response to environmental cues. Many archaea maintain the fluidity and permeability of their membranes by adjusting the number of cyclic moieties within the cores of their glycerol dibiphytanyl glycerol tetraether (GDGT) lipids. Cyclized GDGTs increase membrane packing and stability, which has been shown to help cells survive shifts in temperature and pH. However, the extent of this cyclization also varies with growth phase and electron acceptor or donor limitation. These observations indicate a relationship between energy metabolism and membrane composition. Here we show that the average degree of GDGT cyclization increases with doubling time in continuous cultures of the thermoacidophile Sulfolobus acidocaldarius (DSM 639). This is consistent with the behavior of a mesoneutrophile, Nitrosopumilus maritimus SCM1. Together, these results demonstrate that archaeal GDGT distributions can shift in response to electron donor flux and energy availability, independent of pH or temperature. Paleoenvironmental reconstructions based on GDGTs thus capture the energy available to microbes, which encompasses fluctuations in temperature and pH, as well as electron donor and acceptor availability. The ability of Archaea to adjust membrane composition and packing may be an important strategy that enables survival during episodes of energy stress.\n\nSignificance StatementMicrobial lipid membranes protect and isolate a cell from its environment while regulating the flow of energy and nutrients to metabolic reaction centers within. We demonstrate that membrane lipids change as a function of energy flux using a well-studied archaeon that thrives in acidic hot springs and observe an increase in membrane packing as energy becomes more limited. These observations are consistent with chemostat experiments utilizing a low temperature, neutral pH, marine archaeon. This strategy appears to regulate membrane homeostasis is common across GDGT-producing lineages, demonstrating that diverse taxa adjust membrane composition in response to chronic energy stress.

microbiology↗

bioRxiv2019

Illuminating microbial species-specific effects on organic matter remineralization in marine sediments

Marine microorganisms play a fundamental role in the global carbon cycle by mediating the sequestration of organic matter in ocean waters and sediments. A better understanding of how biological factors, such as microbial community composition, influence the lability and fate of organic matter is needed. Here, we explored the extent to which organic matter remineralization is influenced by species-specific metabolic capabilities. We carried out aerobic time-series incubations of Guaymas basin sediments to quantify the dynamics of carbon utilization by two different heterotrophic marine isolates. Continuous measurement of respiratory CO2 production and its carbon isotopic compositions (13C and 14C) shows species-specific differences in the rate, quantity, and type of organic matter remineralized. Each species was incubated with hydrothermally-influenced vs. unimpacted sediments, resulting in a ~3-fold difference in respiratory CO2 yield across the experiments. Genomic analysis indicated that the observed carbon utilization patterns may be attributed in part to the number of gene copies encoding for extracellular hydrolytic enzymes. Our results demonstrate that the lability and remineralization of organic matter in marine environments is not only a function of chemical composition and/or environmental conditions, but also a function of the microorganisms that are present and active.

microbiology↗

bioRxiv2019

Host engineering for improved valerolactam production in Pseudomonas putida

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

synthetic biology↗