Biology subjects

Farr, A. D.

Publications and source records attributed to Farr, A. D..

8 recordsLinked to original sources

bioRxiv2025

Diverse microbial communities assemble on both recalcitrant and labile carbon sources

Microbial community assembly is shaped by the nature of available resources, with labile carbon sources such as glucose often expected to support low diversity due to rapid growth and competitive exclusion. In contrast, recalcitrant substrates like cellulose are thought to support higher diversity through slower growth and increased niche partitioning. In previous work, we showed that compost-derived microbial communities propagated on cellulose maintained high diversity over nearly a year. To determine whether such diversity is specific to recalcitrant substrates or reflects more general features of assembly, we tracked community dynamics in three environments--cellulose paper, cellulose broth, and glucose--using daily 16S rRNA profiling over eight weeks. Communities were maintained through bi-weekly serial transfers, with five replicates per treatment, yielding a high-resolution dataset of over 800 samples. Despite originating from the same inoculum, communities diverged sharply in both taxonomic and functional composition. Cellulose environments yielded stable communities enriched in specialists, while glucose environments exhibited rapid succession and dominance by generalists. Surprisingly, all environments sustained comparably high levels of taxonomic diversity. Functional inference suggested extensive cross-feeding and resource salvaging in both cases. While the mechanisms maintaining diversity on glucose remain unresolved, our results reveal distinct assembly trajectories under simple carbon regimes and provide a foundation for future mechanistic study.

ecology↗

bioRxiv2024

Experimental capture of genomic islands defines a widespread class of genetic element capable of non-autonomous transfer

Bacteria acquire new genes by horizontal gene transfer (HGT), typically mediated by mobile genetic elements (MGEs). While plasmids, bacteriophages and certain integrative and conjugative elements (ICEs) are well characterized, the broader diversity of MGEs remains poorly understood. Here, we propagated the bacterium Pseudomonas fluorescens SBW25 in the presence of filtrate obtained from garden compost communities. Genome sequencing of derived colonies revealed acquisition of three different mobile elements, each integrated immediately downstream of tmRNA, flanked by direct repeats, and encoding a tyrosine integrase (intY) and putative phage defense systems. Absent are genes with recognized roles in autonomous transfer. Interrogation of DNA sequence databases showed that similar elements are widespread in the genus Pseudomonas and beyond, with Vibrio Pathogenicity Island-1 (VPI-1) from V. cholerae as a notable example. Bioinformatic analyses demonstrate frequent horizontal transfer among diverse hosts. Detailed analysis of a single element, I55, showed that it is transferred between cells by a jumbo phage, and confers a fitness benefit via a type II restriction-modification system. Significance StatementThe impact of horizontal gene transfer on the evolution of bacteria outpaces that driven by spontaneous mutation, but knowledge of the range of mediators, the genes mobilized, and the mechanisms of movement have largely depended on inferences stemming from bioinformatics. Here we describe a real-time evolution experiment in which a single focal strain propagated with filtrate from a complex microbial community captured genetic elements carrying a diverse cargo of genes whose mobility was previously uncharacterized. The elements represent a widespread class of mobile DNA dependent only on a tyrosine integrase targeting a highly conserved genomic site. Genetic analysis of one element shows that it confers a significant fitness advantage via defense against bacteriophages, and hijacks a jumbo bacteriophage for intercellular transfer. Our findings reveal new insights into the hidden diversity and dynamics of MGEs in natural environments. ClassificationBiological Sciences / Microbiology

microbiology↗

bioRxiv2024

An extreme mutational hotspot in nlpD depends on transcriptional induction of rpoS

Mutation rate varies within and between genomes. Within genomes, tracts of nucleotides, including short sequence repeats and palindromes, can cause localised elevation of mutation rate. Additional mechanisms remain poorly understood. Here we report an instance of extreme mutational bias in Pseudomonas fluorescens SBW25 associated with a single base-pair change in nlpD. These mutants frequently evolve in static microcosms, and have a cell-chaining (CC) phenotype. Analysis of 153 replicate populations revealed 137 independent instances of a C565T loss-of-function mutation at codon 189 (CAG to TAG (Q189*)). Fitness measures of alternative nlpD mutants showed molecular parallelism to be unconnected to selective advantage. Recognising that transcription can be mutagenic, and that codon 189 overlaps with a predicted promoter (rpoSp) for the adjacent stationary phase sigma factor, rpoS, transcription across this promoter region was measured. This confirmed rpoSp is induced in stationary phase and that C565T mutation caused significant elevation of transcription. The latter provided opportunity to determine the C565T mutation rate using a reporter-gene fused to rpoSp. Fluctuation assays demonstrate the C565T mutation rate to be 5,700-fold higher than expected. In Pseudomonas, transcription of rpoS requires the positive activator PsrA, which we show also holds for SBW25. Fluctuation assays performed in a {Delta}psrA background showed a 60-fold reduction in mutation rate confirming that the elevated rate of mutation at C565T mutation rate is dependent on induction of transcription. This hotspot suggests a generalisable phenomenon where the induction of transcription causes elevated mutation rates within defining regions of promoters.

evolutionary biology↗

bioRxiv2024

Microbial interactions affect the tempo and mode of antibiotic resistance evolution

The global rise of antibiotic resistance impedes the treatment of bacterial infections. To limit the emergence and evolution of antibiotic resistance it is important to understand how bacterial interactions in multispecies communities affect the course of evolution. We investigated how ecological interactions between microbes derived from polymicrobial urinary tract infections affect the tempo and mode of antibiotic resistance evolution. We show that for representative strains of three uropathogens, Escherichia coli, Klebsiella pneumoniae and Enterococcus faecium, the rate and evolutionary trajectories towards antibiotic resistance differ depending on the conditioned medium mediated interactions with other microbes that alter their growth and antibiotic tolerance. Replicate lineages of the same species evolved under similar ecological conditions show parallel evolutionary trajectories, and resistance mutations and other functional targets selected differed between these conditions. Our findings demonstrate that bacterial interactions differentially affect the evolutionary potential of antibiotic resistance evolution.

ecology↗

bioRxiv2022

Barcoding populations of Pseudomonas fluorescens SBW25

In recent years evolutionary biologists have developed increasing interest in the use of barcoding strategies to study eco-evolutionary dynamics of lineages within evolving populations and communities. Although barcoded populations can deliver unprecedented insight into evolutionary change, barcoding microbes presents specific technical challenges. Here, strategies are described for barcoding populations of the model bacterium Pseudomonas fluorescens SBW25, including the design and cloning of barcoded regions, preparation of libraries for amplicon sequencing, and quantification of resulting barcoded lineages. In so doing, we hope to aid the design and implementation of barcoding methodologies in a broad range of model and non-model organisms.

molecular biology↗

bioRxiv2022

Race to survival during antibiotic breakdown determines the minimal surviving population size

A common strategy used by bacteria to resist antibiotics is enzymatic degradation or modification. Such a collective mechanism also enhances the survival of nearby cells, an effect that increases with the number of bacteria that are present. Collective resistance is of clinical significance, yet a quantitative understanding at the population level is lacking. Here we develop a general theoretical framework of collective resistance under antibiotic degradation. Our modeling reveals that population survival crucially depends on the ratio of timescales of two processes: the rates of population death and antibiotic removal. However, it is insensitive to molecular, biological and kinetic details of the underlying processes that give rise to these timescales. Another important aspect for this race to survival is the degree of cooperativity, which is related to the permeability of the cell wall for antibiotics and enzymes. These observations motivate a coarse-grained, phenomenological model and simple experimental assay to measure the dose-dependent minimal surviving population size. From this model, two dimensionless parameters can be estimated, representing the populations race to survival and single-cell resistance. Our simple model may serve as reference for more complex situations, such as heterogeneous bacterial communities.

microbiology↗

bioRxiv2022

The fitness landscape of TEM-1 β-lactamase is stratified and inverted by sublethal concentrations of cefotaxime

Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit - a concept that may be illustrated by fitness landscapes which map the relationship of genotype space with fitness. Experimentally derived landscapes have demonstrated a predictability to evolution by identifying limited mutational routes that evolution by natural selection may take between low and high-fitness genotypes. However, such studies often utilise indirect measures to determine fitness. We estimated the competitive fitness of each mutant relative to all of its single-mutation neighbours to describe the fitness landscape of three mutations in a {beta}-lactamase enzyme at sub-lethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. We found that in the unstructured environment the antibiotic selected for higher-resistance types - but with an equivalent fitness for subsets of mutants, despite substantial variation in resistance - resulting in a stratified fitness landscape. In contrast, in a structured environment with low antibiotic concentration, antibiotic-susceptible genotypes had a relative fitness advantage, which was associated with antibiotic-induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with sub-inhibitory concentrations of antibiotics, and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes. ImportanceThe evolution of antibiotic resistant bacterial populations underpins the ongoing antibiotic-resistance crisis. We aim to understand how antibiotic-degrading enzymes can evolve to cause increased resistance, how this process is constrained and whether it can be predictable. To this end we performed competition experiments with a combinatorially-complete set of mutants of a {beta}-lactamase gene subject to sub-inhibitory concentrations of the antibiotic cefotaxime. While some mutants confer their hosts with high resistance to cefotaxime, in competition these mutants do not always confer a selective advantage. Similarly, we identified conditions involving spatial structure where mutations causing high resistance result in a selective disadvantage. Together, this work suggests that the relationship between resistance level and fitness at sub-inhibitory concentrations is complex; predicting the evolution of antibiotic resistance requires knowledge of the conditions that select for resistant genotypes and the selective advantage evolved types have over their predecessors.

evolutionary biology↗

bioRxiv2016

Adaptive evolution by spontaneous domain fusion and protein relocalisation

Knowledge of adaptive processes encompasses understanding of the emergence of new genes. Computational analyses of genomes suggest that new genes can arise by domain swapping, however, empirical evidence has been lacking. Here we describe a set of nine independent deletion mutations that arose during the course of selection experiments with the bacterium Pseudomonas fluorescens in which the membrane-spanning domain of a fatty acid desaturase became translationally fused to a cytosolic di-guanylate cyclase (DGC) generating an adaptive phenotype. Detailed genetic analysis of one chimeric fusion protein showed that the DGC domain had become membrane-localised resulting in a new biological function. The relative ease by which this new gene arose along with its profound functional and regulatory effects provides a glimpse of mutational events and their consequences that are likely to play a significant role in the evolution of new genes.

evolutionary biology↗