Biology subjects

Paulsen, I. T.

Publications and source records attributed to Paulsen, I. T..

At least 19 recordsLinked to original sources

bioRxiv2025

A genome-wide approach uncovers the suite of genes important for swarming motility in the biocontrol bacteria Pseudomonas protegens Pf-5

Motility is a crucial bacterial trait, important for competitive success in many contexts. Plant growth promoting rhizobacteria, such as Pseudomonas protegens Pf-5, can protect agricultural crops against disease, relying on motility to access and colonize the plant rhizosphere. Swarming motility is the multicellular movement of bacteria powered by rotating flagella across a semi-solid surface which allows bacteria to move rapidly, search for and acquire food, avoid toxic substances, and colonize new niches. Here we performed genome-wide investigations, using transposon-directed insertion site sequencing (TraDIS) to identify 136 genes involved in P. protegens Pf-5 swarming motility. This method identified flagella biosynthesis, assembly, structure and regulatory genes, as expected. We also identified an additional suite of genes important for swarming, including chemotaxis, c-di-GMP turnover, cell division, peptidoglycan turnover, lipopolysaccharide biosynthesis, metabolism of thiamine (vitamin B1) and transport of pyoverdine. Overall, this study increases our understanding of pseudomonad motility and expands the breadth of lifestyles and habitats examined in relation to swarming motility. Understanding the genetic basis of swarming motility in biocontrol bacteria is important for successful application of these important plant growth promoting microbes in agricultural settings.

genomics↗

bioRxiv2025

Agriculture alters protein evolution of nitrogen cycling genes in soil bacteria at a global scale

Humans are the worlds greatest evolutionary force. Yet, our impacts on the evolution of Earths microbiomes and their biogeochemical processes remain poorly understood. Notably, the overlooked potential for the intensive use of agricultural fertiliser to drive evolutionary changes in soil nutrient cycling genes warrants urgent attention. Here, analysing >2,500 soil metagenomes from across the globe, we identify increased rates of diversifying positive selection on genes involved in the reduction of nitrate (a key component of nitrogen fertilisers) in agricultural, but not natural land systems. Altered selection on genes encoding the respiratory nitrate reductase were specific to Burkholderiales, a major group of denitrifying bacteria. We provide evidence that agriculture is driving evolution of protein regions implicated in substrate access to the enzymes active site, possibly resulting in increased rates of nitrate reduction. We hypothesise that increasing substrate turnover would be evolutionarily advantageous under excess nitrate availability, ultimately enhancing growth rates despite potential enzymatic trade-offs. As Burkholderiales are dominant denitrifiers globally, such evolutionary consequences of agriculture on this lineage could have cascading ecological impacts. These findings indicate that anthropogenic selection can alter protein-level evolution of vital microbial biogeochemical processes.

microbiology↗

bioRxiv2025

Gut transit and gut microbiome changes occur prior to the onset of motor impairment in a mouse model of Machado-Joseph disease

We previously identified microbial shifts prior to the onset of motor and neurological symptoms within a mouse model of the fatal neurodegenerative disease Machado-Joseph disease (MJD). Here, we aimed to explore possible mechanisms contributing to these changes within the microbiome-gut-brain axis, and whether it preceded or followed central neurodegeneration. Here, we report that pre-symptomatic male MJD mice present with significantly different microbiome communities as early as 5-weeks-old. Furthermore, we show that male MJD mice have faster total gut transit times by 9-weeks-old, prior to signs of impaired motor function by 11-weeks-old. To elucidate whether these microbial and colonic functional changes are due to the presence of pathological and morphological changes in the gut, we quantified the formation of ataxin-3 protein aggregates within the gut and examined morphological changes within the gut of pre- and early symptomatic MJD mice relative to proteinopathy in the brain. Interestingly, we observed ataxin-3 aggregates within the brains of pre-symptomatic MJD mice, with significantly more aggregates present in MJD than WT mice from 7-weeks-of-age, an earlier timepoint than previously reported, coinciding with changes within the microbiome. However, we observed no ataxin-3 protein aggregates and no changes in enteric neuron populations or morphology within the gut. Analysis of endocrine factors involved in gut motility and inflammatory markers within the small intestine of 13-week-old males revealed increased expression of genes encoding cholecystokinin (Cck), ghrelin (Ghrl), heme oxygenase (Ho1), interleukin-1 beta (Il1b), and decreased inducible nitric oxide synthase (Nos2). Together, we demonstrate for the first time that colonic dysfunction occurs after gut microbiome changes, but prior to the onset of motor impairments in male MJD mice. Our work suggests that whilst proteinopathy or morphological changes within the gut may not be involved in these changes, inflammation and related endocrine changes could have a role in the interplay between the gut and brain during MJD development, warranting further investigation.

neuroscience↗

bioRxiv2023

The Acinetobacter baumannii virulence factor Omp33 is important for tolerance to benzalkonium chloride

Benzalkonium chloride (BAC) is widely used in many disinfectant solutions in clinical settings to eradicate potential bacterial pathogens, such as Acinetobacter baumannii. We sought to investigate the transcriptomic response of a drug resistant A. baumannii isolate, AB5075-UW, on exposure to a sub-inhibitory concentration of BAC. Our transcriptomic analysis found that BAC caused an increase in the expression of genes associated with protein synthesis, such as translation initiation factors, ribosomal proteins and tRNA synthetases. It also induced the expression of genes associated with energy production and central carbon metabolism. We also observed increased expression of peptidoglycan and rod shape determining genes, which may provide increased mechanical strength to withstand osmotic challenges posed by compounds such as BAC. The most highly expressed genes under BAC stress include those that encode the RND efflux pump AdeABC and the A. baumannii porin Omp33. Mutants of adeABC and its regulator genes adeRS had a higher susceptibility to BAC. Disruption of the gene encoding Omp33 also resulted in higher susceptibility to BAC, and complementation of the mutant with omp33 together with a 450bp upstream region restored tolerance to BAC to parental strain levels (AB5075-UW). Site directed mutagenesis of amino acids associated with Omp33 periplasmic turn (T1), which folds into the lumen of the porin and blocks the channel, suggests that Omp33 may act to prevent entry of BAC into the cell. In previous studies, Omp33 has been described as an important virulence factor in A. baumannii. The results presented in this study describe a novel role for Omp33 in BAC tolerance and reveal that A. baumannii tolerates BAC stress through a combination of mechanisms.

microbiology↗

bioRxiv2023

Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study used coal seam metagenomes to reconstruct important metagenome-assembled genomes (MAGs) using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three taxa were identified with this genetic potential. The first was a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota sp. contained a full set of genes for benzoyl-CoA dearomatisation, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common within this phylum, and thus they may be important organisms for the liberation of recalcitrant carbon on a global scale. Further, two taxa from the Deltaproteobacteria class were also implicated in monoaromatic degradation; two geographically unrelated Syntrophorhabdus aromaticivorans MAGs, and a Syntrophaceae sp. MAG. Each of these three taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides an understanding of their function within the coal seam microbiome, and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic carbon cycling and emissions. ImportanceSubsurface coal seams are highly anoxic and oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, biogenic methane accumulates within many of these coal seams, which implies that the coal seam microbiome can "unlock" this carbon source in situ. For over two decades, researchers have been working to understand which organisms are responsible for these processes. This study provides the first descriptions of these organisms. Here, we report metagenomic insights into the liberation of carbon from aromatic molecules typically found within coal, the degradation pathways involved, and descriptions of the Krumholzibacteriota sp., Syntrophorhabdus aromaticivorans, and Syntrophaceae sp. that contain this genetic potential. Additionally, this is the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatisation of aromatic hydrocarbons. This potential is identified here in numerous taxa within the phylum from other subsurface environments, implicating Krumholzibacteriota in global-scale carbon-cycling processes.

microbiology↗

bioRxiv2023

Stratified microbial communities in Australia's only anchialine cave are taxonomically novel and drive chemotrophic energy production via coupled nitrogen-sulphur cycling

BackgroundAnchialine environments, in which oceanic water mixes with freshwater in coastal aquifers, are characterised by stratified water columns with complex physicochemical profiles. These environments, also known as subterranean estuaries, support an abundance of endemic macro and microorganisms. There is now growing interest in characterising the metabolisms of anchialine microbial communities, which is essential for understanding how complex ecosystems are supported in extreme environments, and assessing their vulnerability to environmental change. However, the diversity of metabolic strategies that are utilised in anchialine ecosystems remains poorly understood. ResultsHere, we employ shotgun metagenomics to elucidate the key microorganisms and their dominant metabolisms along a physicochemical profile in Bundera Sinkhole, the only known continental subterranean estuary in the Southern Hemisphere. Genome-resolved metagenomics suggests that the communities are largely represented by novel taxonomic lineages, with 75% of metagenome-assembled genomes assigned to entirely new or uncharacterised families. These diverse and novel taxa displayed depth-dependent metabolisms, reflecting distinct phases along dissolved oxygen and salinity gradients. In particular, the communities appear to drive nutrient feedback loops involving nitrification, nitrate ammonification, and sulphate cycling. Genomic analysis of the most highly abundant members in this system suggests that an important source of chemotrophic energy is generated via the metabolic coupling of nitrogen and sulphur cycling. ConclusionThese findings substantially contribute to our understanding of the novel and specialised microbial communities in anchialine ecosystems, and highlight key chemosynthetic pathways that appear to be important in these energy-limited environments. Such knowledge is essential for the conservation of anchialine ecosystems, and sheds light on adaptive processes in extreme environments.

microbiology↗

bioRxiv2023

Plastic Leachate Exposure Drives Antibiotic Resistance and Virulence in Marine Bacterial Communities

Plastic pollution is a serious global problem, with more than 12 million tonnes of plastic waste entering the oceans every year. Plastic debris can have considerable impacts on microbial community structure and functions in marine environments, and has been associated with an enrichment in pathogenic bacteria and antimicrobial resistance (AMR) genes. However, our understanding of these impacts is largely restricted to microbial assemblages on plastic surfaces. It is therefore unclear whether these effects are driven by the surface properties of plastics, providing an additional niche for certain microbes residing in biofilms, and/or chemicals leached from plastics, the effects of which could extend to surrounding planktonic bacteria. Here, we examine the effects of polyvinyl chloride (PVC) plastic leachate exposure on the relative abundance of genes associated with bacterial pathogenicity and AMR within a seawater microcosm community. We show that PVC leachate, in the absence of plastic surfaces, drives an enrichment in AMR and virulence genes. In particular, leachate exposure significantly enriches AMR genes that confer multidrug, aminoglycoside and peptide antibiotic resistance. Additionally, enrichment of genes involved in the extracellular secretion of virulence proteins was observed among pathogens of marine organisms. This study provides the first evidence that chemicals leached from plastic particles alone can enrich genes related to microbial pathogenesis within a bacterial community, expanding our knowledge of the environmental impacts of plastic pollution with potential consequences for human and ecosystem health.

microbiology↗

bioRxiv2023

Identifying the suite of genes central to swimming in the biocontrol bacteria Pseudomonas protegens Pf-5

Swimming motility is a key bacterial trait, important to success in many niches, including assisting in colonization of host surfaces. Biocontrol bacteria, such as Pseudomonas protegens Pf-5 are increasingly being used as an agricultural tool to control crop diseases, where motility is a factor in successful colonization of the plant rhizosphere. Swimming motility has been studied in a range of bacteria and typically involves a suite of flagella and chemotaxis genes, however the specific gene set employed for both regulation and biogenesis can differ substantially between organisms. Here we used transposon directed insertion site sequencing (TraDIS), a genome-wide approach, to identify 249 genes involved in P. protegens Pf-5 swimming motility. As expected, flagella and chemotaxis genes comprised a large proportion of these genes. However we also identified a suite of additional genes important for swimming, including genes related to peptidoglycan turnover, O-antigen biosynthesis, cell division, signal transduction, c-di-GMP turnover and phosphate transport, along with 27 conserved hypothetical proteins. Experimental gene knockout mutants and TraDIS data together suggest that defects in the Pst phosphate transporter lead to enhanced swimming motility. Overall, this study expands our knowledge of pseudomonad motility and highlights the utility of a TraDIS-based approach for systematically analyzing the functions of thousands of genes. This work sets a foundation for understanding how swimming motility may be related to the inconsistency in biocontrol bacteria effectiveness and reliability in the field. ImportanceBiocontrol bacteria, such as Pseudomonas protegens Pf-5 are increasingly being used as an agricultural tool to control crop diseases, and motility is a key factor in their successful colonization of plant surfaces. Here we use a high-throughput approach to identify the suite of genes important for swimming motility in P. protegens Pf-5. These included flagella and chemotaxis genes, as well as a variety of cell surface, cell division and signalling genes. We also show that defects in the Pst phosphate transporter lead to enhanced swimming motility, a hitherto unreported link between phosphate transport and swimming motility. Understanding the genetic basis of swimming motility enhances our knowledge of key processes in biocontrol bacteria that are needed to ensure their competitive success. This will contribute to developing strategies to increase the utility of biocontrol bacteria in agricultural settings to prevent crop losses.

genomics↗

bioRxiv2023

AedG, a new weapon in Acinetobacters fight against antimicrobials

A novel multidrug efflux pump, AadT from the Drug:H+ antiporter 2 family, was discovered in Acinetobacter multidrug resistance plasmids. Here, we profiled the antimicrobial resistance potential and examined the distribution of this gene. Putative homologs of this efflux pump were encoded in many Acinetobacter species and other Gram-negative species, and were genetically associated with novel variants of adeAB(C), which encodes a major tripartite efflux pump in Acinetobacter. The AadT pump conferred decreased susceptibility to at least eight diverse antimicrobials, including antibiotics erythromycin, tetracycline; biocides chlorhexidine; and dyes ethidium bromide and DAPI. These results show that AadT is a new determinant in the Acinetobacter resistance arsenal and may cooperate with variants of AdeAB(C).

microbiology↗

bioRxiv2022

Intermittent fasting has a diet-specific impact on the gut microbiota and colonic mucin O-glycosylation of mice

The colonic mucus layer and microbiota adhered to it are vital for mediating host metabolic, immune, and gut health. Yet, how intermittent fasting impacts these microbial communities and O-glycosylation of mucin proteins, the predominant component of the colonic mucus layer, remains largely unexplored. Here, using a C57BL/6J mouse model fed either a high-fat diet or normal chow, we examined the impact of a two-day a week fasting regimen on host physiology, faecal and colonic mucosal microbiota, and mucin O-glycosylation. Our results demonstrated distinct diet-specific impacts of intermittent fasting on host physiology; mice fed the high-fat diet had a lower body weight and improved glucose tolerance upon fasting, whilst there were no significant changes in mice fed the normal chow. This was observed despite the similar feed and energy intake between groups with and without fasting. There were significant changes in the faecal and colonic mucosal microbiota community structure and composition, and mucin O-glycosylation upon fasting in both dietary groups, but the specific nature of these alterations was diet-dependent. Correlation analysis revealed significant associations between fasting-mediated changes in the abundance of specific mucosal bacteria and O-glycan structures. While intermittent fasting is a popular means of extending healthy life expectancy, there is a lack of information on its impacts on the mucosal microbiota and colonic mucus layer, which are key determinants of gut health. Our study addresses this knowledge gap and serves as the first report on how intermittent fasting influences colonic mucin O-glycosylation and the associations between mucosal glycans and bacteria.

microbiology↗

bioRxiv2022

Laboratory evolution and polyploid SCRaMbLE reveal genomic plasticity to synthetic chromosome defects and rearrangements

We have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyces cerevisiae chromosome XIV as part of the international Sc2.0 project. We showed that certain synthetic loxPsym recombination sites can interfere with mitochondrial protein localization, that the deletion of one intron (NOG2) reduced fitness, and that a reassigned stop codon can lead to a growth defect. In parallel to these rational debugging modifications, we used Adaptive Laboratory Evolution to generate a general growth defect suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss. The presence of wild-type chromosomes in the hybrid tetraploids increased post-SCRaMbLE viability and heterologous DNA integration, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

synthetic biology↗

bioRxiv2021

The effect of root exudates on the transcriptome of rhizosphere Pseudomonas spp.

Plants live in association with microorganisms that positively influence plant development, vigor, and fitness in response to pathogens and abiotic stressors. The bulk of the plant microbiome is concentrated belowground at the plant root-soil interface. Plant roots secrete carbon-rich rhizodeposits containing primary and secondary low molecular-weight metabolites, lysates, and mucilages. These exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. We addressed this gap by focusing on the molecular dialogue between eight well-characterized beneficial strains of the Pseudomonas fluorescens group and Brachypodium distachyon, a model for economically important food, feed, forage, and biomass crops of the grass family. We collected and analyzed root exudates of B. distachyon and demonstrated the presence of multiple carbohydrates, amino acids, organic acids and phenolic compounds. The subsequent screening of bacteria by Biolog Phenotype MicroArrays revealed that many of these metabolites provide carbon and energy for the Pseudomonas strains. RNA-seq profiling of bacterial cultures amended with root exudates revealed changes in the expression of genes encoding numerous catabolic and anabolic enzymes, transporters, transcriptional regulators, stress response, and conserved hypothetical proteins. Almost half of the differentially expressed genes mapped to the variable part of the strains pangenome, reflecting the importance of the variable gene content in the adaptation of P. fluorescens to the rhizosphere lifestyle. Our results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant hosts.

microbiology↗

bioRxiv2020

Yeast synthetic minimal biosensors for evaluating protein production

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer cell development, the induction of the UPR has a significant impact on recombinant protein production yields in microbial cell factories, including the industrial workhorse Saccharomyces cerevisiae. Being able to accurately detect and measure this ER stress response in single cells, enables the rapid optimisation of protein production conditions and high-throughput strain selection strategies. Current methodologies to monitor the UPR in S. cerevisiae are often temporally and spatially removed from the cultivation stage, or lack updated systematic evaluation. To this end we constructed and systematically evaluated a series of high-throughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The native promoters of DER1 and ERO1 were identified to have suitable UPR biosensor properties and served as an expression level guide for orthogonal sensor benchmarking. Our best synthetic minimal sensor, SM1, was only 98 bp in length, had minimal homology to other native yeast sequences and displayed superior sensor characteristics. Using this synthetic minimal UPR sensor, we demonstrate its ability to accurately discriminate between cells expressing different heterologous proteins and at varying production levels. Our sensor is thus a novel high-throughput tool for determining expression/engineering strategies for optimal heterologous protein production.

synthetic biology↗

bioRxiv2020

The molecular basis of Acinetobacter baumannii cadmium toxicity and resistance

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, soil and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Although metal ion homeostasis in the hospital pathogen Acinetobacter baumannii is known to contribute to pathogenesis, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. Further, we show that the CzcCBA heavy metal efflux system also contributes to cadmium efflux. Analysis of the A. baumannii metallome under cadmium stress showed zinc depletion and copper enrichment, which are likely to influence cellular fitness. Overall, this work expands our understanding of the role of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCECadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, which includes Acinetobacter baumannii. Here we show that A. baumannii utilises a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux, to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be the primary means by which cadmium exerts toxicity upon the bacterium.

microbiology↗

bioRxiv2020

Spermidine and spermine are the natural substrates of the Acinetobacter baumannii AmvA multidrug efflux pump

Multidrug efflux pumps are important drivers of antibiotic resistance in Acinetobacter baumannii and other pathogens, however their natural roles beyond transport of clinical antimicrobials are poorly described. Polyamines are an ancient class of molecules with broad roles in all three kingdoms of life, and are the likely natural substrate of at least one efflux pump family. We have defined the transcriptome of A. baumannii following treatment with high levels of the polyamines putrescine, cadaverine, spermidine and spermine. These molecules influenced expression of multiple gene classes in A. baumannii including those associated with virulence, and the four polyamines induced distinct but overlapping transcriptional responses. Polyamine shock also induced expression of the MFS-family efflux pump gene amvA and its repressor gene amvR. Loss of amvA dramatically reduced tolerance to the long-chain triaamine spermidine, but caused only modest changes in resistance to known AmvA substrates such as acriflavine. We confirmed reduced accumulation of spermidine in amvA-deficient A. baumannii, and showed that its expression is induced by long-chain polyamines through its cognate regulator AmvR. Our findings suggest that the conserved A. baumannii efflux pump AmvA has evolved to export spermidine from the cell, but that its substrate recognition promiscuity also allows activity against clinically-important biocides and antibiotics. ImportanceAMR genes, including multidrug efflux pumps, evolved long before the ubiquitous use of antimicrobials in medicine and infection control. Multidrug efflux pumps often transport metabolites, signals and host-derived molecules in addition to antibiotics or biocides. Understanding the ancestral physiological roles of multidrug efflux pumps could help to inform the development of strategies to subvert their activity. In this study, we investigated the response of Acinetobacter baumannii to polyamines, a widespread, abundant class of amino acid-derived metabolites, which led us to identify long-chain polyamines as natural substrates of the disinfectant efflux pump AmvA. A second clinically-important efflux pump, AdeABC, also contributed to polyamine tolerance. Our results suggest that the disinfectant resistance capability that allows A. baumannii to survive in hospitals may have evolutionary origins in the transport of polyamine metabolites.

microbiology↗

bioRxiv2020

Elucidating essential genes in plant-associated Pseudomonas protegens Pf-5 using transposon insertion sequencing

Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacteria that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used in this species. Here we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses and transport were under-represented. Comparison of the essential gene sets of Pf-5 and P. aeruginosa PA14, an opportunistic human pathogen, provides insight into the biological processes important for their different lifestyles. Key differences include cytochrome c biogenesis, formation of periplasmic disulfide bonds, lipid biosynthesis, ribonuclease activity, lipopolysaccharides and cell surface structures. Comparison of the Pf-5 in silico predicted and in vitro determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles can improve our understanding of the biological processes important for survival and growth in different environmental niches. ImportanceEssential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens, but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analysis of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of P. aeruginosa PA14, an opportunistic human pathogen, provides insight into differences in gene essentiality which may be linked to their different lifestyles.

genomics↗

bioRxiv2020

Characterising the mechanism of action of an ancient antimicrobial, honey, using modern transcriptomics

Manuka honey has broad-spectrum antimicrobial activity and unlike traditional antibiotics, resistance to its killing effects has not been reported. However, its mechanism of action remains unclear. Here we investigated the mechanism of action of manuka honey and its key antibacterial components using a transcriptomic approach in a model organism, Pseudomonas aeruginosa. We show that no single component of honey can account for its total antimicrobial action, and that honey affects the expression of genes in the SOS response, oxidative damage and quorum sensing. Manuka honey uniquely affects genes involved in the explosive cell lysis process and in maintaining the electron transport chain, causing protons to leak across membranes and collapsing the proton motive force; and induces membrane depolarisation and permeabilisation in P. aeruginosa. These data indicate that the activity of manuka honey comes from multiple mechanisms of action that do not engender bacterial resistance. ImportanceThe threat of antimicrobial resistance to human health has prompted interest in complex, natural products with antimicrobial activity. Honey has been an effective topical wound treatment throughout history, predominantly due to its broad-spectrum antimicrobial activity. Unlike traditional antibiotics, honey-resistant bacteria have not been reported, however, honey remains underutilised in the clinic in part due to a lack of understanding of its mechanism of action. Here we demonstrate that honey affects multiple processes in bacteria, and this is not explained by its major antibacterial components. Honey also uniquely affects bacterial membranes and this can be exploited for combination therapy with antibiotics that are otherwise ineffective on their own. We argue that honey should be included as part of the current array of wound treatments due to its effective antibacterial activity that does not promote resistance in bacteria.

microbiology↗

bioRxiv2019

Native and synthetic methanol assimilation in Saccharomyces cerevisiae

Microbial fermentation for chemical production is becoming more broadly adopted as an alternative to petrochemical refining. Fermentation typically relies on sugar as a feedstock, however, one-carbon compounds like methanol are an attractive alternative as they can be derived from organic waste and natural gas. This study focused on engineering methanol assimilation in the yeast Saccharomyces cerevisiae. Three methanol assimilation pathways were engineered and tested: a synthetic xylulose monophosphate (XuMP), a hybrid methanol dehydrogenase-XuMP, and a bacterial ribulose monophosphate (RuMP) pathway, with the latter identified as the most effective at assimilating methanol. Additionally, 13C-methanol tracer analysis uncovered a native capacity for methanol assimilation in S. cerevisiae, which was optimized using Adaptive Laboratory Evolution. Three independent lineages selected in liquid methanol-yeast extract medium evolved premature stop codons in YGR067C, which encodes an uncharacterised protein that has a predicted DNA-binding domain with homology to the ADR1 transcriptional regulator. Adr1p regulates genes involved in ethanol metabolism and peroxisomal proliferation, suggesting YGR067C has a related function. When one of the evolved YGR067C mutations was reverse engineered into the parental CEN.PK113-5D strain, there were up to 5-fold increases in 13C-labelling of intracellular metabolites from 13C-labelled methanol when 0.1 % yeast extract was a co-substrate, and a 44 % increase in final biomass. Transcriptomics and proteomics revealed that the reconstructed YGR067C mutation results in down-regulation of genes in the TCA cycle, glyoxylate cycle, and gluconeogenesis, which would normally be up-regulated during growth on a non-fermentable carbon source. Combining the synthetic RuMP and XuMP pathways with the reconstructed Ygr067cp truncation led to further improvements in growth. These results identify a latent methylotrophic metabolism in S. cerevisiae and pave the way for further development of native and synthetic one-carbon assimilation pathways in this model eukaryote.

synthetic biology↗