Applied and Environmental Microbiology最新文献

Veillonella spp. are nitrate-reducing bacteria with anaerobic respiratory activity that reduce nitrate to nitrite. They are obligate anaerobic, Gram-negative cocci that ferment lactate as the main carbon source and produce short-chain fatty acids (SCFAs). Commensal Veillonella reside in the human body site where lactate level is, however, limited for Veillonella growth. In this study, nitrate was shown to promote the anaerobic growth of Veillonella in the lactate-deficient media. We aimed to investigate the underlying mechanisms and the metabolism involved in nitrate respiration. Nitrate (15 mM) was demonstrated to promote Veillonella dispar growth and viability in the tryptone-yeast extract medium containing 0.5 mM L-lactate. Metabolite and transcriptomic analyses revealed nitrate enabled V. dispar to actively utilize glutamate and aspartate from the medium and secrete tryptophan. Glutamate or aspartate was further supplemented to a medium to investigate individual catabolism during nitrate respiration. Notably, nitrate was demonstrated to elevate SCFA production in the glutamate-supplemented medium, and further increase tryptophan production in the aspartate-supplemented medium. We proposed that the increased consumption of glutamate provided reducing power for nitrate respiration and aspartate served as a substrate for fumarate formation. Both glutamate and aspartate were incorporated into the central metabolic pathways via reverse tricarboxylic acid cycle and were linked with the increased production of acetate, propionate, and tryptophan. This study provides further understanding of the promoted growth and metabolic mechanisms by commensal V. dispar utilizing nitrate and specific amino acids to adapt to the lactate-deficient environment.IMPORTANCENitrate is a pivotal ecological factor influencing microbial community and metabolism. Dietary nitrate provides health benefits including anti-diabetic and anti-hypertensive effects via microbial-derived metabolites such as nitrite. Unraveling the impacts of nitrate on the growth and metabolism of human commensal bacteria is imperative to comprehend the intricate roles of nitrate in regulating microbial metabolism, community, and human health. Veillonella are lactate-utilizing, nitrate-reducing bacteria that are frequently found in the human body site where lactate levels are low and nitrate is at millimolar levels. Here, we comprehensively described the metabolic strategies employed by V. dispar to thrive in the lactate-deficient environment using nitrate respiration and catabolism of specific amino acids. The elevated production of SCFAs and tryptophan from amino acids during nitrate respiration of V. dispar further suggested the potential roles of nitrate and Veillonella in the promotion of human health.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia, and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus, Gordonia, and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene; Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons; and Nocardia neocaledoniensis (and many other Nocardia spp.) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.

Biofilm formation is a common adaptation enabling bacteria to thrive in various environments and withstand external pressures. In the context of host-microbe interactions, biofilms play vital roles in establishing microbiomes associated with animals and plants and are used by opportunistic microbes to facilitate survival within hosts. Investigating biofilm dynamics, composition, and responses to environmental stressors is crucial for understanding microbial community assembly and biofilm regulation in health and disease. In this study, we explore in vivo colonization and in vitro biofilm formation abilities of core members of the honey bee (Apis mellifera) gut microbiota. Additionally, we assess the impact of glyphosate, a widely used herbicide with antimicrobial properties, and a glyphosate-based herbicide formulation on growth and biofilm formation in bee gut symbionts as well as in other biofilm-forming bacteria associated with diverse animals and plants. Our results demonstrate that several strains of core bee gut bacterial species can colonize the bee gut, which probably depends on their ability to form biofilms. Furthermore, glyphosate exposure elicits variable effects on bacterial growth and biofilm formation. In some instances, the effects correlate with the bacteria's ability to encode a susceptible or tolerant version of the enzyme inhibited by glyphosate in the shikimate pathway. However, in other instances, no such correlation is observed. Testing the herbicide formulation further complicates comparisons, as results often diverge from glyphosate exposure alone, suggesting that co-formulants influence bacterial growth and biofilm formation. These findings highlight the nuanced impacts of environmental stressors on microbial biofilms, with both ecological and host health-related implications.

Importance: Biofilms are essential for microbial communities to establish and thrive in diverse environments. In the honey bee gut, the core microbiota member Snodgrassella alvi forms biofilms, potentially aiding the establishment of other members and promoting interactions with the host. In this study, we show that specific strains of other core members, including Bifidobacterium, Bombilactobacillus, Gilliamella, and Lactobacillus, also form biofilms in vitro. We then examine the impact of glyphosate, a widely used herbicide that can disrupt the bee microbiota, on bacterial growth and biofilm formation. Our findings demonstrate the diverse effects of glyphosate on biofilm formation, ranging from inhibition to enhancement, reflecting observations in other beneficial or pathogenic bacteria associated with animals and plants. Thus, glyphosate exposure may influence bacterial growth and biofilm formation, potentially shaping microbial establishment on host surfaces and impacting health outcomes.

Various environmental factors, including H₂ availability, metabolic tradeoffs, optimal growth temperature, stochasticity, and hydrology, were examined to determine if they affect microbial competition between three autotrophic thermophiles. The thiosulfate reducer Desulfurobacterium thermolithotrophum (T_opt72°C) was grown in mono- and coculture separately with the methanogens Methanocaldococcus jannaschii (T_opt82°C) at 72°C and Methanothermococcus thermolithotrophicus (T_opt65°C) at 65°C at high and low H₂ concentrations. Both methanogens showed a metabolic tradeoff shifting from high growth rate-low cell yield at high H₂ concentrations to low growth rate-high cell yield at low H₂ concentrations and when grown in coculture with the thiosulfate reducer. In 1:1 initial ratios, D. thermolithotrophum outcompeted both methanogens at high and low H₂, no H₂S was detected on low H₂, and it grew with only CO₂ as the electron acceptor indicating a similar metabolic tradeoff with low H₂. When the initial methanogen-to-thiosulfate reducer ratio varied from 1:1 to 10⁴:1 with high H₂, D. thermolithotrophum always outcompeted M. jannaschii at 72°C. However, M. thermolithotrophicus outcompeted D. thermolithotrophum at 65°C when the ratio was 10³:1. A reactive transport model that mixed pure hydrothermal fluid with cold seawater showed that hyperthermophilic methanogens dominated in systems where the residence time of the mixed fluid above 72°C was sufficiently high. With shorter residence times, thermophilic thiosulfate reducers dominated. If residence times increased with decreasing fluid temperature along the flow path, then thermophilic methanogens could dominate. Thermophilic methanogen dominance spread to previously thiosulfate-reducer-dominated conditions if the initial ratio of thermophilic methanogen-to-thiosulfate reducer increased.

Importance: The deep subsurface is the largest reservoir of microbial biomass on Earth and serves as an analog for life on the early Earth and extraterrestrial environments. Methanogenesis and sulfur reduction are among the more common chemolithoautotrophic metabolisms found in hot anoxic hydrothermal vent environments. Competition between H2-oxidizing sulfur reducers and methanogens is primarily driven by the thermodynamic favorability of redox reactions with the former outcompeting methanogens. This study demonstrated that competition between the hydrothermal vent chemolithoautotrophs Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Desulfurobacterium thermolithotrophum is also influenced by other overlapping factors such as staggered optimal growth temperatures, stochasticity, and hydrology. By modeling all a

The variation in the soil microbial community along the altitude gradient has been widely documented. However, the structure and function of the microbial communities distributed along the altitude gradient in the crater still need to be determined. We gathered soil specimens from different elevations within the Nushan volcano crater to bridge this knowledge gap. We investigated the microbial communities of bacteria and fungi in the soil. It is noteworthy that the microbial alpha diversity peaks in the middle of the crater. However, network analysis shows that bacterial (nodes 760 vs 613 vs 601) and fungal (nodes 328 vs 224 vs 400) communities are most stable at the bottom and top of the crater, respectively. Furthermore, the soil microbial network exhibited a decline, followed by an increase across varying altitudes. The core microorganisms displayed the highest correlation with pH and alkaline phosphatase (AP, as determined through redundancy analysis (RDA) and Mantel tests for correlation analysis. The fungal community has a higher number of core microorganisms, while the bacterial core microorganisms demonstrate greater susceptibility to environmental factors. In conclusion, we utilized Illumina sequencing techniques to assess the disparities in the structure and function of bacteria and fungi in the soil.IMPORTANCEThese findings serve as a foundation for future investigations on microbial communities present in volcanic soil.

Hyperosmotic stress tolerance is crucial for Saccharomyces cerevisiae in producing value-added products from renewable feedstock. The limited understanding of its tolerance mechanism has impeded the application of these microbial cell factories. Previous studies have shown that Med3 plays a role in hyperosmotic stress in S. cerevisiae. However, the specific function of Med3 in hyperosmotic stress tolerance remains unclear. In this study, we showed that the deletion of the mediator Med3 impairs S. cerevisiae growth under hyperosmotic stress. Phenotypic analyses and yeast two-hybrid assays revealed that Med3 interacts with the transcription factor Stb5 to regulate the expression of the genes gnd1 and ald6, which are involved in NADPH production under hyperosmotic stress conditions. The deletion of med3 resulted in a decrease in intracellular NADPH content, leading to increased oxidative stress and elevated levels of intracellular reactive oxygen species under hyperosmotic stress, thereby impacting bud formation. These findings highlight the significant role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.IMPORTANCEHyperosmotic stress tolerance in the host strain is a significant challenge for fermentation performance in industrial production. In this study, we showed that the S. cerevisiae mediator Med3 is essential for yeast growth under hyperosmotic conditions. Med3 interacts with the transcription factor Stb5 to regulate the expression of genes involved in the NADPH-generation system during hyperosmotic stress. Adequate NADPH ensures the timely removal of excess reactive oxygen species and supports bud formation under these conditions. This work highlights the crucial role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.

The continued emergence of antibiotic resistance among bacterial pathogens remains a significant challenge. Indeed, the enhanced antibiotic resistance profiles of contemporary pathogens often restrict the number of suitable molecular tools that are available. We have constructed a series of plasmids that confer resistance to two infrequently used antibiotics with variants of each plasmid backbone incorporating several regulatory control systems. The regulatory systems include both commonly used systems based on the lac- and arabinose-controlled promoters found in Escherichia coli, as well as less frequently used systems that respond to tetracycline/anhydrotetracycline and toluic acid. As a test case, we demonstrate the utility of these plasmids for regulated and tunable gene expression in a multidrug-resistant (MDR) isolate of Acinetobacter baumannii, strain AB5075-UW. The plasmids include derivatives of a freely replicating, broad-host-range plasmid allowing for inducible gene expression as well as a set of vectors for introducing genetic material at the highly conserved Tn7-attachment site. We also modified a set of CRISPR-interference plasmids for use in MDR organisms, thus allowing researchers to more readily interrogate essential genes in currently circulating clinical isolates. These tools will enhance molecular genetic analyses of bacterial pathogens in situations where existing plasmids cannot be used due to their antibiotic resistance determinants or lack of suitable regulatory control systems.

Importance: Clinical isolates of bacterial pathogens often harbor resistance to multiple antibiotics, with Acinetobacter baumannii being a prime example. The drug-resistance phenotypes associated with these pathogens represent a significant hurdle to researchers who wish to study modern isolates due to the limited availability of plasmid tools. Here, we present a series of freely replicating and Tn7-insertion vectors that rely on selectable markers to less frequently encountered antibiotics, apramycin, and hygromycin. We demonstrate the utility of these plasmid tools through a variety of experiments looking at a multidrug-resistant strain of A. baumannii, strain AB5075. Strain AB5075 is an established model strain for present-day A. baumannii, due in part to its genetic tractability and because it is a representative isolate of the globally disseminated multidrug-resistant clade of A. baumannii, global clone 1. In addition to the drug-selection markers facilitating use in strains resistant to more commonly used antibiotics, the vectors allow for controllable expression driven by several regulatory systems, including isopropyl β-D-1-thiogalactopyranoside (IPTG), arabinose, anhydrotetracycline, and toluic acid.

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid croplands competition between human food and animal feed. Agricultural co-products such as soybean meals have become important components of the circular economy thanks to their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet of monogastric animals, especially fungal carbohydrate-active enzymes (CAZymes). Here, we describe a time-course production and analysis of Aspergillus terreus secretomes for the identification of CAZymes able to enhance the digestibility of soybean meals. Functional assays revealed that the release of nutrients and the degradation of pectins in soybean meals can be tightly interconnected. Using a comparative proteomics approach, we identified several fungal pectin-degrading enzymes leading to increased assimilable nutrients in the soluble fraction of soybean meals. Our results reinforce the importance of deconstructing pectic polysaccharides in feedstuffs and contribute to sharpen our understanding of the fungal enzymatic interplays involved in pectin hydrolysis.IMPORTANCEIn the present study, we developed a strategy to identify the key fungal enzymatic activities involved in the improvement of soybean meal (SBM) digestibility. Our data unravel the importance of pectin degradation for the release of nutrients from SBM and provide some insights regarding the degradation of rhamnogalacturonan-I (RG-I) by ascomycetes. Indeed, the hydrolysis of pectins and RG-I by human microbiota is well documented in the literature, but our knowledge of the fungal CAZymes at play for the degradation of soybean pectins remains hitherto underexplored. Due to its wide use in animal feed, improving the digestibility of SBM by enzymatic treatments is a current challenge for feed additive suppliers. Since non-starch polysaccharides and pectins have often been reported for their anti-nutritional role in SBM, we believe this study will provide new avenues toward the improvement of enzymatic cocktails for animal nutrition and health.

With the rising demand for sustainable renewable resources, microorganisms capable of producing bioproducts such as bioplastics are attractive. While many bioproduction systems are well-studied in model organisms, investigating non-model organisms is essential to expand the field and utilize metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple non-sulfur bacterium capable of producing bioplastics. To increase bioplastic production, genes encoding the putative regulatory protein PhaR and the depolymerase PhaZ of the polyhydroxyalkanoate (PHA) biosynthesis pathway were deleted. Genes associated with pathways that might compete with PHA production, specifically those linked to glycogen production and nitrogen fixation, were deleted. Additionally, RuBisCO form I and II genes were integrated into TIE-1's genome by a phage integration system, developed in this study. Our results show that deletion of phaR increases PHA production when TIE-1 is grown photoheterotrophically with butyrate and ammonium chloride (NH₄Cl). Mutants unable to produce glycogen or fix nitrogen show increased PHA production under photoautotrophic growth with hydrogen and NH₄Cl. The most significant increase in PHA production was observed when RuBisCO form I and form I & II genes were overexpressed, five times under photoheterotrophy with butyrate, two times with hydrogen and NH₄Cl, and two times under photoelectrotrophic growth with N₂ . In summary, inserting copies of RuBisCO genes into the TIE-1 genome is a more effective strategy than deleting competing pathways to increase PHA production in TIE-1. The successful use of the phage integration system opens numerous opportunities for synthetic biology in TIE-1.IMPORTANCEOur planet has been burdened by pollution resulting from the extensive use of petroleum-derived plastics for the last few decades. Since the discovery of biodegradable plastic alternatives, concerted efforts have been made to enhance their bioproduction. The versatile microorganism Rhodopseudomonas palustris TIE-1 (TIE-1) stands out as a promising candidate for bioplastic synthesis, owing to its ability to use multiple electron sources, fix the greenhouse gas CO₂, and use light as an energy source. Two categories of strains were meticulously designed from the TIE-1 wild-type to augment the production of polyhydroxyalkanoate (PHA), one such bioplastic produced. The first group includes mutants carrying a deletion of the phaR or phaZ genes in the PHA pathway, and those lacking potential competitive carbon and energy sinks to the PHA pathway (namely, glycogen biosynthesis and nitrogen fixation). The second group comprises TIE-1 strains that overexpress RuBisCO form I or form I & II genes inserted via a phage integration system. By studying numerous metabolic mutants and overexpression strains, we conclude that genetic mod

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proB_m1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GK^D118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proB_m1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GK^D118N/D119N and γ-GK^D154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GK^D107N and γ-GK^E143A, the γ-GK^D118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.