Root inoculation with rhizobacteria is an emerging strategy to enhance plant resistance to aphid herbivory, yet the microbial functional traits underpinning these responses remain poorly characterised. Here, we present a comparative genomic analysis of five rhizobacteria (Acidovorax radicis N35, Bacillus subtilis B171, Bacillus velezensis FZB42, Rhizobium radiobacter F4 and Pseudomonas simiae WCS417r) that suppress aphids when inoculated onto barley. As expected, functional variation largely reflected phylogenetic relatedness; however, candidate traits implicated in modulation of plant immune defences were conserved across all strains, including biosynthesis of 2,3-butanediol, riboflavin and salicylic acid. Additional shared functions, linked to plant defence signalling, included phytoene and squalene biosynthesis (absent in P. simiae) and N-acyl homoserine lactone quorum sensing (absent in Bacillus spp.). Strain-specific traits were also identified, including surfactin production in Bacillus spp. and hydrogen cyanide biosynthesis in A. radicis and P. simiae. Comparison with a broader collection of rhizobacteria revealed that many putative plant-beneficial functions identified were widely conserved, including among closely related phytopathogens. This extensive functional overlap suggests aphid suppression cannot be explained solely by presence or absence of broad functional traits, but rather by specific trait combinations, regulatory differences, or context-dependent expression. This highlights the need for genome-informed approaches for bioinoculant discovery.
Iron is an important micronutrient required by marine microorganisms. The Southern Ocean is an ecosystem where primary production is limited by the availability of iron, but how microorganisms in this region adapt to low-iron conditions or respond to episodic iron inputs is understudied. We tested how external iron additions would impact bacterial production of siderophores during phytoplankton growth using shipboard incubation experiments under a range of iron conditions. Similar heterotrophic bacteria communities and phytoplankton biomass were observed whether iron was added from the continental margin or as inorganic iron. Under both high- and low-iron conditions, the heterotrophic bacteria community remained the same but produced distinct iron-binding siderophores. Siderophores were detected in the highest concentrations when phytoplankton and heterotrophic bacteria were iron-limited, and unique compounds were produced in low-iron conditions compared to when iron was added. Hydrophobic siderophores dominated when iron concentrations were low, and hydrophilic compounds when iron concentrations were higher, and this was likely due to environmental factors rather than changes in the microbial siderophore producers. Our results also highlight that phytoplankton growth likely played an important role in stimulating siderophore production across both low- and high-iron conditions, with important implications for phytoplankton and bacteria interactions.
Bacterial chemotaxis enables cells to move in gradients of environmental signals that are sensed by chemoreceptors. A major limitation in the field consists currently in the lacking information on the signal(s) or chemoeffectors recognized by the majority of chemoreceptors, which in turn hampers understanding of how the environment has shaped chemotactic capabilities. A number of different approaches for signal identification have been reported that are often labour-intensive. Here, we developed a straightforward NADH-coupled CheA ATPase assay to systematically screen potential chemoeffectors. Obtained results are complemented with differential scanning fluorimetry and isothermal titration calorimetry analyses, as well as structural protein modelling to study the chemoreceptors of Pseudomonas parafulva PSR09-11288. Using this integrated approach, we identified L-malate, citrate, L-glutamine, and L-asparagine as chemoattractants and revealed that chemoreceptor B2J77_03605 specifically recognizes L-malate and citrate, whereas chemoreceptor B2J77_13170 binds L-glutamine and L-asparagine. The universality of this approach is shown by the fact that both chemoreceptors belong to different families and possess sensor domains that belong to the HBM and dCache families, respectively. This pipeline is a scalable workflow for chemoeffector identification, permitting systematic exploration of bacterial chemotactic capabilities.
Natural microbial communities generally have complex compositions and unclear metabolic interactions, posing constraints on their applications. Clarifying these intricate interactions within microbial communities is challenging for traditional experiment-based methods. Here, we developed a simulation-based approach to design synthetic communities (SynComs) by simplifying complex microbial communities through metabolic modelling. We constructed genome-scale metabolic models (GSMMs) and curated them based on data obtained from straightforward experiments, ensuring these models precisely characterized metabolic features of each strain. By simulations utilizing multi-strain metabolic models encompassing various strain combinations, we identified helper strains capable of enhancing the degradation efficiency of degrader strains and predicted optimal strain combinations that achieved a simplified community structure while maintaining high pollutant-degrading efficiency. The simulations also unravelled cross-feeding of glucosamine, amino acids and organic acids between the degrader and helper strains, which boosted the pollutant-degrading efficiency of SynComs. Furthermore, helper strains rapidly degraded the toxicant intermediate, thereby alleviating its inhibitory effect on degrader strains. These predictions were further verified experimentally, demonstrating the accuracy and feasibility of metabolic model-based simulations. Our study establishes a framework for designing simplified SynComs without sacrificing degradation efficiency and highlights the often-underestimated role of microbial interactions in biodegradation.
The rapid evolution of pesticide resistance in Plutella xylostella has reduced the effectiveness of conventional pest control methods in cruciferous crops. This study investigated the virulence of Serratia marcescens PXG6 against P. xylostella and identified two putative virulence factors, ser (serralysin) and shlB (hemolysin transporter). Whole-genome sequencing revealed a GC content of 59.65%, 4650 predicted genes, multiple secretion systems (types I, V, and VI), and 715 putative virulence-related genes. Bioinformatic analyses predicted type IV pili, serralysin, hemolysin, and flagella as pathogenic determinants. Purified prodigiosin exhibited concentration-dependent insecticidal activity, whereas bacterial proteins alone caused little larval mortality, indicating that full virulence depends on live bacterial cells. Gene knockout mutants Δser and ΔshlB exhibited altered growth, motility, biofilm formation, and stress tolerance. Deletion of shlB significantly reduced hemolytic activity, protein secretion, and virulence against P. xylostella, whereas Δser had minimal impact on larval survival. Overall, PXG6 pathogenicity involves multiple bacterial components, with shlB playing a key role in virulence. These findings improve our understanding of the pathogenic mechanisms of PXG6 and support the development of S. marcescens-based biocontrol strategies against P. xylostella.
Intensive nitrogen fertilization in Lei bamboo (Phyllostachys praecox) plantations has increased productivity but has also reduced nitrogen-use efficiency (NUE), accelerated nutrient losses, and contributed to soil degradation. How nutrient-release strategies influence rhizosphere microbiome assembly and ecosystem functioning remains poorly understood. Here, we developed a bamboo shoot-specific controlled-release fertilizer (CRF) and evaluated different urea-CRF blending ratios to identify sustainable fertilization strategy for Lei bamboo production. Mixed CRF-urea treatments outperformed both sole-fertilizer applications and the unfertilized control. T2 (30% urea + 70% CRF) achieved the highest shoot yield, whereas T3 (50% urea + 50% CRF) enhanced NUE. Optimized fertilization improved soil nutrient availability and organic matter accumulation without significantly affecting soil pH. Metagenomic analysis revealed the enrichment of taxa associated with nutrient transformation, organic matter turnover, and plant growth. However, pathway-level analysis revealed shifts in carbon, nitrogen, and sulfur cycling activities under optimized nutrient-release regimes. Our results demonstrate that synchronizing nitrogen release with plant and microbial demand enhances rhizosphere function, productivity and NUE. T2 delivered the strongest overall agronomic performance by maximizing shoot yield while maintaining improved nutrient retention and ecological stability. These findings provide a mechanistic link between fertilization strategy, rhizosphere microbial dynamics, and ecosystem function, supporting sustainable Lei bamboo production.
Subterranean estuaries (STEs) are key bioreactors regulating the quantity and chemical composition of groundwater-derived nitrogen (N) reaching coastal ecosystems. Yet, the microbial controls on N-cycling within these groundwater-seawater mixing zones remain poorly understood. We investigated the spatio-temporal variations in microbial communities and their N-cycling potential within an alluvial Mediterranean STE with high N concentration. We explored changes in microbial abundance, heterotrophic activity, taxonomic composition, and the abundance of N-cycling genes across groundwater samples collected at several depths and distances from the shoreline in winter and summer. Microbial abundance, activity, and diversity varied strongly across hydrochemical zones according to physicochemistry and aquifer depth but showed limited seasonality. Functional predictions suggested a complex, spatially structured suite of N pathways encoded by diverse taxa occupying different STE zones, and quantitative-PCR revealed niche partitioning between ammonia-oxidizing archaea, prevalent in fresh-groundwater, and bacterial denitrifiers enriched in deep-saline layers. Multiple linear model predictions showed a stronger fit for NO2 - and NH4 + concentrations when using microbial properties than when using environmental variables, highlighting their importance for understanding N cycling in STEs. Our results suggest that the functional potential of the STE microbiome is complex and spatially structured across hydrochemical zones, explaining spatial variations in STE N-cycling.
Reptile microbiotas remain poorly understood despite their importance for host ecology and evolution. This study investigated how diet, climate and evolutionary history shape the gut microbiota of 234 Australian freshwater turtles spanning 10 species, four climatic zones and contrasting trophic strategies. It was found that turtles' microbiotas were dominated by Pseudomonadota, Actinobacteriota and Bacteroidota. Microbial richness and evenness varied significantly among species, diets and climates. Carnivorous turtles exhibited greater bacterial diversity than omnivores, while individuals from oceanic and humid subtropical zones had higher diversity than those from semi-arid and Mediterranean regions. Canonical Correspondence Analysis confirmed diet and climate as significant predictors of community composition, together explaining 2.76% of total variation. Omnivorous turtles were enriched in Bacteroidota, Myxococcota, Cyanobacteriota and Bacillota, whereas climatic effects drove distinct phylum-level signatures across habitats. Host phylogeny showed a weak but significant signal of phylosymbiosis, indicating an evolutionary imprint on microbial structure. Collectively, these findings reveal that turtle microbiotas are shaped by a complex interplay between ecological and evolutionary forces, underscoring the importance of integrating microbial data into conservation strategies for Australia's increasingly threatened freshwater turtles.
Ester-containing pesticides, such as diphenyl ether herbicides, aryloxyphenoxypropionate herbicides, and strobilurin fungicides, are widely applied in agriculture, but their residues pose significant risks to ecosystems. Functional microorganisms can degrade ester-containing pesticides in the natural environment. However, the microbial mechanisms responsible for their degradation in soils remain unclear. A novel hydrolase gene triS from the strain S113 encoded a broadly active carboxylesterase that hydrolyzes a wide range of ester-containing pesticides. TriS exhibited a maximum sequence similarity of 34.92% to proteins in the Swiss-Prot (UniProt) database. Gene redundancy was observed in strain S113, with TriS showing higher catalytic activity than its homologues. The enzyme contains a conserved Ser226-Glu346-His456 catalytic triad, and its substrate-binding pocket can accommodate complex substrates, which contributes to its broad substrate spectrum and enhanced catalytic efficiency. Genomic analyses revealed that triS resides in a conserved locus, whereas related paralogs occupy more variable regions, suggesting potential horizontal dissemination. Bioinformatics-based taxonomic profiling showed widespread occurrence of TriS homologues, mainly in agricultural soils, and also in forests and wetlands, indicating spread beyond pesticide-treated fields. This study elucidated the function and distribution pattern of triS, enhancing our understanding of microbial ester-containing pesticide degradation in the environment.
Microplastics (MPs) have established complex bidirectional interactions with bacteria, encompassing both mutualism and antagonism. This review advances a unified 'Stress-Habitat-Degradation' triangular model that systematically integrates three interconnected dimensions: (1) MPs-induced biotoxic stress on bacterial communities in water, soil, guts and plant tissues; (2) the plastisphere as a colonisation substrate that enriches pathogens and facilitates ARG spread; and (3) enzymatic biodegradation by functional taxa like Pseudomonas via specific enzymatic pathways. This framework explicitly links microbial community dynamics to functional outcomes, demonstrating that the same MPs-bacteria interplay simultaneously drives toxicity/pathogen enrichment and beneficial degradation. Environmental factors (temperature, oxygen, soil organic matter) profoundly shape these interactions by regulating gene expression and community succession. Crucially, we identify and resolve apparent contradictions-such as differential Gram-positive/Gram-negative responses and conflicting diversity trends-by attributing them to variations in polymer type, particle size, concentration, exposure time, host species and test conditions. Overall, this synthesis provides a critical integration of MPs-bacterial interactions and proposes a novel pollution control strategy centred on these interactions, redefining environmental risk assessment and bioremediation approaches.
Coastal environments are increasingly recognised as reservoirs of known antibiotic resistance genes (ARGs), but are less frequently identified as sources of novel ARGs. Here, we investigated class A β-lactamases circulating in European coastal environments used for oyster farming. We examined their diversity, function, and the ecological factors associated with their geographic distribution and environmental dynamics. A high diversity of carbenicillinases was detected in the culturable microbiome of European oysters. The Harveyi and Splendidus clades were key Vibrio lineages structuring the geography of carbenicillinase diversity. The Harveyi clade was primarily associated with the circulation of known carbenicillinases in Mediterranean samples, whereas the Splendidus clade contributed previously uncharacterized carbenicillinase sequences across all Europe. A one-year seasonal monitoring revealed that Vibrio alginolyticus drives the circulation of blaCARB-42 in the Mediterranean Thau lagoon, with dynamics strongly associated with seawater temperature. blaCARB-42 conferred intrinsic resistance to both carboxypenicillins and aminopenicillins in V. alginolyticus, which was found in most other species of the Harveyi clade with additional resistances to aztreonam, third-generation cephalosporins and aminoglycosides. Since the Harveyi clade includes major human pathogens, these findings have direct implication for environmental and One Health surveillance, as rising seawater temperatures may increase coastal exposure to antibiotic-resistant Harveyi clade Vibrio.
ABSTRACT The production of medium‐chain carboxylates (MCC) by mixed culture fermentation of complex organic feedstocks is often constrained by the availability of reducing equivalents. Therefore, mixotrophic fermentation processes, which integrate diverse carbon and reducing equivalents sources, are gaining more attention. However, the microbial communities evolving in such complex environments, where organic and inorganic substrates interact, remain insufficiently understood. To identify the key microorganisms involved in mixotrophic MCC production, two bioreactors were initially fed with brewer's spent grain as organic feedstock and supplemented daily with H 2 and CO 2 as inorganic substrates. Time‐course analyses of net metabolite production rates and microbial community composition identified Megasphaera spp. (likely M. elsdenii and M. hexanoica ) as the main chain elongators in the system. The results suggest that these Megasphaera species metabolize short‐chain carboxylates, H 2 and CO 2 , with potential cross‐feeding interactions with acetogens (likely Clostridium ljungdahlii and Clostridium luticellarii ). The homoacetogenic acetate produced is subsequently elongated, likely by Megasphaera species, using H 2 and CO 2 as electron and carbon sources for production of elongated carboxylates. This study paves the way for the development of MCC production strategies that integrate organic waste valorization with direct CO 2 utilization, using H 2 as an electron donor, a process that aligns with low‐carbon‐footprint technologies.
Methanol derived from pectin degradation is common in oxic and anoxic sediments and is used by various aerobic and anaerobic microorganisms such as acetogenic bacteria or methanogenic archaea as a carbon and energy source. Acetogens use the Wood-Ljungdahl pathway to disproportionate methanol to CO2 and acetate. Methanol metabolism has been studied primarily in mesophilic acetogens such as Acetobacterium woodii and Eubacterium callanderi, that do not have cytochromes or quinones. Moorella thermoacetica, the model organism for elucidation of acetogenesis, is a thermophilic, cytochrome- and quinone-containing acetogen which is commonly found in terrestrial soil. Here, we elucidated the physiology and bioenergetics of M. thermoacetica growing on methanol by performing physiological and biochemical experiments as well as genome-wide transcription analysis. Thereby, we identified genes and enzymes involved in acetogenesis from methanol. We will present a comprehensive model for carbon and energy flow from methanol to acetate and line out the bioenergetics of methanol-based acetogenesis in M. thermoacetica. Furthermore, we show that in the presence of DMSO as an alternative electron sink, no acetate is produced from methanol.
Ferromanganese (Fe-Mn) concretions are porous accumulations of iron and manganese (hydr)oxides. While recent studies suggest that microbes contribute to metal accumulation in Baltic Sea concretions, the detailed composition of microbial communities and their impact on metal enrichment across different concretion morphotypes remain unexplored. We investigated how microbes influence the accumulation and release of trace metals and rare-earth elements in Fe-Mn concretions from the Gulf of Finland through 15-week microcosm incubation experiments with biotic and abiotic treatments, focusing on three main concretion morphotypes: crust, discoidal, and spheroidal. Elemental analysis showed that microbes enhanced metal incorporation in discoidal and spheroidal morphotypes. Characterisation of microbial composition revealed that all three morphologies host distinct communities. Discoidal and spheroidal morphotypes had a higher relative abundance of Gammaproteobacteria and sulfate-reducing bacteria, and a lower abundance of Entotheonellaeota, compared to crusts. In all morphotypes, the bacterial phylum Pseudomonadota dominated, with several genera of Fe- and Mn-oxidisers and reducers. Fe-Mn concretions also host communities involved in methane oxidation and nitrogen cycling, consistent with decreased methane and increased nitrous oxide, nitrite, and nitrate concentrations in the microcosms. Our findings underscore that distinct microbial communities are associated with different concretion morphotypes, potentially influencing nutrient and metal cycling on the seafloor.
Quorum quenching (QQ) is an effective biological strategy for mitigating membrane biofouling in membrane bioreactors (MBRs), yet the persistence of QQ bacteria under harsh industrial wastewater conditions remains poorly understood. Here, the induction, resuscitation and functional recovery of the viable but nonculturable (VBNC) state in the efficient QQ bacterium Brucella sp. ZJ1 was investigated under salinity and combined phenol-salinity stress. Combined stress markedly accelerated VBNC formation (36 h vs. 10 days under salinity alone) and caused greater oxidative damage, metabolic suppression, structural deterioration and loss of QQ activity. Salinity-induced VBNC cells recovered following stress removal, whereas phenol-salinity-induced cells required resuscitation-promoting factor (Rpf) for efficient revival. Rpf-mediated resuscitation substantially restored both QQ activity and biofilm inhibition capacity. Transcriptomic analysis revealed that VBNC formation was accompanied by coordinated repression of genes involved in central metabolism, DNA replication and protein biosynthesis, together with activation of osmotic adaptation, membrane transport, quorum sensing and oxidative stress response pathways. These findings demonstrate that the VBNC state is an active adaptive strategy that preserves the potential for functional recovery and provides new insights for improving the antifouling performance of QQ-based MBR systems treating high-strength industrial wastewater.
Lactococci are best known to thrive in acidic conditions. While their physiology in an acidic environment is well researched, their stress response towards an alkaline environment remains to be explored. In this study, we habituated Lactococcus lactis FM03 to pH 6, 7 and 8 in chemostats with a fixed dilution rate of 0.2 h-1 and defined the alkaline-induced stress proteome. Based on an enrichment analysis of GO-terms, we identified key physiological adaptations, including cell wall reinforcement, an increase in the uptake and metabolism of peptides and amino acids, as well as elevated levels of translation-related proteins. Transmission electron microscopy revealed a doubling in cell wall thickness at pH 8 compared with pH 6, and we measured higher release of multiple amino acids in the supernatant at pH 8. Further, the compatible solute uptake system for glycine betaine (OpuA) was upregulated, indicating osmotic stress conceivably linked to low cyclic-di-AMP levels. Selected global stress proteins were upregulated at pH 8 (GroEL, HtrA, FtsH), and RelA increased, suggesting the activation of the stringent response, whereas acid resistance systems (F0F1-ATPases, ADI, GAD) decreased. Together, this work defined a distinct alkaline stress remodelling of the proteome in L. lactis FM03 under sustained alkaline growth.
Fluoride is abundant in the Earth's crust but is rarely used by biological systems and is toxic inside cells. Most prokaryotic genomes contain genes encoding fluoride export channels, known as CrcB, or fluoride/proton antiporters, known as CLCF. Prokaryotes rely on one type only. In this study, Pseudomonas putida ATCC 12633 that natively expressed a chromosomally-encoded CrcB was engineered with plasmids containing a CLCF exporter gene. The addition of CLCF and subsequent adaptive evolution made the cells resistant to > 500 mM sodium fluoride and able to degrade 150 mM 2-fluoropropionic acid and export 150 mM fluoride. In the absence of CLCF and adaption, only 1 mM 2-fluoropropionic was degraded and culture density decreased. The adaption of multiple cell lines occurred uniformly. All increased their CLCF gene copy and incurred mutations in the native CrcB. Two of those point mutations and a designed deletion were introduced into the unadapted wild-type strain and confirmed as CrcB knockouts. In total, the CLCF antiporter was selected for under the conditions used and was necessary for biodegrading high concentrations of an organofluorine compound. Sustaining viability at high fluoride levels is relevant for engineering prokaryotes to biodegrade fluorinated chemicals and installing fluoride into compounds by biosynthesis.
Xanthoria parietina is one of the most widespread and ecologically versatile lichens, yet the diversity of its fungal and algal symbionts and their contribution to its broad ecological niche remain poorly understood. Genetic diversity and phylogenies of both lichen symbionts were inferred from nrITS data. Mycobiont-phycobiont interaction networks were constructed, and ecological niches of associated Trebouxia species were modelled using 19 bioclimatic variables. Phylogenetic analyses revealed high diversity within Xanthoria parietina and clarified the placement of poorly studied species within the genus Xanthoria (e.g., X. monofoliosa and X. aureola s. lat.), and revealed a novel lineage (Xanthoria sp. 'hydra'). All the photobionts belonged to Trebouxia clade A; comprising nine Trebouxia species-level lineages, including the newineage Trebouxia sp. A56. Trebouxia decolorans (A33) was the most frequent photobiont and exhibited the broadest climatic niche, whereas T. solaris (A35) and T. tabarcae (A48) occupied narrower iches. Species-distribution models predicted widespread suitability for T. decolorans across Europe and coastal-Mediterranean suitability for T. tabarcae. Xanthoria parietina displays remarkable symbiont flexibility in associating with multiple Trebouxia lineages within clade A. This flexibility likely broadens its ecological niche and enhances its ability to thrive across heterogeneous Mediterranean environments.
Microorganism-mineral interaction is crucial for understanding the degradation of organic matter involved in the electron exchange in marine sediments. Widespread metal-reducing bacteria Shewanella spp. have a unique ability of extracellular electron transfer (EET); however, their EET activity and underlying mechanisms under high-salinity stress in the deep sea remain poorly explored. Here, we studied the EET process and the underlying metabolic mechanism based on a deep-sea bacterium Shewanella piezotolerans WP3. S. piezotolerans WP3 has comparable electroactivity to the model strain S. oneidensis MR-1, achieving a maximum current density of 9.7 ± 0.7 μA/cm2 at 0.6 V vs. Ag/AgCl. Multiheme-cytochrome OmcA-MtrCAB complex contributed to the direct EET, with mtrB, mtrA and omcA-1 upregulated and the redundant omcA genes (i.e., omcA-4, omcA-3) exhibiting low expression. Riboflavin, synthesised from guanosine triphosphate under high-salinity conditions, was secreted to facilitate EET. Enhanced glycolysis and TCA cycle activities under high anode potential (0.6 V) were confirmed by the downregulation of intermediate metabolites (e.g., phosphoenolpyruvate) and the upregulation of corresponding genes (e.g., pyk), supporting the high energy yield for the EET process. Our findings provide new insights into the EET mechanisms of marine Shewanella, paving the way for the development of bioelectronic sensors and biotechnology applications in high-salinity wastewater.
Microorganisms play essential roles in global ecosystems, yet much of their diversity, particularly among fungi, remains unexplored due to challenges in culturing and genomic characterisation. Trichomycetes, an early-diverging lineage of obligate gut symbionts of aquatic insects, exemplify this 'microbial dark matter', as most taxa cannot be maintained in axenic culture. Here, we present the first culture-independent genome assembly of Ejectosporus trisporus, an unculturable Harpellales fungus isolated from the hindgut of a winter stonefly (Allocapnia sp.) in Rouge National Urban Park, Canada. Using a single-thallus genomic approach based on multiple displacement amplification and Illumina short-read sequencing, we generated a 29.3 Mb genome assembly with 76.6% BUSCO completeness, comparable to existing culture-based Harpellales genomes. Phylogenomic analyses using 1241 conserved orthologs placed E. trisporus in a well-supported clade with Zancudomyces culisetae and Capniomyces stellatus, confirming its taxonomic position. Scanning electron microscopy further revealed detailed ultrastructural features of thalli, trichospores, and zygospores. This study demonstrates the feasibility of single-thallus genomics for unculturable fungi and provides the first genomic resource for an unculturable trichomycete species. Our study establishes a valuable basis for future large-scale genomic investigations of early-diverging fungi, enabling further exploration of the symbiosis and ecological roles of these cryptic gut-dwelling fungi.
