Applied and Environmental Microbiology最新文献

Microbiome composition and function often change throughout a host's life cycle, reflecting shifts in the ecological niche of the host. The mechanisms that establish these relationships are therefore important dimensions of host ecology and evolution; yet, their nature remains poorly understood. Here, we sought to investigate the microbial communities associated with the complex life cycle of the dung beetle Onthophagus taurus and the relative contributions of host life stage, sex, and environment in determining microbiome assembly. We find that O. taurus plays host to a diverse microbiota that undergo drastic community shifts throughout host development, influenced by host life stage, environmental microbiota, and, to a lesser degree, sex. Contrary to predictions, we found that egg and pupal stages-despite the absence of a digestive tract or defined microbe-storing organs-do not constrain microbial maintenance, while host-constructed environments, such as a maternally derived fecal pellet or the pupal chamber constructed by late larvae, may still serve as complementary microbial refugia for select taxa. Lastly, we identify a small community of putative core microbiota likely to shape host development and fitness. Our results provide important insights into mechanisms employed by solitary organisms to assemble, maintain, and adjust beneficial microbiota to confront life-stage-specific needs and challenges.

Importance: As the influence of symbionts on host ecology, evolution, and development has become more apparent so has the importance of understanding how hosts facilitate the reliable maintenance of their interactions with these symbionts. A growing body of work has thus begun to identify diverse behaviors and physiological mechanisms underpinning the selective colonization of beneficial symbionts across a range of host taxa. Yet, how organisms with complex life cycles, such as holometabolous insects, establish and maintain key symbionts remains poorly understood. This is particularly interesting considering the drastic transformations of both internal and external host morphology, and the ecological niche shifts in diet and environment, that are the hallmark of metamorphosis. This work investigates the dynamic changes of the microbiota associated with the complex life cycle and host-constructed environments of the bull-headed dung beetle, Onthophagus taurus, a useful model for understanding how organisms may maintain and modulate their microbiota across development.

Natural chondroitin sulfate (CS), extracted from animal cartilage, is widely used in the pharmaceuticals and foods. However, contamination with animal-derived heteropolysaccharides presents significant challenges, including potential immune responses. To address this, we developed a green and efficient method for synthesizing chondroitin sulfate E (CSE) via enzymatic synthesis, identifying EcCHST15, a sulfotransferase that catalyzes the conversion of chondroitin sulfate A (CSA) to CSE. We investigated the novel catalytic mechanism of CHST15 through quantum mechanical (QM) calculations and experimental validation, confirming its alignment with the SN2 reaction mechanism. Subsequently, we enhanced the catalytic efficiency of CHST15 using protein engineering, improving the catalytic efficiency from 18.1% in the wild type (WT) to 62.5% in the M7 mutant-a 3.5-fold increase. Finally, we constructed a six-enzyme cascade whole-cell catalyst, achieving a 72.2% conversion of 15 g/L CSA to produce CSE within 24 h. These findings offer a promising strategy for the industrial production of CSE.IMPORTANCECurrent methods for obtaining chondroitin sulfate (CS) primarily rely on tissue extraction and chemical synthesis. However, these approaches are hindered by contamination risks from animal-derived heteropolysaccharides and the technical challenges inherent in complex chemical synthesis, limiting the scalability of industrial CS production. To address this, we developed a green and efficient enzymatic biosynthesis method for chondroitin sulfate E (CSE). By identifying and engineering the sulfotransferase CHST15 from Erpetoichthys calabaricus, we created a mutant (EcCHST15^M7) with a 3.5-fold increase in catalytic efficiency toward chondroitin sulfate A (CSA) compared to the wild-type enzyme. Additionally, we constructed a six-enzyme cascade whole-cell biocatalyst, achieving a 72.2% conversion rate from CSA to CSE. This study opens new avenues for the industrial-scale production of CSE through sustainable enzymatic processes.

Aedes aegypti mosquitoes are well adapted to dry climates and can retain their eggs for extended periods in the absence of suitable habitat. Wolbachia strains transferred from other insects to mosquitoes can be released to combat dengue transmission by blocking virus replication and spreading through populations, but host fitness costs imposed by Wolbachia, particularly under some environments, can impede spread. We, therefore, assessed the impact of two Wolbachia strains being released for dengue control (wAlbB and wMelM) on fecundity and egg viability following extended egg retention (up to 24 days) under laboratory conditions. Egg viability following retention decreased to a greater extent in females carrying wMelM compared to uninfected or wAlbB females. Fertility fully recovered in uninfected females following a second blood meal after laying retained eggs, while wMelM females experienced only partial recovery. Effects of wMelM on egg retention were similar regardless of whether females were crossed to uninfected or wMelM males, suggesting that fitness costs were triggered by Wolbachia presence in females. The fecundity and hatch proportions of eggs of wMelM females declined with age, regardless of whether females used stored sperm or were recently inseminated. Costs of some Wolbachia strains during egg retention may affect the invasion and persistence of Wolbachia in release sites where larval habitats are scarce and/or intermittent.IMPORTANCEWolbachia mosquito releases are expanding around the world with substantial impacts on dengue transmission. Releases have succeeded in many locations, but the establishment of Wolbachia has been challenging in some environments, and the factors contributing to this outcome remain unresolved. Here, we explore the effects of Wolbachia on a novel trait, egg retention, which is likely to be important for the persistence of mosquito populations in locations with intermittent rainfall. We find substantial impacts of the Wolbachia strain wMelM on the quality of retained eggs but not the wAlbB strain. This cost is driven by the Wolbachia infection status of the female and can partially recover following a second blood meal. The results of our study may help to explain the difficulty in establishing Wolbachia strains at some field release sites and emphasize the need to characterize Wolbachia phenotypes across a variety of traits and strains.

Advances in DNA metabarcoding have greatly expanded our knowledge of microbial communities in recent years. Pipelines and parameters have been tested extensively for bacterial metabarcoding using the 16S rRNA gene and best practices are largely established. For fungal metabarcoding using the internal transcribed spacer (ITS) gene, however, only a few studies have considered how such pipelines and parameters can affect community prediction. Here, we report a novel bias uncovered during ITS region 2 (ITS2) sequencing of Trichoderma-infected ant fungus gardens and confirmed this bias using mock communities. Abnormally low forward read quality caused Trichoderma ITS2 reads to be computationally filtered before and during read pair merging, thus almost entirely eliminating Trichoderma amplicon sequence variants from the resulting fungal community profiles. Sliding window quality trimming before filtering allowed most of these reads to pass filtering and merge successfully, producing community profiles that now correlated with visual signs of Trichoderma infection and matched the composition of the mock communities. Applying such sliding window trimming to a previously generated environmental ITS2 data set increased the detected fungal diversity and again overcame read quality biases against Trichoderma to detect it in nearly every sample instead and often at high relative abundances. This analysis additionally identified a similar, but distinct, bias against a second fungal genus Meyerozyma. The prevalence of such quality biases against other fungal ITS sequences is unknown but may be widespread. We, therefore, advocate for the routine use of sliding window quality trimming as a best practice in ITS2 metabarcoding analysis.

Importance: Metabarcode sequencing produces DNA abundance profiles that are presumed to reflect the actual microbial composition of their corresponding input samples. However, this assumption is not always tested, and taxon-specific biases are often not apparent, especially for low-abundance taxa in complex communities. Here, we identified internal transcribed spacer region 2 (ITS2) read quality aberrations that caused dramatic reductions in the relative abundances of specific taxa in multiple data sets characterizing ant fungus gardens. Such taxon-specific biases in read quality may be widespread in other environments and for other fungal taxa, thereby causing incorrect descriptions of these mycobiomes.

Wohlfahrtiimonas chitiniclastica is an emerging zoonotic pathogen associated with bacteremia, myiasis, and soft tissue infections. It is insufficiently identified and underestimated due to reasons, such as shortcomings of the traditional identification techniques and language barriers in local case reports from different regions. In this review, we summarize the currently available literature. In particular, we added previously overlooked cases from Chinese and other medical communities. The clinical characteristics, identification, and treatment of W. chitiniclastica are discussed. This work provides a complete review of the previous work including cases from human, animal, and other sources.

Cadmium (Cd) and chromium (Cr) are frequently encountered toxicants, while iron (Fe) plays a crucial role in bacterial survival under conditions of heavy metal stress. However, intracellular Fe ion depletion by heavy metals leads to a state of Fe starvation. Therefore, it is imperative to investigate the mechanism through which bacteria maintain a balance between heavy metal detoxification and Fe homeostasis. This study demonstrates Cd(II) immobilization and Cr(VI) reduction abilities of Stenotrophomonas sp. SY1, while proteomics reveals the upregulation of heme metabolism in response to Cd(II) and Cr(VI) exposure. The expression of the heme-uptake system in Escherichia coli can enhance Cd(II) immobilization and facilitate Cr(VI) reduction. The ferruginous hemeprotein HhuH exhibits the ability to chelate Cd(II) and reduce Cr(VI). The presence of Cd(II) and Cr(VI) in strain SY1 initially led to Fe starvation. Subsequently, the hemeprotein HhuH facilitated Cd(II) adsorption and Cr(VI) reduction, thereby restoring normal cellular Fe homeostasis. Our findings explain the hemeprotein-mediated mechanism for Cd(II) adsorption and Cr(VI) reduction, providing further insights into the correlation between heavy metal and Fe metabolism.IMPORTANCEIron (Fe) is an indispensable trace element for many organisms, and virtually, all bacteria require Fe as a cofactor in enzymes to facilitate redox reactions involved in fundamental cellular processes during periods of heavy metal stress. Understanding bacterial response to Fe in heavy metal contamination is essential. Therefore, our study elucidates Cd(II) adsorption and Cr(VI) reduction processes mediated by the Fe-bearing hemeprotein HhuH. It is a unique trifunctional protein capable of chelating Cd(II) and reducing Cr(VI), demonstrating significant potential in the environmental remediation of heavy metals.

Soil microbial communities are pivotal to plant health and nutrient acquisition. It is becoming increasingly clear that many interactions, both among and between microbes and plants, are governed by small bioactive molecules or "secondary metabolites" that can aid in communication, competition, and nutrient uptake. Yet, secondary metabolite biogeography - who makes what, where, and why-is in its infancy. Further, secondary metabolite biosynthesis genes are often silent or weakly expressed under standard laboratory conditions, making it incredibly difficult to study these small molecules. To begin to address these dual challenges, we focused on redox-active metabolites (RAMs), a specific class of small molecules, and took advantage of recent findings that many RAMs aid in acquiring phosphorus and that their production is frequently stimulated by stress for this macronutrient. We developed a screen for RAM-producing bacteria that leverages phosphorus limitation to stimulate metabolite biosynthesis and uses a colorimetric (ferrozine) iron-reduction assay to identify redox activity. We isolated 557 root-associated bacteria from grasses collected at sites across the United States (Santa Rita Experimental Range [AZ], Konza Prairie Biological Station [KS], and Harvard Forest [MA]) and from commercial tomato plants and screened them for RAM production. We identified 128 soil isolates of at least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes that produced RAMs under phosphorus stress. Our work reveals that the production of RAMs under phosphorus stress is common across diverse soil bacteria and provides an approach to screen for these small molecules rapidly.IMPORTANCEBy secreting secondary metabolites, bacteria at the plant root can defend against diseases and help acquire essential nutrients. However, the genes that synthesize secondary metabolites are typically inactive or are weakly expressed under standard laboratory conditions. This fact makes it difficult to study these small molecules and hinders the discovery of novel small molecules that may play crucial roles in agricultural and biomedical settings. Here, we focus on redox-active metabolites (RAMs), a class of secondary metabolites that can help bacteria solubilize phosphorus and are often produced when phosphorus is limited. We developed a screen that rapidly identifies RAM-producing bacteria by utilizing a colorimetric iron-reduction assay in combination with phosphorus limitation to stimulate biosynthesis. The screen reveals that RAM-producing bacteria are far more prevalent in soil than previously appreciated and that this approach can be used to identify RAM producers.

Bacterial cellulose (BC) is an extracellular polysaccharide produced by bacteria that has wide applications in the food industry, tissue engineering, and battery manufacturing. Genome editing of BC-producing Komagataeibacter species is expected to optimize BC production and its properties. However, the available technology can target only one gene at a time and requires foreign DNA templates, which may present a regulatory hurdle for genetically modified organisms. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-guided base editing method for Komagataeibacter species using Cas9 nickase and cytidine deaminase. Without foreign DNA templates, C-to-T conversions were performed within an 8 bp editing window with 90% efficiency. Double- and triple-gene editing was achieved with 80%-90% efficiency. Fusing uracil-DNA glycosylase with the base editor enabled C-to-G editing. The base editor worked efficiently with various Komagataeibacter species. Finally, mannitol metabolic genes were investigated using base-editing-mediated gene inactivation. This study provides a powerful tool for multiplex genome editing of Komagataeibacter species.

Importance: Komagataeibacter, a bacterial genus belonging to the family Acetobacteraceae, has important applications in food and material biosynthesis. However, the genome editing of Komagataeibacter relies on traditional homologous recombination methods. Therefore, only one gene can be manipulated in each round using foreign DNA templates, which may present a regulatory hurdle for genetically modified organisms when microorganisms are used in the food industry. In this study, a powerful base editing technology was developed for Komagataeibacter species. C-to-T and C-to-G base conversions were efficiently implemented at up to three loci in the Komagataeibacter genome. This base editing system is expected to accelerate basic and applied research on Komagataeibacter species.

Cronobacter sakazakii is a foodborne pathogen linked to severe infections in infants and often associated with contaminated powdered infant formula. The RecA protein, a key player in DNA repair and recombination, also influences bacterial resilience and virulence. This study investigated the impact of recA deletion on the pathogenicity and environmental stress tolerance of C. sakazakii BAA-894. A recA knockout mutant displayed impaired growth, desiccation tolerance, and biofilm formation. In a rat model, the mutant demonstrated significantly reduced virulence evidenced by higher host survival rates and lower bacterial loads in blood and tissues compared to the wild-type strain. Proteomic analysis revealed extensive disruptions in protein expression, particularly downregulation of carbohydrate metabolism and respiration-related proteins, alongside increased protein deamidation and oxidation. Functional assays identified fructose-bisphosphate aldolase as a target of oxidative and deamidative damage, resulting in reduced enzymatic activity and glycolytic disruption. These findings highlight the critical role of RecA in maintaining protein homeostasis, environmental resilience, and pathogenicity in C. sakazakii, providing valuable insights for developing targeted interventions against this pathogen.IMPORTANCECronobacter sakazakii poses significant risks due to its ability to persist in low-moisture environments and cause severe neonatal infections. This study identifies RecA as a key factor in environmental resilience and virulence, making it a promising target for mitigating infections and contamination. Inhibiting RecA function could sensitize C. sakazakii to stress during production and sterilization processes, reducing its persistence in powdered infant formula. Future research on RecA-specific inhibitors may lead to innovative strategies for enhancing food safety and preventing infections caused by this pathogen.