Applied and Environmental Microbiology最新文献

The spaceflight environment imparts unique selective pressures on the plants and microbes of plant growth chambers on the International Space Station (ISS), which generally manifests through genetic signatures associated with a heightened response to stress. Terrestrially, a baseline understanding of the gene fitness response for any plant growth-promoting microbe when in a tripartite relationship with host and pathogen is currently unknown and is important to characterize before closed-environment spaceflight implementation. To that end, this study evaluated the behavior of an ISS plant habitat isolate of Burkholderia contaminans as tomato seeds transitioned to seedlings and assessed gene fitness during challenge with Fusarium oxysporum f. sp. lycopersici (FOL), the causal agent of Fusarium wilt. Using a seed film delivery method vetted for spaceflight, B. contaminans was applied to Solanum lycopersicum cv. Red Robin seeds. Green fluorescent protein (GFP)-tagged B. contaminans was primarily found to localize at the shoot-root junction and was detected on shoots. Upon FOL challenge, B. contaminans population levels remained stable, and despite harboring antifungal and plant growth-promoting capacity, these properties were not conferred in response to FOL in the tissue culture environment. To probe mechanisms underlying the bacterial-fungal interaction between B. contaminans and FOL in the tomato root zone, a genome-wide transposon mutant library was developed for the B. contaminans isolate. Transposon sequencing (Tn-Seq) analysis revealed that the type II secretion system (T2SS) was critical for root zone establishment, whereas a Nudix hydrolase was specifically important for responding to FOL infection and provided further confirmation that antifungal and siderophore-producing gene clusters were not.IMPORTANCEThis study is the first to evaluate the genetic fitness of a Burkholderia contaminans International Space Station (ISS) isolate in the plant root zone in association with the obligate pathogen Fusarium oxysporum f. sp. lycopersici (FOL). This isolate of B. contaminans establishes in the tomato root zone, does not confer plant growth promotion in tissue culture, but is persistent in the tomato root zone when challenged with FOL through stress-adaptation mechanisms rather than direct antifungal antagonism. The response of B. contaminans in the host root zone when in the presence of the pathogen suggests the microbe is primed to counter stress, which may further confer an advantage in the spaceflight environment.

Laccases are present as isozymes in white-rot fungi, yet their evolutionary history and functional role in lignin degradation remain controversial. Trametes hirsuta, a ubiquitous fungus in forest ecosystems, can completely break down cellulose, hemicellulose, and lignin in wood. Based on bioinformatic and biochemical characterization, we have shown that five laccase isozyme genes (lacA-E) in Trametes hirsuta AH28-2 were derived from a single ancestral laccase gene, lacF, with lacA and lacB originating from disparate evolutionary branches. The syringyl-type (S-type) lignin model compounds significantly induced the expression of lacA-lacE at both transcriptional and expression levels. Furthermore, in vitro and in vivo analyses demonstrated that the later-emerging laccases, LacA and LacB, primarily contribute to oxidizing S-type lignin present in hardwood, whereas laccase LacF plays a primary role in oxidizing guaiacyl-type (G-type) lignin found in softwood. Finally, evolutionary analysis of ancestral laccases from Agaricomycetes fungi also revealed a shift from better oxidation of G-type lignin in softwood by earlier ancestral laccases to better oxidation of S-type lignin in hardwood by later ancestral laccases. These findings indicate that laccase evolution in Agaricomycetes fungi is consistent with lignin synthesis. We have demonstrated the direct involvement of laccases at different evolutionary stages in preferentially oxidizing different types of lignin.IMPORTANCELaccases in white-rot fungi always exist in the form of isozymes. However, the evolutionary history and functional diversification of laccase isozymes remain controversial. Our study demonstrates that the six laccase isozymes in Trametes hirsuta AH28-2 belong to three distinct evolutionary branches. Among them, LacF represents an earlier-emerging branch and primarily contributes to oxidizing the G-type units of gymnosperm lignin. In contrast, LacA and LacB, which are later-emerging, primarily contribute to oxidizing the S-type units in angiosperm lignin. Interestingly, ancestral laccases reconstructed at different evolutionary nodes also display distinct lignin oxidation preferences. This suggests that the evolution of laccases in Agaricomycetes fungi is closely linked to the emergence of S-type lignin units in angiosperms. These findings reveal the co-evolutionary relationship between lignin structural changes and fungal laccase diversification, providing new insights into the evolutionary mechanisms and biological functions of laccase isozymes.

The current understanding of carbohydrate substrate degradation is largely derived from incubation experiments involving specific substrates. In estuaries, carbohydrates are often grouped together with other sources of carbon, for analytical purposes, and measured as total and fractional organic matter. Here, we describe putative carbohydrate degradation at the polysaccharide level by the prokaryotic community in an estuary. Samples were collected along a freshwater-to-marine salinity gradient from both the water column and underlying benthic sediments. Metagenomic and metatranscriptomic data were used to determine carbohydrate-active enzyme (CAZyme)-encoding metagenome-assembled genomes and associated transcriptional activity across the gradient. Previous work demonstrated assimilation of xylan (a component of hemicellulose) in estuaries. We show the genetic mechanisms associated with the degradation of xylan, as well as arabinogalactan (also from hemicellulose), and various other glycans were widespread among estuarine taxa and actively expressed. In addition, results show different carbohydrate degradation strategies between planktonic and benthic organisms. For example, results indicate that sediment communities harbored a greater variety and density of CAZyme-encoding genes and capacity to degrade complex plant biomass (cellulose and hemicellulose) and dedicated more gene transcription overall to CAZymes than planktonic communities. In contrast, planktonic prokaryotes tended to express a greater fraction of their CAZyme-encoding gene repertoires. The transcription of gene clusters associated with degrading beta-1,3-glucans such as laminarin was prevalent in the water column. Microbial activity to degrade alpha-glucans such as glycogen was predicted to be ubiquitous but was greatest in planktonic communities. Taken together, results highlight differences in the capacity of planktonic and benthic communities to degrade carbohydrates, which reflect differences in substrate availability and complexity.IMPORTANCEEstuaries are productive ecosystems that combine various forms of organic carbon from autochthonous (e.g., algal primary producers and mangroves) and allochthonous (e.g., terrestrial plant) sources. The degradation and recycling of this organic carbon is driven by heterotrophic bacteria that are expected to harbor diverse genetic mechanisms for carbohydrate degradation to match the diversity and complexity of organic carbon encountered in the environment. Results here illustrate the diversity of carbohydrate-active enzymes (notably glycosyl hydrolases) encoded by estuarine communities and the different substrate prioritizations of planktonic and benthic communities.

Despite the promise of phages as antibiotic alternatives, their efficacy is often undermined by the rapid emergence of bacterial resistance. Phage-derived enzymes, particularly depolymerases, offer a compelling strategy to overcome this limitation and enhance antibacterial therapy. Focusing on Vibrio pathogens, the major threats to global aquaculture, our bioinformatic analysis revealed that 79.4% of cultured and 46.2% of uncultured Vibrio phages encode putative depolymerases, underscoring a vast but underexploited antibacterial resource. We further isolated and characterized VnaP, a depolymerase-encoding phage (novel genus, Caudovircetes) that forms distinctive halo plaques indicative of depolymerase activity. Genome analysis identified ORF193, encoding a novel polysaccharide depolymerase lacking sequence or structural homology to any characterized depolymerases. Heterologously expressed Dep193 efficiently degraded Vibrio surface polysaccharides and exhibited potent antibiofilm activity. While Dep193 exhibits modest standalone antibacterial activity, its synergistic combination with VnaP significantly enhances bacterial clearance and delays resistance emergence across multiple Vibrio species. As the first biochemically validated Vibrio phage depolymerase, Dep193 broadens the known diversity of these enzymes and establishes an effective strategy for Vibrio control in aquaculture.IMPORTANCEThe rapid emergence of antibiotic-resistant Vibrio strains threatens global aquaculture sustainability, necessitating alternative antimicrobial strategies. This study identifies and characterizes Dep193, a novel phage-encoded depolymerase with polysaccharide-degrading and antibiofilm activities that enhances phage therapy efficacy through a previously unreported mechanism. The Dep193-phage VnaP combination exhibits broad-spectrum activity against multiple Vibrio species, demonstrating strong potential as a therapeutic strategy for aquaculture. Notably, Dep193 lacks any recognizable functional domains found in characterized depolymerases, representing the first validated member of a novel evolutionary clade. These findings expand the known diversity of phage depolymerases and provide a promising avenue for the targeted control of Vibrio infections in aquaculture.

To mitigate bacterial contamination in underground farmland, a comprehensive understanding of the transport and adhesion mechanisms of phytopathogenic bacteria in porous media is crucial for safeguarding soil and groundwater. This study aims to elucidate the effects of Pseudomonas amygdali pv. tabaci 6605 flagella (wild type, ΔfliC strain) and their glycosylation (Δfgt1 and Δfgt2 strains) on bacterial transport and deposition in sandy porous media through a combination of experimental observations and numerical simulations. Flagella play a key role in bacterial transport and deposition dynamics through its surface properties. Their intrinsic hydrophobicity enhances bacterial adhesion and promotes deposition onto sandy grains while simultaneously limiting transport through the porous medium. However, glycosylation of flagellin introduces hydrophilic glycans, which counteract this effect by increasing the overall hydrophilicity of the bacterial surface. As a result, glycosylated flagella facilitate bacterial mobility and improve recovery in the effluent while reducing retention within the sand matrix. These findings highlight the critical influence of flagellar biochemical modifications on bacterial behavior in porous environments. They provide valuable insights for understanding and managing microbial contamination in subsurface systems.IMPORTANCEThis work, conducted using homogeneous laboratory sand, could be extended to other types of abiotic media found in natural environments, such as clay, heterogeneous sands, and soils. Our study highlights the impact of flagellar glycosylation on bacterial behavior, an essential factor for assessing the risk posed by phytopathogenic bacteria in agricultural settings and for developing effective soil bioremediation strategies. Moreover, this study provides valuable insights into the mechanisms governing bacterial transport and deposition at the macroscopic (column) scale under dynamic flow conditions. Investigating unsaturated flow conditions, which better approximate real field scenarios, may further our understanding of bacterial interactions at air-solid-water interfaces. Future research should explore bacterial movement across different spatial scales. In particular, pore-scale experiments can provide direct evidence of processes such as attachment and motility. This could significantly enhance our understanding of microbial dynamics in complex environments.

Accumulation of harmful metal(loid)s in acidic pit lakes (APLs) is a serious environmental issue in mining districts. This lab-based study evaluated a novel method to stimulate dissimilatory sulfate reduction to promote the formation of sparingly soluble metal(loid)-sulfide minerals in the permanently stratified deep layer of Cueva de la Mora (CM), an APL in the Iberian Pyrite Belt in Spain. Solid-phase biomass was selected because it can be pressed into high-density forms that are dense enough to settle into the deep layer of a lake. This "direct delivery" of electron donor overcomes the current "indirect method" to stimulate algae growth in the upper layer and wait for algae to die and settle into the deep layer. We added the microalgae Coccomyxa onubensis (predominant in CM), Euglena gracilis (another acid-tolerant microalgae), and Lemna obscura (duckweed), as well as model biocomponents (amino acids, monosaccharides, and lipids) as substrates to stimulate biological sulfide production (biosulfidogenesis). We found that compared with biocomponents, high-density biomass required a shorter lag time before it was utilized. Temporal patterns of the production of sulfide and volatile fatty acids with high-density biomass were similar to patterns with amino acids, suggesting that amino acids may be the preferred substrate among the biocomponent monomers for the microbial community. Biosulfidogenesis led to the complete removal of metal(loid)s (Zn and As) contaminants from solution, mimicking the chemical composition of the deep layer. Desulfosporosinus, the only acid-tolerant sulfate-reducing bacteria (SRB) identified in situ, was significantly enriched in the laboratory setup and presumably responsible for biosulfidogenesis.IMPORTANCERemediation of high concentrations of harmful metal(loid)s in acidic pit lakes is challenging. This research presents a novel strategy by supplying high-density biomass as a carbon source and electron donor to stimulate biological dissimilatory sulfate reduction in acidic pit lakes in the Iberian Pyrite Belt. The formation of biogenic sulfide precipitates dissolved metal(loid)s in the acidic pit lakes. This approach is feasible in meromictic acidic pit lakes, where precipitated metal(loid)s would remain sequestered in bottom sediments. However, the deep layer of acidic pit lakes is often oligotrophic with respect to organic carbon. Pelletized high-density biomass can be added to the top layer of the lake and transported to the deep layer. This strategy offers practical and adaptable guidance for the bioremediation of persistent metal(loid) contamination in acidic pit lakes.

Diverse Bifidobacterium animalis subsp. lactis strains are widely used as commercial probiotics. While proof-of-concept studies have shown that some strains can be edited using several CRISPR-Cas approaches, this species remains difficult to engineer, hindering functional genomic studies to establish their molecular mode of action and enhance their probiotic functionalities. Here, we show that >95% of available B. lactis genomes harbor a conserved Type I-G CRISPR-Cas system, which we leverage to develop and validate a broadly applicable genome editing framework. We redesigned backbone plasmids with different replicons and antibiotic resistance markers and evaluated performance across six commercial strains for transformation efficiency. A vector carrying the pBC1 origin coupled with a chloramphenicol resistance marker improved transformation in most strains. Using synthetic CRISPR arrays with self-targeting spacers in combination with homologous editing templates, we tested multiple spacers and evaluated short (600 bp) versus long (1,000 bp) homology arms. To demonstrate applicability, we generated knockouts in three glycoside hydrolases within the Balac 1593-1601 cluster, readily cured editing plasmids in non-selective medium, and performed iterative genome editing. Growth phenotyping across carbohydrates confirmed that the GH36 α-galactosidase Balac 1601 knockout abolished melibiose and raffinose utilization, and that deletions within Balac 1596 and Balac 1593 carbohydrate hydrolases produced non-canonical phenotypes, suggestive of a modulatory role associated with shift in carbon use and compensation by other pathways. These results establish a practical toolkit for editing diverse B. lactis strains, unravel the genomics underlying probiotic attributes, and provide a blueprint for genome engineering in other non-model probiotic bacteria.IMPORTANCEBifidobacterium animalis subsp. lactis strains are prominent probiotics widely formulated in foods and dietary supplements, yet remain difficult to engineer, limiting efforts to connect genes to probiotic traits and to build strains with enhanced functions. Here, we harness the native Type I-G CRISPR Cas system to enable genome editing across commercial B. lactis strains by optimizing a compact plasmid backbone, testing multiple spacers to achieve efficient editing, and selecting homology arms of the appropriate length for recombination. With this framework, we generate knockouts at multiple, functionally distinct loci, demonstrating target-agnostic applicability, and we cure the CRISPR-editing vectors efficiently, enabling sequential edits. This toolkit enables systematic genotype-to-phenotype mapping in B. lactis and provides a practical framework for strain improvement in organisms of industrial relevance.

Salmonella Enteritidis (S. Enteritidis) is a major foodborne pathogen, and its effective control is critical for food safety. Although slightly acidic electrolyzed water (SAEW) is widely adopted as an eco-friendly bactericidal agent in food systems, its impact on beneficial microbiota remains poorly characterized, despite their importance for food quality. To address this gap, we systematically compared SAEW tolerance between Bacillus subtilis (B. subtilis, a model probiotic) and S. Enteritidis in monoculture and co-culture systems. Notably, B. subtilis retained viability at 70 mg/L ACC (from 7.82 to 3.99 log CFU/mL, P < 0.05), whereas S. Enteritidis was completely inactivated at 40 mg/L. In co-culture, B. subtilis maintained consistently higher viable counts (P < 0.05), demonstrating a significant survival advantage under SAEW stress. Furthermore, B. subtilis demonstrated rapid recovery in co-culture, attaining 8.0 log CFU/mL within 32 h post-exposure, while S. Enteritidis was eradicated. Mechanistically, SAEW disrupted membrane integrity in both strains but triggered divergent stress responses: S. Enteritidis exhibited significant ROS accumulation (P < 0.05), ATP depletion (P < 0.05), and suppressed the activities of key antioxidant enzymes (P < 0.05). Conversely, B. subtilis showed significant upregulation of these enzymes (SOD, CAT, GSH-Px, P < 0.05) with stable ROS levels. Consequently, SAEW enables selective pathogen inactivation while preserving probiotic strains, supporting its targeted application in food systems.IMPORTANCEThe increasing adoption of slightly acidic electrolyzed water (SAEW) as an eco-friendly disinfectant in food safety highlights the need for a deeper understanding of its selective bactericidal mechanisms. This study addresses a critical gap in the literature by demonstrating that SAEW effectively targets harmful pathogens, such as Salmonella Enteritidis, while preserving beneficial probiotics, such as Bacillus subtilis. By elucidating the differential stress responses of these microorganisms, our findings provide valuable insights into the ecological dynamics of food systems. The ability of SAEW to selectively inactivate pathogens without disrupting beneficial microbiota supports its targeted application in enhancing food safety and quality. This research not only advances the scientific understanding of SAEW's mechanisms but also has practical implications for developing safer food preservation methods, ultimately contributing to public health and food security.

The boom of microbiome research in agriculture over the past several decades allows scientists, growers, policymakers, and businesses to collaborate on a unique opportunity-deploying microbiomes and microbiome attributes for the improvement of crop production. The idea of translational microbiomes is well established in the medical field; however, this framework is relatively new to agriculture. In this review, we discuss a series of methodologies grounded in microbiome science to enhance crop health. These include diagnostic approaches (pathogen and toxin detection and the monitoring of stress-related community ecology patterns) and intervention strategies (synthetic communities, microbiome-aware crop management practices, passaging microbiomes, and exploiting the vertical and lateral transmission of microbiomes to seeds). Developing and implementing these approaches remain challenging due, in part, to a shortage of long-term in situ studies demonstrating the robustness and effectiveness of translational microbiome efforts against the background of heterogeneity and ecological complexity of agricultural systems. Moreover, the cost and availability of 'omics methods central to microbiome analysis, disparate standards for microbiome product development, and limited longstanding relationships with stakeholders have slowed down the application of microbiome-based solutions. However, the increasing cost-effectiveness of microbiome approaches in crop management makes translational microbiomes likely assets in the movement toward precision agriculture. This "personalized treatment" for plants holds promise for improved food security and environmental sustainability, by reducing commonplace synthetic amendments and promoting native microbial biodiversity.