Applied and Environmental Microbiology最新文献

Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers in certain areas are utilizing denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci (PULs). However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to identify microbial drivers for lignocellulose transformation in woodchip bioreactors and their active enzymes. Our findings highlight a microbial community enriched for (ligno)cellulose-degrading denitrifiers with key players from the taxa Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota). A wide substrate specificity is observed among the many expressed carbohydrate-active enzymes (CAZymes) including PULs from Bacteroidetes. This suggests a broad degradation of lignocellulose subfractions, including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions.

Importance: Freshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota) playing significant roles in lignocellulose transformation and showcasing a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improve water quality, protect aquatic ecosystems, and reduce greenhouse gas emissions from WBRs.

Managing water quality with microbial impairment caused by Enterococcus poses unique challenges regarding the determination of fecal host origin. Most water monitoring is performed based on Enterococcus counts that neither detect the location of the introduction of pollution nor identify the type of contaminating Enterococcus. The use of sequenced-based microbial source tracking could allow for identification of fecal origin and potential remediation of pollution. The state of Delaware has numerous waterways with high microbial impairment from unknown sources, so we used sequence-based microbial source tracking to investigate potential microbial pollution in three watersheds with significant variation in land use and population density. In this study, we use a 16S rRNA sequence reference library of microbial communities from relevant fecal sources (wild animal, domestic animal, sediment, and septic/wastewater) to determine the most likely sources of microbial impairment in three Delaware watersheds. This study assigned sources of microbial contamination to mostly human-related sources (septic and wastewater) or unknown sources indicating that waste infrastructure may have a larger influence on microbial community structure in Delaware watersheds than previously considered. Our results suggest that long-term source tracking is valuable for ruling out native or domesticated animals as contributors to water pollution.IMPORTANCETraditional microbial pollution monitoring utilizes specific fecal indicator bacteria that need to grow in the laboratory for detection. Here, we show the use of sequence information from whole microbial communities and an expanded reference library in microbial source tracking. Expanding the host detection range by including the whole microbial community may allow for a wider range of potential fecal origin identification even when specific fecal indicators are absent or in low concentration. We show that many Delaware waterways bear the signature of human influence compared to natural sources. In addition, the robust reference library built in this study can be used to conduct source tracking studies in the mid-Atlantic USA.

Candida albicans is an opportunistic pathogen and colonizer of the human gut and mucosal membranes. C. albicans exhibits morphological plasticity, which is crucial for its fitness within the host and virulence. Morphogenesis in C. albicans is regulated, in part, by its production of farnesol, an autoregulatory molecule that inhibits filamentation. Morphogenesis is also regulated in response to external cues, such as serum, which stimulates hyphal formation by C. albicans. The precise mechanism by which serum stimulates hyphal formation is unknown. The most abundant serum protein is albumin. The binding affinity of albumin for nonpolar, fatty-acid-like molecules suggests that it may interact directly with exogenous farnesol and influence morphogenesis through sequestration of free farnesol. To test this hypothesis, we assessed whether albumin and albumin devoid of fatty acids (i) stimulated farnesol secretion and (ii) influenced the farnesol threshold required to inhibit filamentation. We found that albumin promoted farnesol secretion and filamentation, and the extent of its ability to do so was based on the presence or absence of bound fatty acids. We hypothesize that albumin not bound to fatty acids has the capacity to bind to farnesol and sequester it from C. albicans, encouraging filamentation.IMPORTANCEFor at least 50 years, researchers have wondered about the mechanisms by which serum stimulates germ tube formation (GTF) and hyphal growth in C. albicans. Here, we tested a model (Nickerson et al., Microbiol Mol Biol Rev 88:e00081-22, 2024, https://doi.org/10.1128/mmbr.00081-22) that serum promotes GTF and farnesol synthesis in part by extracting internal farnesol (F_i) from the cells toward the excess binding capacity of the albumins. The data presented here suggests that albumin not bound by fatty acids sequesters free farnesol thereby modulating filamentation and farnesol secretion by altering the equilibrium of internal vs external farnesol. We expect that the influence of secreted farnesol on cell morphology will differ during pathogenesis depending on location within the body, but sequestration of farnesol in the blood could mediate immune cell recruitment and promote hyphal formation.

The ability of microorganisms to decompose brown algae has attracted attention. This study aims to clarify the characteristics of marine microbial communities in which prokaryotic and eukaryotic microorganisms interact via the metabolism of brown algae carbohydrates. Amplicon-based microbiome analysis revealed the predominance of the genera Marinomonas and Vibrio in seawater and seaweed samples mixed with alginate and mannitol, which are the primary carbohydrates in brown algae. Three Vibrio species and Candida intermedia were isolated via alginate enrichment culture. Although C. intermedia did not utilize alginate as a nutrient source, the yeast grew in the spent alginate medium in which Vibrio algivorus had been cultured. Coculture with C. intermedia and the Vibrio isolates, especially V. algivorus, also enhanced the growth of the yeast on alginate. These results suggested that C. intermedia grew because of the supply of nutrients via alginate metabolism by Vibrio species. In the coculture medium, the amount of phosphatidylserine increased in the early phase but decreased with the growth of C. intermedia, indicating that phosphatidylserine secreted by Vibrio is involved in the putative mutualistic interaction. We examined whether such interaction is applicable to the production of useful substances and succeeded in lipid production by oleaginous marine yeast Yarrowia lipolytica through coculture with V. algivorus. Our study suggested the potential of mutualistic interaction via degradation of alginate by marine Vibrio for production of industrially useful substances in yeast cells.IMPORTANCEIn this study, we analyzed the microbiome of seawater and seaweed in the presence of brown algae carbohydrates and reconstructed the putative mutualistic relationship of marine Vibrio and Candida intermedia mediated by metabolism of brown algae in the ocean.

Soil microorganisms play a crucial role in suppressing soil-borne diseases. Although the composition of microbial communities in healthy versus diseased soils is somewhat understood, the interplay between microbial interactions and disease incidence remains unclear. This study used 16S rRNA and fungal internal transcribed spacer (ITS) sequencing to investigate the bacterial and fungal community composition in three soil types: forest soil (Z), soil from healthy banana plantations (H), and soil from diseased banana plantations (D). Principal coordinate analysis revealed significant differences among the bacterial and fungal community structures of the three soil types. Compared with those in forest soil, bacterial and fungal diversities significantly decreased in diseased banana soil. Key microorganisms, including the bacteria Chloroflexi and Pseudonocardia and the fungi Mortierellomycota and Moesziomyces, were significantly increased in soil from diseased banana plantations. Redundancy analysis revealed that total nitrogen and available phosphorus were the primary drivers of the soil microbial community structure. The neutral community model posited that the bacterial community assembly in banana plantations is predominantly governed by stochastic processes, whereas the fungal community assembly in banana plantations is primarily driven by deterministic processes. Furthermore, co-occurrence network analysis revealed that the proportion of positive edges in the fungal network of soil from diseased banana plantations was 5.92 times lower than that in soil from healthy banana plantations, and its fungal network structure was sparse and simple. In conclusion, reduced interactions within the fungal network were significantly linked to the epidemiology of Fusarium wilt. These findings underscore the critical role of soil fungal communities in modulating pathogens.

Importance: Soil microorganisms are pivotal in mitigating soil-borne diseases. The intricate mechanisms underlying the interactions among microbes and their impact on disease occurrence remain enigmatic. This study underscores that a reduction in fungal network interactions correlates with the incidence of soil-borne Fusarium wilt.

Marine yeasts play a crucial role in marine microbial ecology, facilitating the biogeochemical cycling of carbon and nitrogen in marine ecosystems, while also serving as important reservoirs of bioactive compounds with extensive applications in pharmaceuticals, agriculture, and various industries. Intertidal flats, characterized by their complex ecological dynamics, are postulated to harbor a wealth of yeast resources. This study employed a culture-dependent approach to assess the diversity, spatio-temporal distribution, and biotechnological potential of yeast communities residing within the intertidal sediments and seawater of Aoshan Bay. A total of 392 yeast strains were identified from 20 distinct genera, encompassing 43 recognized species and four candidate novel species. Notably, 17 of these species were identified as novel occurrences in marine environments, underscoring the rich yeast biodiversity of the Aoshan Bay ecosystem, with Candida emerging as the dominant genus in both sedimentary and aqueous habitats. Yeast community composition exhibited significant spatial and temporal variation, with peak diversity and abundance observed in autumn, the subtidal zone, and the surface soil layer. No clear pattern, however, emerged linking these shifts to specific changes in community composition, highlighting the complex interactions between microbial communities, environmental variables, and anthropogenic disturbance. Although several yeast species isolated here have been previously recognized for their biotechnological potential, their diverse and abundant extracellular enzyme profiles were characterized, further highlighting their crucial role in organic matter decomposition and nutrient cycling within the tidal ecosystem, as well as their potential applicability in the food, fine chemical, textile, and pharmaceutical industries.IMPORTANCEThis study presents groundbreaking insights into the yeast diversity of Aoshan Bay, offering invaluable information on their spatial and temporal distribution patterns, as well as their biotechnological potential in the tidal environment. The findings reveal that the eutrophic intertidal flat is a rich repository of yeasts with abundant extracellular enzymatic activity and an important role in nutrient cycling and decomposition processes. Also, these yeasts serve as crucial indicators of ecosystem health and function and are excellent candidates for biotechnological and industrial applications. Collectively, this study not only expands our knowledge of the diversity and distribution of intertidal yeasts but also highlights their promising potential for biotechnological applications.

Gas fermentation using acetogenic bacteria requires a chemically defined minimal medium to be established. This approach not only helps in creating a cost-effective medium but also allows for a thorough exploration of their metabolic potential. In this study, the auxotrophy of the acetogen Clostridium sp. AWRP was investigated through genomic analysis and growth performance in formulated media. It was found that the strain needs pantothenate and biotin and that substituting vitamin B₆ from pyridoxine to pyridoxamine or pyridoxal-5'-phosphate is crucial for growth. The determined chemically defined minimal medium supported both heterotrophic (using fructose as a substrate) and autotrophic (using syngas as a substrate) growth of the AWRP strain. To overcome the vitamin B₆ auxotrophy, the pdxST genes responsible for vitamin B₆ biosynthesis were introduced into the AWRP strain using plasmid-based gene expression system and CRISPR/Cas12a genome-editing technology. As a result, the genetically engineered strains were able to grow successfully without vitamin B₆. This chemically defined minimal medium will enhance the fermentation performance of AWRP.

Importance: The identification of auxotrophy in Clostridium sp. AWRP underpins subsequent investigations into its physiology and metabolism. Additionally, the development of a chemically defined minimal medium specific to this acetogenic bacterium will enable reproducible industrial processes. This innovation is particularly significant for the bioconversion of carbon monoxide and/or dioxide into commercially valuable chemicals through the process of gas fermentation.

Sourdoughs are maintained by back-slopping over long time periods. To determine strain-level persistence of bacteria, we characterized four sourdoughs from bakeries over a period of 3.3, 11.0, 18.0, and 19.0 years. One sourdough included isolates of Levilactobacillus spp. and Fructilactobacillus spp. that differed by fewer than 10 single-nucleotide polymorphisms (SNPs) from the isolates obtained 3.3 years earlier and thus likely represent the same strain. Isolates of Levilactobacillus parabrevis differed by 200-300 SNPs; their genomes were under positive selection, indicating transmission from an external source. In two other sourdoughs, isolates of Fructilactobacillus sanfranciscensis that were obtained 11 and 18 years apart differed by 19 and 29 SNPs, respectively, again indicating repeated isolation of the same strain. The isolate of Fl. sanfranciscensis from the fourth sourdough differed by 45 SNPs from the isolate obtained 19 years previously. We thus identified strain-level persistence in three out of four long-term back-slopped sourdoughs, making it possible that strains persisted over periods that are long enough to allow bacterial speciation and domestication.IMPORTANCEThe assembly of microbial communities in sourdough is shaped by dispersal and selection. Speciation and domestication of fermentation microbes in back-slopped food fermentations have been documented for food-fermenting fungi including sourdough yeasts but not for bacteria, which evolve at a slower rate. Bacterial speciation in food fermentations requires strain-level persistence of fermentation microbes over hundreds or thousands of years. By documenting strain-level persistence in three out of four sourdoughs over a period of up to 18 years, we demonstrate that persistence over hundreds or thousands of years is possible, if not likely. We thus not only open a new perspective on fermentation control in bakeries but also support the possibility that all humans, despite their cultural diversity, share the same fermentation microbes.

The Lyme disease spirochete Borrelia burgdorferi cycles between immature black-legged ticks (Ixodes scapularis) and vertebrate reservoir hosts, such as rodents. Larval ticks acquire spirochetes from infected hosts, and the resultant nymphs transmit the spirochetes to naïve hosts. This study investigated the impact of immunocompetence and host tissue spirochete load on host-to-tick transmission (HTT) of B. burgdorferi and the spirochete load inside immature I. scapularis ticks. Wild-type (WT) C57BL/6J mice and mice with severe combined immunodeficiency (SCID) were experimentally infected with B. burgdorferi. To measure HTT, WT and SCID mice were repeatedly infested with I. scapularis larvae, and ticks were sacrificed at three different developmental stages: engorged larvae, 1-month-old, and 12-month-old nymphs. The spirochete loads in immature ticks and mouse tissues were estimated using qPCR. In WT mice, HTT decreased from 90% to 65% over the course of the infection, whereas in the SCID mice, HTT was always 100%. Larvae that fed on SCID mice acquired a much larger dose of spirochetes compared to larvae that fed on WT mice. This difference in spirochete load persisted over tick development where nymphs that fed as larvae on SCID mice had significantly higher spirochete loads compared to their WT counterparts. HTT and the tick spirochete loads were strongly correlated with the mouse tissue spirochete loads. Our study shows that the host immune system (e.g., the presence of antibodies) influences HTT of B. burgdorferi and the spirochete load in immature I. scapularis ticks.IMPORTANCEThe tick-borne spirochete Borrelia burgdorferi causes Lyme disease in humans. This pathogen is maintained in nature by cycles involving black-legged ticks and wildlife hosts. The present study investigated the host factors that influence the transmission of B. burgdorferi from infected hosts to feeding ticks. We infected immunocompetent mice and immunocompromised mice (that cannot develop antibodies) with B. burgdorferi and repeatedly infested these mice with ticks. We determined the percentage of infected ticks and their spirochete loads. This percentage was 100% for immunocompromised mice but decreased from 90% to 65% over time (8 weeks) for immunocompetent mice. The tick spirochete load was much higher in ticks fed on immunocompromised mice compared to ticks fed on immunocompetent mice. In summary, the host immune system influences the transmission of B. burgdorferi from infected hosts to ticks and the spirochete loads in those ticks, which, in turn, determines the risk of Lyme disease for people.