Applied and Environmental Microbiology最新文献

Paenibacillus polymyxa, a Gram-positive bacterium commonly found in soil and plant roots, plays an important role in the environment due to its nitrogen-fixing ability and is renowned for producing antibiotics like polymyxin. In this study, we present a robust framework for investigating the evolutionary and taxonomic connections of strains belonging to P. polymyxa available at the National Center for Biotechnology Information, as well as five new additional strains isolated at the University of Camerino (Italy), through pangenome analysis. These strains can produce secondary metabolites active against Staphylococcus aureus and Klebsiella pneumoniae. Employing techniques such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) estimation, OrthoFinder, and ribosomal multilocus sequence typing, we consistently divided these P. polymyxa strains into four clusters, which differ significantly in terms of ANI and dDDH percentages, both considered as reference indices for separating bacterial species. Moreover, the strains of Cluster 2 were re-classified as belonging to the Paenibacillus ottowii species. By comparing the pangenomes, we identified the core genes of each cluster and analyzed them to recognize distinctive features in terms of biosynthetic/metabolic potential. The comparison of pangenomes also allowed us to pinpoint differences between clusters in terms of genetic variability and the percentage of the genome dedicated to core and accessory genes. In conclusion, the data obtained from our analyses of strains belonging to the P. polymyxa species converge toward a necessary reclassification, which will require a fundamental contribution from microbiologists in the near future.

Importance: The development of sequencing technologies has led to an exponential increase in microbial sequencing data. Accurately identifying bacterial species remains a challenge because of extensive intra-species variability, the need for multiple identification methods, and the rapid rate of taxonomic changes. A substantial contribution to elucidating the relationships among related bacterial strains comes from comparing their genomic sequences. This comparison also allows for the identification of the "pangenome," which is the set of genes shared by all individuals of a species, as well as the set of genes that are unique to subpopulations. Here, we applied this approach to Paenibacillus polymyxa, a species studied for its potential as a biofertilizer and biocontrol agent and known as an antibiotic producer. Our work highlights the need for a more efficient classification of this bacterial species and provides a better delineation of strains with different properties.

Between 2% and 15% of pregnant women unknowingly experience asymptomatic bacteriuria (ASB), defined as ≥10⁵ CFU per milliliter of urine in the absence of symptoms. ASB increases the risk of adverse pregnancy outcomes including pyelonephritis, preterm labor, and low-birth weight infants. While pregnant women in the United States are routinely screened for ASB, those in developing countries with limited resources and funding lack an accurate mechanism for ASB screening. Aquagenx water quality test kits detect Escherichia coli, the most common causative agent of ASB, and total coliform bacteria in drinking water via colorimetric and fluorescent indicators. We found that the Aquagenx system is compatible with human urine and then proceeded to develop an ASB screening protocol using disposable inoculating loops. Our protocol diagnosed artificial ASB^- samples (10⁴ CFU/mL E. coli) with a false positive (FP) rate of 33% (n = 18) and ASB⁺ (10⁵ CFU/mL E. coli) with a false negative (FN) rate of 5.6% (n = 18). Clinical sample testing with our protocol revealed a FP rate of 0% in ASB^- samples (n = 28) and a FN rate of 0% in ASB⁺ samples caused by coliforms (n = 13). Aquagenx did not detect ASB in nine clinical samples with non-coliform etiological agents due to the limitations of the technology. However, with very high accuracy for detection of E. coli and other causative agents that collectively account for 90.1% of ASB cases, these kits could be used as a diagnostic ASB screening tool in developing countries in which there is currently no alternative to urine culture.IMPORTANCEAsymptomatic bacteriuria (ASB) affects 2%-15% of pregnant women and can result in adverse maternal and fetal outcomes if left undetected and untreated. In the United States and other developed nations, pregnant women are regularly screened for ASB via urine culture. However, in low-resource countries where bacterial culture is not available, dipstick testing is used. Although accurate in cases of symptomatic bacteriuria, dipstick detection is ineffective for detecting ASB. Here, we made use of an existing water quality field test for ASB urine screening, which would be readily deployable in low-resource settings. We optimized a dilution protocol for sampling patient urine within the detection limits of the Aquagenx kit technology. Overall, we were able to detect ASB samples with Gram-negative pathogens that collectively account for 90% of all ASB cases. Utilization of this repurposed technology for proactive medical screening may help prevent adverse pregnancy and birth outcomes due to ASB.

Formaldehyde is a known toxic compound, and functional formaldehyde detoxification is crucial for the survival of all living cells. Such detoxification systems are of particular importance for methylotrophic microorganisms that rely on formaldehyde as a central metabolite in their one-carbon metabolism. Understanding formaldehyde dissimilation pathways in non-methylotrophic industrial microorganisms is necessary for ongoing research aiming at engineering methylotrophy into their metabolism (synthetic methylotrophy). There is a variety of formaldehyde dissimilation pathways across microorganisms, often based on the activity of formaldehyde dehydrogenases. In this study, we investigated the role of the yycR gene of Bacillus subtilis putatively encoding a novel, uncharacterized zinc-type alcohol dehydrogenase-like protein. We showed that the B. subtilis ΔyycR mutant displayed a reduced formaldehyde tolerance level and confirmed the enzymatic activity of recombinantly produced and purified YycR as formaldehyde dehydrogenase in vitro. Biochemical analyses demonstrated that YycR activity is optimal at 40°C, with the highest measured activity at pH 9.5, formaldehyde is the preferred substrate, and the kinetic constants are K_m of 0.19 ± 0.05 mM and V_max of 2.24 ± 0.05 nmol min^-1. Altogether, we showed that YycR is a novel formaldehyde dehydrogenase with a role in formaldehyde detoxification in B. subtilis, providing valuable insights for future research on synthetic methylotrophy in this organism.

Importance: Formaldehyde is a key metabolite in methanol assimilation for many methylotrophic microorganisms, and at the same time, it is toxic to all living cells, which means its intracellular concentrations must be tightly controlled. An in-depth understanding of methanol detoxification systems in industrially relevant microorganisms is a prerequisite for the introduction of methanol utilization pathways into their metabolism (synthetic methylotrophy). Bacillus subtilis, an industrial workhorse conventionally used for the production of enzymes, is known to possess two formaldehyde detoxification pathways. Here, we identify a novel formaldehyde dehydrogenase in this bacterium as a path towards creating innovative prospect strategies for strain engineering towards synthetic methylotrophy.

Candida auris, an emerging fungal pathogen characterized by multidrug resistance and high-mortality nosocomial infections, poses a serious global health threat. However, the precise and rapid identification and characterization of C. auris remain a challenge. Here, we employed Raman spectroscopy combined with machine learning to identify C. auris isolates and its closely related species as well as to predict antifungal resistance and key virulence factors at the single-cell level. The average accuracy of identification among all Candida species was 93.33%, with an accuracy of 98% for the clinically simulated samples. The drug susceptibility of C. auris to fluconazole and amphotericin B was 99% and 94%, respectively. Furthermore, the phenotypic prediction of C. auris yielded an accuracy of 100% for aggregating cells and 97% for filamentous cells. This proof-of-concept methodology not only precisely identifies C. auris at the clade-specific level but also rapidly predicts the antifungal resistance and biological characteristics, promising a valuable medical diagnostic tool to combat this multidrug-resistant pathogen in the future.

Importance: Currently, combating Candida auris infections and transmission is challenging due to the lack of efficient identification and characterization methods for this species. To address these challenges, our study presents a novel approach that utilizes Raman spectroscopy and artificial intelligence to achieve precise identification and characterization of C. auris at the singe-cell level. It can accurately identify a single cell from the four C. auris clades. Additionally, we developed machine learning models designed to detect antifungal resistance in C. auris cells and differentiate between two distinct phenotypes based on the single-cell Raman spectrum. We also constructed prediction models for detecting aggregating and filamentous cells in C. auris, both of which are closely linked to its virulence. These results underscore the merits of Raman spectroscopy in the identification and characterization of C. auris, promising improved outcomes in the battle against C. auris infections and transmission.

Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling and are promising bioproduction chassis. Here, we isolate two novel cyanobacteria with 4.6Mbp genomes, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO₂. We describe complete genome sequences for both isolates and, focusing on UTEX 3222 due to its planktonic growth in liquid, characterize biotechnologically relevant growth and biomass characteristics. UTEX 3222 outpaces other fast-growing model strains on a solid medium. It can double every 2.35 hours in a liquid medium and grows to high density (>31 g/L biomass dry weight) in batch culture, nearly double that of Synechococcus sp. PCC 11901, whose high-density growth was recently reported. In addition, UTEX 3222 sinks readily, settling more quickly than other fast-growing strains, suggesting favorable economics of harvesting UTEX 3222 biomass. These traits may make UTEX 3222 a compelling choice for marine carbon dioxide removal (CDR) and photosynthetic bioproduction from CO₂. Overall, we find that bio-prospecting in environments with naturally elevated CO₂ may uncover novel CO₂-metabolizing organisms with unique characteristics.

Importance: Cyanobacteria provide a potential avenue for both biomanufacturing and combatting climate change via high-efficiency photosynthetic carbon sequestration. This study identifies novel photosynthetic organisms isolated from a unique geochemical environment and describes their genomes, growth behavior in culture, and biochemical composition. These cyanobacteria appear to make a tractable research model, and cultures are made publicly available alongside information about their culture and maintenance. Application of these organisms to carbon sequestration and/or biomanufacturing is discussed, including unusual, rapid settling characteristics of the strains relevant to scaled culture.

Associations with soil microorganisms are crucial for plants' overall health and functioning. While much work has been done to understand drivers of rhizosphere microbiome structure and function, the relative importance of geography, climate, soil properties, and plant genetics remains unclear, as results have been mixed and comprehensive studies across many sites and genotypes are limited. Rhizosphere microbiomes are crucial for crop resistance to pathogens, stress tolerance, nutrient availability, and ultimately yield. Here, we quantify the relative roles of plant genotype, environment, and their interaction in shaping soil rhizosphere communities, using 16S and ITS gene sequencing of rhizosphere soils from 10 genotypes of cultivated sunflower (Helianthus annuus) at 15 sites across the Great Plains of the United States. While site generally outweighed genotype overall in terms of effects on archaeal, bacterial, and fungal richness, community composition, and taxa relative abundances, there was also a significant interaction such that genotype exerted a significant influence on archaeal, bacterial, and fungal microbiomes in certain sites. Site effects were attributed to a combination of spatial distance and differences in climate and soil properties. Microbial taxa that were previously associated with resistance to the fungal necrotrophic pathogen Sclerotinia were present in most sites but differed significantly in relative abundance across sites. Our results have implications for plant breeding and agronomic microbiome manipulations for agricultural improvement across different geographic regions.IMPORTANCEDespite the importance of plant breeding in agriculture, we still have a limited understanding of how plant genetic variation shapes soil microbiome composition across broad geographic regions. Using 15 sites across the Great Plains of North America, we show that cultivated sunflower rhizosphere archaeal, bacterial, and fungal communities are driven primarily by site soil and climatic differences, but genotype can interact with site to influence the composition, especially in warmer and drier sites with lower overall microbial richness. We also show that all taxa that were previously found to be associated with resistance to the fungal pathogen Sclerotinia sclerotiorum were widespread but significantly affected by site, while a subset was also significantly affected by genotype. Our results contribute to a broader understanding of rhizosphere archaeal, bacterial, and fungal community assembly and provide foundational knowledge for plant breeding efforts and potential future microbiome manipulations in agriculture.

Dietary fibers play a crucial role in shaping the gut microbiome and influencing gastrointestinal (GI) physiology. Grain-based diets (GBDs) are widely used in rodent studies, but their utility is limited due to batch-to-batch variability resulting from inconsistent ingredients. Purified diets (PDs) are composed of only known and refined ingredients and offer a solution to the constraints of GBDs. This study aimed to identify a combination of dietary fibers in a purified diet (PD) that promotes optimal murine gut morphometry and a diverse intestinal microbial community. Male C57BL/6J mice were fed either two grain-based diets (GBDs) or four PDs with varying fiber compositions for 28 days. Mice consuming PDs lacking soluble fiber had more gonadal fat (P < 0.05), shorter small intestines (P < 0.05), and lighter ceca (P < 0.05) compared with those fed the LabDiet 5001 GBD. Increasing the proportion of soluble fibers in PDs progressively reduced microbial diversity in the cecum and colon. Multidimensional scaling analysis revealed distinct microbial communities in the cecum and colon between mice fed GBDs and PDs (P < 0.05). Differential abundance analysis identified relatively more Family XII UCG 001 and less Lactococcus in mice fed GBDs relative to mice consuming PDs (P < 0.05). While no PD recapitulated the gut microbial composition of GBDs, PDs with high soluble fiber content best preserved GI morphometry. These findings underscore the importance of considering diet as an experimental variable and highlight the need for a PD formulation that combines the benefits of GBDs on GI health and microbial richness.

Importance: Dietary fibers are essential for maintaining gut health. Insoluble fibers aid in fecal bulking and water retention while soluble fiber is a fermentative substrate for intestinal microbial communities. Grain-based diets (GBDs) are commonly used in preclinical research but the variability in ingredients across batches impedes reproducibility. Purified diets (PDs), which are composed of highly refined ingredients, pose a potential solution but the most widely used low-fat control PDs contain no soluble fiber. This study intended to identify a PD with a combination of fibers that promotes murine gut health and microbial diversity. A PD with optimal fiber composition would aid in the standardization and reproducibility of studies investigating intestinal physiology and the gut microbiota.

Fungi generate a diverse array of bioactive compounds with significant pharmaceutical applications. However, the chemical diversity of natural products in fungi remains largely unexplored. Here, we present a paradigm for specifically discovering diverse and bioactive compounds from fungi by integrating genome mining with building block molecular network and coculture analysis. Through pangenome and sequence similarity network analysis, we identified a rare type I polyketide enzyme from Penicillium sp. ZJUT-34. Subsequent building block molecular network and coculture strategy led to the identification and isolation of a pair of novel polyketides, (±)-peniphenone E [(±)-1], three known polyketides (2-4), and three precursor compounds (5-7) from a combined culture of Penicillium sp. ZJUT-34 and Penicillium sp. ZJUT23. Their structures were established through extensive spectroscopic analysis, including NMR and HRESIMS. Chiral HPLC separation of compound 1 yielded a pair of enantiomers (+)-1 and (-)-1, with their absolute configurations determined using calculated ECD methods. Compound (±)-1 is notable for its unprecedented structure, featuring a unique 2-methyl-hexenyl-3-one moiety fused with a polyketide clavatol core. We proposed a hypothetical biosynthetic pathway for (±)-1. Furthermore, compounds 2, 5, and 6 exhibited strong antioxidant activity, whereas (-)-1, (+)-1, 3, and four exhibited moderate antioxidant activity compared to the positive control, ascorbic acid. Our research demonstrates a pioneering strategy for uncovering novel polyketides by merging genome mining, metabolomics, and cocultivation methods. This approach addresses the challenge of discovering natural compounds produced by rare biosynthetic enzymes that are often silent under conventional conditions due to gene regulation.IMPORTANCEPolyketides, particularly those with complex structures, are crucial in drug development and synthesis. This study introduces a novel approach to discover new polyketides by integrating genomics, metabolomics, and cocultivation strategies. By combining genome mining, building block molecular networks, and coculturing techniques, we identified and isolated a unique polyketide, (±)-peniphenone E, along with three known polyketides and three precursor compounds from Penicillium sp. ZJUT-34 and Penicillium sp. ZJUT23. This approach highlights the potential of using combined strategies to explore fungal chemical diversity and discover novel bioactive compounds. The successful identification of (±)-peniphenone E, with its distinctive structure, demonstrates the effectiveness of this integrated method in enhancing natural product discovery and underscores the value of innovative approaches in natural product research.

Microbial lipopeptides are synthesized by nonribosomal peptide synthetases and are composed of a hydrophobic fatty acid chain and a hydrophilic peptide moiety. These structurally diverse amphiphilic molecules can interact with biological membranes and possess various biological activities, including antiviral properties. This study aimed to evaluate the cytotoxicity and antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of 15 diverse lipopeptides to understand their structure-activity relationships. Non-ionic lipopeptides were generally more cytotoxic than charged ones, with cationic lipopeptides being less cytotoxic than anionic and non-ionic variants. At 100 µg/mL, six lipopeptides reduced SARS-CoV-2 RNA to undetectable levels in infected Vero E6 cells, while six others achieved a 2.5- to 4.1-log reduction, and three had no significant effect. Surfactin, white line-inducing principle (WLIP), fengycin, and caspofungin emerged as the most promising anti-SARS-CoV-2 agents. Detailed analysis revealed that these four lipopeptides affected various stages of the viral life cycle involving the viral envelope. Surfactin and WLIP significantly reduced viral RNA levels in replication assays, comparable to neutralizing serum. Surfactin uniquely inhibited viral budding, while fengycin impacted viral binding after pre-infection treatment of the cells. Caspofungin demonstrated a lower antiviral effect compared to the others. Key structural traits of lipopeptides influencing their cytotoxic and antiviral activities were identified. Lipopeptides with a high number of amino acids, especially charged (preferentially anionic) amino acids, showed potent anti-SARS-CoV-2 activity. This research paves the way for designing new lipopeptides with low cytotoxicity and high antiviral efficacy, potentially leading to effective treatments.

Importance: This study advances our understanding of how lipopeptides, which are molecules mostly produced by bacteria, with both fat and protein components, can be used to fight viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By analyzing 15 different lipopeptides, researchers identified key structural features that make some of these molecules particularly effective at reducing viral levels while being less harmful to cells. Specifically, lipopeptides with certain charged amino acids were found to have the strongest antiviral effects. This research lays the groundwork for developing new antiviral treatments that are both potent against viruses and safe for human cells, offering hope for better therapeutic options in the future.

Environmental microorganisms have evolved a variety of strategies to survive fluctuations in environmental conditions, including the production of biofilms and differentiation into spores. Myxococcus xanthus are ubiquitous soil bacteria that produce starvation-induced multicellular fruiting bodies filled with environmentally resistant spores (a specialized biofilm). Isolated spores have been shown to be more resistant than vegetative cells to heat, ultraviolet radiation, and desiccation. The evolutionary advantage of producing spores inside fruiting bodies is not clear. Here, we examine a hypothesis that the fruiting body provides additional protection from environmental insults. We developed a high-throughput method to compare the recovery (outgrowth) of distinct cell types (vegetative cells, free spores, and spores within intact fruiting bodies) after exposure to ultraviolet radiation or desiccation. Our data indicate that haystack-shaped fruiting bodies protect spores from extended UV radiation but do not provide additional protection from desiccation. Perturbation of fruiting body morphology strongly impedes recovery from both UV exposure and desiccation. These results hint that the distinctive fruiting bodies produced by different myxobacterial species may have evolved to optimize their persistence in distinct ecological niches.IMPORTANCEEnvironmental microorganisms play an important role in the production of greenhouse gases that contribute to changing climate conditions. It is imperative to understand how changing climate conditions feedback to influence environmental microbial communities. The myxobacteria are environmentally ubiquitous social bacteria that influence the local microbial community composition. Defining how these bacteria are affected by environmental insults is a necessary component of predicting climatic feedback effects. When starved, myxobacteria produce multicellular fruiting bodies filled with spores. As spores are resistant to a variety of environmental insults, the evolutionary advantage of building a fruiting body is not clear. Using the model myxobacterium, Myxococcus xanthus, we demonstrate that the tall, haystack-shaped fruiting body morphology enables significantly more resistance to UV exposure than the free spores. In contrast, fruiting bodies are slightly detrimental to recovery from extended desiccation, an effect that is strongly exaggerated if fruiting body morphology is perturbed. These results suggest that the variety of fruiting body morphologies observed in the myxobacteria may dictate their relative resistance to changing climate conditions.