Biology subjects

Malone, J. G.

Publications and source records attributed to Malone, J. G..

13 recordsLinked to original sources

bioRxiv2025

A Novel Stress Response Pathway Mediates Antibiotic Tolerance and Architecture in Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa is a multidrug-resistant opportunistic pathogen, with chronic infections often associated with biofilm formation. Here, we investigate the previously uncharacterized gene PA3049, which is upregulated under biofilm conditions, to determine its role in infection, biofilm formation, and antimicrobial tolerance. We show that the small uncharacterised protein PA3049, renamed as Biofilm antibiotic tolerance Regulator (BatR), promotes biofilm establishment and enhances bacterial survival in sub-inhibitory concentrations of antibiotics. Proteomic analysis revealed that BatR influences the R2/F2 pyocin cluster, which drives explosive cell lysis and extracellular DNA (eDNA) release during biofilm development. We further identify a specific interaction between BatR and PA0486 (SrkA), an uncharacterised Ser/Thr protein kinase. We show that SrkA controls biofilm and pyocyanin production, and lysis-mediated eDNA release through regulation of the R2/F2 pyocin cluster and activation of bacteriophage Pf1. Our findings support a model in which SrkA directly regulates key biofilm-associated phenotypes, while BatR acts as a modulatory partner that tunes SrkA activity under specific conditions. Finally, BatR function was tested in high- validity infection models, including the ex vivo pig lung model of cystic fibrosis infection and a synthetic chronic-wound model. In these models, BatR contributes to biofilm architecture and antibiotic tolerance, and modulates pyocyanin production. Our study implicates the BatR/SrkA system in the response of P. aeruginosa biofilms to antibiotic challenge in lung infections.

microbiology↗

bioRxiv2025

Arabidopsis arenosa influences its microbiome as a serpentine soil adaptation strategy

There is growing evidence that microbes can facilitate plant growth in metal-rich soils. However, our current understanding of how plants recruit their microbiomes under abiotic stress remains incomplete. Serpentine soils are elementally skewed, with high concentrations of magnesium and nickel, often accompanied by other heavy metals, which can limit calcium availability and present unique challenges to plant growth. These soils are also nutrient-poor, prone to erosion, and have low water-holding capacity. To date, the mechanisms by which plants adapt to serpentine soils remain poorly understood and the role of plant-associated microbiomes in this process has not been described. Here, we focus on Arabidopsis arenosa populations adapted to serpentine conditions and investigate their bacterial and fungal microbiomes to better understand the role of plant-associated microbes in serpentine adaptation. We show that serpentine soils harbour distinct plant-associated microbiomes across different plant niches and that the plant genetic background plays a key role in shaping microbial community composition. Finally, we identify serpentine-specific bacterial and fungal variants that may contribute to plant adaptation under these challenging soil conditions.

ecology↗

bioRxiv2025

Cyclic-di-GMP controls Type III effector export and symptom development in Pseudomonas syringae infections via the export ATPase HrcN

Pseudomonas syringae is a destructive bacterial pathogen that infects a wide variety of plants. Following apoplastic entry, P. syringae uses its type 3 injectisome (T3I) to secrete host-specific effectors into the cytoplasm, enabling tissue wetting and immune suppression and leading to bacterial proliferation, chlorosis and necrosis. P. syringae strains encode dozens of highly specialised effectors, whose composition defines strain specificity and host range. Effective plant infection depends on the tight temporal and hierarchical control of effector delivery through the T3I. Effector secretion is driven by HrcN, an ATPase complex that interacts with the base of the T3I and is essential for plant infection. HrcN binds specifically to the bacterial signalling molecule cyclic-di-GMP, although the impact of binding on T3I function and P. syringae virulence is currently unknown. To address this, we examined the influence of mutating the predicted cyclic-di-GMP-hrcN binding site on plant infection and effector secretion. Despite maintaining effective bacterial proliferation in Arabidopsis thaliana leaves, two hrcN mutants showed severely compromised disease symptoms, a phenotype linked to reduced translocation of a specific subset of T3I-effectors, with HopAA1-2 particularly important for symptom development. We propose that cyclic-di-GMP binding may represent a novel regulatory mechanism for effector secretion during bacterial infections.

microbiology↗

bioRxiv2025

Plasticity in a bacterial global regulatory switch that drives a shift in antibiotic resistance and virulence

Antibiotic resistance and expression of virulence factors can impact the outcome of infection by Pseudomonas aeruginosa. Pathogenesis is often modelled using the P. aeruginosa PAO1 reference strain but laboratory lineages vary in the sequence and activity of MexT, a global regulator impacting virulence, biofilm formation and ciprofloxacin resistance. We defined the impact of active versus inactive MexT in PAO1 and observed global transcriptomic changes affecting the expression of 900 genes. Phenotyping revealed altered metabolism, antibiotic resistance and virulence - resulting in striking variation across a single model organism. We propose that antibiotic-resistance, introduced during work with the original PAO1 strain, has caused plasticity in mexT that accounts for variation across lineages. To test this, we introduced antibiotic resistance into clinical P. aeruginosa isolates and observed downstream mutations in mexT when selective pressure was removed, supporting the proposed evolutionary pathway. Overall, we have demonstrated the transcriptomic basis of MexT as a phenotypic switch in PAO1 and implicated antibiotic resistance as a cause of downstream changes in mexT in P. aeruginosa. Furthermore, MexS/MexT-regulated efflux is implicated in the antibiotic stress response and virulence, helping identify the mechanisms for rapid phenotypic switching through mexT and confirming that PAO1 is a very different organism compared to most isolates. Improved understanding of the regulatory changes linked to antibiotic resistance is particularly relevant to P. aeruginosa where cycles of antibiotic treatment are common.

microbiology↗

bioRxiv2024

Understanding the biosynthesis, metabolic regulation, and anti-phytopathogen activity of 3,7-dihydroxytropolone in Pseudomonas spp.

The genus Pseudomonas is a prolific source of specialized metabolites with significant biological activities, including siderophores, antibiotics, and plant hormones. These molecules play pivotal roles in environmental interactions, influencing pathogenicity, inhibiting microorganisms, responding to nutrient limitation and abiotic challenges, and regulating plant growth. These properties mean that pseudomonads are candidates as biological control agents against plant pathogens. Multiple transposon-based screens have identified a Pseudomonas biosynthetic gene cluster (BGC) associated with potent antibacterial and antifungal activity that produces 7-hydroxytropolone (7-HT). In this study, we show that this BGC also makes 3,7-dihydroxytropolone (3,7-dHT), which has strong antimicrobial activity towards Streptomyces scabies, a potato pathogen. Both molecules exhibit broad biological activities, suggesting roles in competitive soil and plant microbial communities. Through metabolomics and reporter assays, we unveil the involvement of cluster-situated genes in generating phenylacetyl-coenzyme A, a key precursor for tropolone biosynthesis via the phenylacetic acid catabolon. The clustering of these phenylacetic acid genes within tropolone BGCs is unusual in other Gram-negative bacteria. Our findings support the interception of phenylacetic acid catabolism via an enoyl-CoA dehydratase encoded in the BGC, as well as highlighting an essential biosynthetic role for a conserved thioesterase. Biochemical assays were used to show that this thioesterase functions after a dehydrogenation-epoxidation step catalysed by a flavoprotein. We use this information to identify diverse uncharacterised BGCs that encode proteins with homology to flavoproteins and thioesterases involved in tropolone biosynthesis. This study provides insights into tropolone biosynthesis in Pseudomonas, laying the foundation for further investigations into the ecological role of tropolone production.

microbiology↗

bioRxiv2023

Barley cultivars shape the abundance, phenotype, genotype and gene expression of their associated microbiota by differential root exudate secretion

Plant associated microbes play vital roles in promoting plant growth and health, with plants secreting root exudates into the rhizosphere to attract beneficial microbes. Exudate composition defines the nature of microbial recruitment, with different plant species attracting distinct microbiota to enable optimal adaptation to the soil environment. To more closely examine the relationship between plant genotype and microbial recruitment, we analysed the rhizosphere microbiomes of landrace (Chevallier) and modern (NFC Tipple) barley cultivars. Distinct differences were observed between the plant-associated microbiomes of the two cultivars, with the plant-growth promoting rhizobacterial genus Pseudomonas substantially more abundant in the Tipple rhizosphere. Striking differences were also observed between the phenotypes of recruited Pseudomonas populations, alongside distinct genotypic clustering by cultivar. Cultivar-driven Pseudomonas selection was driven by root exudate composition, with the greater abundance of hexose sugars secreted from Tipple roots attracting microbes better adapted to growth on these metabolites, and vice versa. Cultivar-driven selection also operates at the molecular level, with both gene expression and the abundance of ecologically relevant loci differing between Tipple and Chevallier Pseudomonas isolates. Finally, cultivar-driven selection is important for plant health, with both cultivars showing a distinct preference for microbes selected by their genetic siblings in rhizosphere transplantation assays.

microbiology↗

bioRxiv2022

Compensatory mutations reducing the fitness cost of plasmid carriage occur in plant rhizosphere communities

Plasmids drive bacterial evolutionary innovation by transferring ecologically important functions between lineages, but acquiring a plasmid often comes at a fitness cost to the host cell. Compensatory mutations, which ameliorate the cost of plasmid carriage, promote plasmid maintenance in simplified laboratory media across diverse plasmid-host associations. Whether such compensatory evolution can occur in more complex communities inhabiting natural environmental niches where evolutionary paths may be more constrained is, however, unclear. Here we show a substantial fitness cost of carrying the large conjugative plasmid pQBR103 in Pseudomonas fluorescens in the plant rhizosphere. This plasmid fitness cost could be ameliorated by compensatory mutations affecting the chromosomal global regulatory system gacA/gacS, which arose rapidly in plant rhizosphere communities and were exclusive to plasmid carriers. These findings expand our understanding of the importance of compensatory evolution in plasmid dynamics beyond simplified lab media. Compensatory mutations contribute to plasmid survival in bacterial populations living within complex microbial communities in their natural environmental niche.

microbiology↗

bioRxiv2021

Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance and epiphytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these pathways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we analysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT-PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature and surface association strongly affect the expression of different polysaccharides under the control of the signalling proteins ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides and WapQ-mediated lipopolysaccharide production are important for leaf adhesion, while -glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection. HighlightPseudomonas syringae uses the coordinated deployment of polysaccharides to address environmental challenges during plant colonization. Functional redundancy renders individual polysaccharides dispensable during plant infection, but their combined loss impedes pathogenicity.

microbiology↗

bioRxiv2021

Biophysical and proteomic analyses suggest functions of Pseudomonas syringae pv tomato DC3000 extracellular vesicles in bacterial growth during plant infection

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

plant biology↗

bioRxiv2020

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PA01 and define its role in abiotic stress protection. We show that trehalose metabolism in PA01 is integrated with the biosynthesis of branched -glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and -glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is vital to the PA01 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PA01 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived -glucan, with deletion of the glgE synthase gene compromising PA01 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.

microbiology↗

bioRxiv2020

A low-cost pipeline for soil microbiome profiling

BackgroundCommon bottlenecks in environmental microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in PCR inhibitors. To address this, we have established a low-cost genomic DNA extraction method for inhibitor rich samples alongside an Illumina-compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil(R) and MP Biomedicals FastDNA SPIN) and a recently published non-commercial extraction method by Zou et al. (2017). Our benchmarking experiment used four different soil types (coniferous, broad leafed, and mixed forest plus a standardised cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analysing sequence variants of 16S V4 and ITS rRNA amplicons. ResultsWe found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil(R) kit, which relies on silica column-based DNA extraction with extensive washing delivered the cleanest genomic DNA e.g. best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals FastDNA SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. ConclusionHere we present a low-cost genomic DNA extraction method for inhibitor rich sample types such as soil that can be coupled to an Illumina-compatible simple two step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high quality genomic DNA at a fraction of the cost of commercial kits and enables cost-effective, large scale amplicon sequencing projects. Notably our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, Dovetail genomics etc.

genomics↗

bioRxiv2020

Second messenger control of mRNA translation by dynamic ribosome modification

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.

microbiology↗

bioRxiv2019

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.

microbiology↗