Applied and Environmental Microbiology最新文献

Filamentous fungi play important roles in the development of surface-ripened cheese microbial communities and contribute to the aesthetics and flavors of these products. Much is known about the diversity and ecology of desirable cheese fungi, but our understanding of the natural history of cheese spoilage molds is limited. The goal of this work was to characterize the genomic diversity of Aspergillus species contaminating artisan cheeses and to identify how the abiotic environment of cheese (the substrate itself and temperature) may constrain the growth of Aspergillus. Comparative genomics identified two main species of Aspergillus, A. westerdijkiae and A. ostianus, as the spoilage molds across three different facilities in the Northeastern United States that experienced contamination events. Multiple genomic types of A. westerdijkiae were found across the different cheese production facilities, indicating that these contamination events are not caused by a single clonal strain. All A. westerdijkiae isolates produced ochratoxin A, but concentrations varied greatly across strains. RNA-sequencing of A. westerdijkiae on nutrient-rich lab media (malt extract agar) versus cheese curd agar identified a suite of pathways enriched in expression on cheese, including degradation of amino and fatty acids. Experiments measuring growth over a range of temperatures identified that spoilage Aspergillus species have a higher optimal growth temperature compared to desirable fungal species in cheese rinds and are outcompeted by Penicillium species at temperatures lower than 15°C. Global fungal metabarcoding databases suggest that A. westerdijkiae is not normally found in natural habitats of the Northeastern United States, and it may be introduced to this region.IMPORTANCEOver the past decade, disruptive contamination events of Aspergillus spoilage molds have occurred at cheese production facilities in Massachusetts, Connecticut, and Vermont in the United States, causing aesthetic, flavor, and potential safety issues. Our work highlights independent introductions of different strains of A. westerdijkiae into multiple cheese facilities and suggests that temperature could be used to control the abundance of Aspergillus spoilage molds. Based on our analysis of the global distribution of A. westerdijkiae, it is not invading cheese facilities from local fungal populations and may be a contaminant in materials used for cheese production.

YggS (COG0325 family) was widely conserved across the three domains of life and had been implicated in pyridoxal 5'-phosphate (PLP) homeostasis. However, transcriptional regulators that directly controlled yggS had remained unknown. Here, using Alcaligenes faecalis JQ135, PhpR, a previously unrecognized MocR-family regulator, was identified as a transcriptional regulator that directly controlled yggS. Deletion of yggS in strain JQ135 impaired cell growth and caused intracellular PLP imbalance. PhpR (PLP homeostasis-related protein regulator, phpR) directly repressed yggS transcription by binding a conserved non-palindromic promoter motif in a PLP-dependent manner. Comparative genomic analyses further revealed that the adjacent yggS-phpR gene pair was divergently transcribed and highly conserved, but restricted to Alcaligenes species. Together, these findings established the first direct transcriptional regulatory mechanism for a YggS-family protein and revealed a PLP-responsive regulatory circuit linking cofactor homeostasis in bacteria.

Importance: Pyridoxal 5'-phosphate (PLP), the main catalytically active form of vitamin B6, was an essential cofactor for many enzymes, particularly those involved in amino acid metabolism. PLP deficiency could impair the activity of PLP-dependent enzymes and disrupt cellular metabolism, whereas excessive accumulation of free PLP could also cause metabolic imbalance. YggS-family proteins were widely conserved PLP homeostasis factors, yet how their expression was regulated had remained largely unknown. Here, we identified PhpR as a previously uncharacterized MocR-family transcriptional regulator that directly repressed the expression of YggS in Alcaligenes, establishing a direct link between intracellular PLP availability and yggS expression. These findings revealed a previously unknown regulatory mechanism governing bacterial PLP homeostasis and provided new insight into how PLP-responsive transcriptional regulation coordinated cofactor balance with cellular metabolism.

Coral holobionts are typically studied at colony or fragment scale, erasing the spatial context of the microenvironments which microbial life occupies. Recent microsensor work (Q. Zhang, E. Bollati, and M. Kühl, Appl Environ Microbiol 92:e02441-25, 2026, https://doi.org/10.1128/aem.02441-25) shows the coral gastrovascular cavity (GVC) sustains active anaerobic metabolism, reflecting patterns of symbiotic state and heterotrophic feeding. These findings recast the GVC as a discrete, gut-like ecosystem with its own redox regime, substrate supply, and bacterial community, calling for spatially explicit approaches in coral research to resolve metabolism at microscopic scales.

Bile acid (BA) transformation relies on gut microbiota and is vulnerable to Toxoplasma gondii infection, yet feline microbial BA-transforming capacity upon toxoplasmosis remains unclear. Here, we constructed a catalog of 2,474 nonredundant feline gut microbial genomes and integrated serum metabolomic data to verify BA transformation alterations. The results revealed that the feline gut microbiome harbored widespread genetic potential for BA transformation but lacked a complete 7α-dehydroxylation pathway due to the absence of the key gene baiE. The BA transformation-related genomes (2,045 in total) were predominantly from the phyla Bacillota_A and Actinomycetota, among which only 37 encoded baiB, all belonging to Bacillota_A. The distribution of BA transformation-related genes varied across intestinal regions: genes encoding 7α-HSDH were primarily enriched in the small intestine, whereas genes encoding 3α-HSDH, baiCD, and baiH were more abundant in the large intestine. Additionally, the abundance of genes encoding BSH and 3α-HSDH increased significantly in the small intestine on day 3 post-infection, accompanied by increases in the phylum Bacillota_C and genera such as Blautia_A, Enterococcus_E, and Ligilactobacillus. Serum metabolomics revealed a significant increase in cholesterol levels post-infection, supporting the impact of T. gondii infection on intestinal BA transformation. These findings illustrated that the feline gut microbiota played an important role in BA transformation and that T. gondii infection disrupted the microbial potential for secondary BA transformation. This study provided new insights into gut microbiota-associated metabolic perturbations during feline toxoplasmosis.

Importance: Bile acid (BA) transformation plays a critical role in host metabolism and immune regulation. Although studies on BA transformation are increasing, the capacity for BA transformation within the feline gut microbiota and the impact of Toxoplasma gondii infection on this capacity remain unclear. To bridge this gap, we constructed a catalog of 2,474 nonredundant feline gut microbial genomes and integrated serum metabolomic data to verify BA transformation alterations. Our findings revealed that the feline gut microbiome lacked a complete 7α-dehydroxylation pathway, and the specific functions involved in BA transformation may differ between the small and large intestines. Furthermore, integrated metagenomic and serum metabolomic analyses suggested that T. gondii infection disrupted BA transformation capacity in the small intestine. This study provided new insights into gut microbiota-associated metabolic perturbations during feline toxoplasmosis.

In vitro viability assays are essential for studying the survival of Borrelia burgdorferi after exposure to stressful conditions. In contrast to the cumbersome and tedious manual microscopic counting method, fluorescence-based assays are more rapid and convenient and thus can be used in a high-throughput format. In the SYBR Green I/propidium iodide (PI) assay, spirochetes are labeled with different fluorescent dyes, and fluorescence ratios are then compared to calculate the ratio between live and dead spirochetes. Stained spirochetes are also manually counted under a fluorescence microscope or analyzed via flow cytometry to obtain the live/dead ratio. In this study, we compared these methods to determine the most accurate technique for quantifying spirochete viability. Our results showed that, compared with the SYBR Green I/PI assay that carries inherent caveats, both the fluorescent microscopic counting method and the flow cytometry method provide more reliable and accurate results for B. burgdorferi viability assays, and flow cytometry offers significant advantages in speed and reduced labor compared to manual microscopic counting. Leveraging the flow cytometry-based viability assay, we investigated the role of gene bbi29, encoding a putative virulence-associated lipoprotein, in B. burgdorferi stress response. The contribution of bbi29 to B. burgdorferi mammalian infection was also examined using a murine model of Lyme disease.

Importance: Lyme disease, caused by Borrelia burgdorferi, is the most common tick-borne disease in the United States. Assessment of B. burgdorferi growth and viability has been traditionally challenging due to the slow growth and unique morphology of the spirochete. Recently, an optimized SYBR Green I/PI assay was developed and has since been used by a number of laboratories to assess B. burgdorferi viability. We herein surprisingly found that the SYBR Green I/PI assay has significant caveats and does not provide accurate quantification of B. burgdorferi viability. Rather, our data showed that flow cytometry is a superior alternative to quantify spirochete viability, providing a valuable methodological framework for future studies. Additionally, by investigating the role and requirement for gene bbi29, we have refined our understanding of hypothetical proteins in B. burgdorferi pathogenesis. Together, this work highlights the importance of developing and validating experimental approaches and functionally characterizing B. burgdorferi genes with unknown functions.

Biogeochemical cycles exert significant influences on the fate and behavior of antibiotics in estuarine and coastal environments; however, ferrous sulfide (FeS)-mediated enrichment and dissemination of antibiotic resistance genes (ARGs) remain poorly understood. Here, we conducted microcosm experiments and field investigations to examine the relationships of FeS amendment, microbial communities, functional potentials in relation to elemental cycling, and ARG abundance. The positive effects of FeS on ARG abundance were aerobic dependent, which were accompanied by a significant enrichment of Proteobacteria, as the crucial hosts of ARGs. Proteobacteria were associated with systematic metabolic pathways for carbon fixation (Calvin-Benson-Bassham [CBB] cycle), N₂ fixation, and FeS oxidation. In addition, field samples from China estuarine and coastal wetlands further showed that CBB cycle, N₂ fixation, and sulfur oxidation pathways were significantly correlated with the relative abundance of ARGs. These findings collectively indicate that FeS oxidation facilitates enrichment and dissemination of ARGs through selective enrichment of Proteobacteria and consequent enhancement of metabolic synergies between sulfur, iron, carbon, and nitrogen cycles. However, FeS-mediated oxidation may also degrade microbial DNA and suppress ARG proliferation, indicating that the net outcome of ARG enrichment depends on oxygen conditions and microbial community filtering. The cascade processes of environmental regulation-community restructuring-metabolic synergy are of great significance to enhance ARG enrichment and dissemination. Therefore, redox fluctuations can enhance elemental cycling and further promote significant enrichment and dissemination of ARGs. These findings highlight the importance of redox dynamics in ARG monitoring and risk assessment in estuarine and coastal wetlands.IMPORTANCEEstuarine and coastal wetlands are important ecosystems for antibiotic resistance gene (ARG) enrichments. This study investigated FeS oxidation-mediated biogeochemical cycling and ARG dissemination. FeS oxidation increased enrichment and dissemination of the ARGs in estuarine and coastal sediments. The FeS oxidation reshaped microbial community structure and selectively enriched the critical Proteobacteria, which played the crucial hosts of the ARGs. The enrichment and dissemination of ARGs in the presence of FeS oxidation were due mainly to the enhanced metabolic coupling of sulfur oxidation, nitrogen fixation, and carbon fixation. CBB cycle, N₂ fixation, and sulfur oxidation pathways were also significantly correlated with ARG abundance along the latitudinal gradient. Therefore, enhanced elemental metabolic pathways under FeS oxidation exacerbated enrichment and dissemination of ARGs, highlighting the coupling mechanisms of biogeochemical cycle and ARGs in estuarine and coastal wetlands.

Anaerobic degradation of polycyclic aromatic hydrocarbons (PAHs) proceeds through CoA-activated intermediates and β-oxidation-like reactions; however, the enzymes responsible for key hydration and hydrolytic steps remain insufficiently defined. In the sulfate-reducing enrichment culture N47, the thn operon encodes multiple putative hydratases and hydrolases proposed to participate in anaerobic naphthalene degradation. Here, we cloned, heterologously expressed, and purified six candidate enzymes (ThnA, ThnH, ThnI, ThnL, ThnM, and ThnU) to elucidate their substrate specificities. Among them, ThnL showed robust activity toward 2-carboxycyclohexylideneacetyl-CoA, catalyzing rapid hydration of the enoyl-CoA double bond to form the tertiary alcohol 1-(1-hydroxy-2-carboxycyclohexyl)acetyl-CoA. ThnL exhibited kinetic parameters of Kₘ = 0.11 mM and Vₘₐₓ = 350 µmol min⁻¹ mg⁻¹. Two additional hydratases, ThnU and ThnI, converted the substrate only weakly (0.26% and 0.08% of ThnL activity), indicating promiscuous rather than physiological roles. ThnL also catalyzed the hydrolytic C1-C2 ring cleavage of 2-oxocyclohexane-1-carbonyl-CoA to pimelyl-CoA, revealing a bifunctional hydratase/hydrolase activity, although this reaction is unlikely to be physiologically relevant based on growth assays. This study shows that the ThnL-catalyzed hydration of 2-carboxycyclohexylideneacetyl-CoA to the tertiary alcohol 1-(1-hydroxy-2-carboxycyclohexyl)acetyl-CoA completes the final preparatory step before the second-ring cleavage in anaerobic naphthalene degradation.IMPORTANCEPolycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are widespread environmental pollutants that persist in oxygen-limited environments, such as marine sediments and contaminated aquifers. Although microorganisms are known to degrade these compounds anaerobically, the enzymatic mechanisms underlying key steps of these pathways remain poorly understood. In particular, the enzymes responsible for hydration reactions preceding ring cleavage in anaerobic naphthalene degradation have not been experimentally verified. In this study, we characterized several candidate enzymes encoded by the thn operon from a sulfate-reducing enrichment culture and identified ThnL as the enzyme catalyzing the hydration of 2-carboxycyclohexylideneacetyl-CoA. This finding clarifies a crucial preparatory step before the second-ring cleavage during anaerobic naphthalene metabolism and advances our understanding of microbial PAH degradation in anoxic environments.