Applied and Environmental Microbiology最新文献

The Gram-negative marine bacterium Vibrio anguillarum is able to cause vibriosis with hemorrhagic septicemia in many fish species, and iron acquisition is a critical step for virulence. Despite the fact that genes specific to certain processes of iron transport have been studied, the iron-regulated circuits of the V. anguillarum strains remain poorly understood. In this study, we showed that in V. anguillarum strain 775, iron could affect the expression of a number of critical metabolic pathways and virulence factors. The global iron uptake regulator VaFur is the major actor to control these processes for the bacterium to respond to different iron conditions. A VaFur binding motif was identified to distinguish directly and indirectly regulated targets. The absence of VaFur resulted in the aberrant expression of most iron acquisition determinants under rich-iron conditions. A similar regulation pattern was also observed in the transcription of genes coding for the type VI secretion system. The expression of peroxidase genes is positively controlled by VaFur to prevent iron toxicity, and the deletion of Vafur caused impaired growth in the presence of iron and H₂O₂. VaFur also upregulates some virulence factors under limited-iron conditions, including metalloprotease EmpA and motility, which are likely critical for the high virulence of V. anguillarum 775. The deletion of VaFur led to reduced swimming motility and decreased extracellular protease activity under limited-iron conditions, thereby leading to attenuated pathogenicity. Our study provides more evidence to better understand the VaFur regulon and its role in the pathogenesis of V. anguillarum.IMPORTANCEVibriosis, the most common disease caused by marine bacteria belonging to the genus Vibrio, leads to massive mortality of economical aquatic organisms in Asia. Iron is one of the most important trace elements, and its acquisition is a critical battle occurring between the host and the pathogen. However, excess iron is harmful to cells, so iron utilization needs to be strictly controlled to adapt to different conditions. This process is mediated by the global iron uptake regulator Fur, which acts as a repressor when iron is replete. On the other hand, free iron in the host is limited, so the reduced virulence of the Δfur mutant should not be directly caused by abnormally regulated iron uptake. The significance of this work lies in uncovering the mechanism by which the deletion of Fur causes reduced virulence in Vibrio anguillarum and identifying the critical virulence factors that function under limited-iron conditions.

Antimicrobial resistance is a global public and animal health concern. Antimicrobial resistance genes (ARGs) have been detected in dairy farm environments globally; however, few longitudinal studies have utilized shotgun metagenomics for ARG surveillance in pasture-based systems. This 15-month study aimed to undertake a baseline survey using shotgun metagenomics to assess the relative abundance and diversity of ARGs in two pasture-based dairy farm environments in New Zealand with different management practices. There was no statistically significant difference in overall ARG relative abundance between the two dairy farms (P = 0.321) during the study period. Compared with overseas data, the relative abundance of ARG copies per 16S rRNA gene in feces (0.08-0.17), effluent (0.03-0.37), soil (0.20-0.63), and bulk tank milk (0.0-0.12) samples was low. Models comparing the presence or absence of resistance classes found in >10% of all feces, effluent, and soil samples demonstrated no statistically significant associations (P > 0.05) with "season," and only multi-metal (P = 0.020) and tetracycline (P = 0.0003) resistance were significant at the "farm" level. Effluent samples harbored the most diverse ARGs, some with a recognized public health risk, whereas soil samples had the highest ARG relative abundance but without recognized health risks. This highlights the importance of considering the genomic context and risk of ARGs in metagenomic data sets. This study suggests that antimicrobial resistance on pasture-based dairy farms is low and provides essential baseline ARG surveillance data for such farming systems.IMPORTANCEAntimicrobial resistance is a global threat to human and animal health. Despite the detection of antimicrobial resistance genes (ARGs) in dairy farm environments globally, longitudinal surveillance in pasture-based systems remains limited. This study assessed the relative abundance and diversity of ARGs in two New Zealand dairy farms with different management practices and provided important baseline ARG surveillance data on pasture-based dairy farms. The overall ARG relative abundance on these two farms was low, which provides further evidence for consumers of the safety of New Zealand's export products. Effluent samples harbored the most diverse range of ARGs, some of which were classified with a recognized risk to public health, whereas soil samples had the highest ARG relative abundance; however, the soil ARGs were not classified with a recognized public health risk. This emphasizes the need to consider genomic context and risk as well as ARG relative abundance in resistome studies.

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.

Horizontal gene transfer (HGT) is a major process by which genes are transferred between microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant-associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provide the first demonstration of gene transfer via a triparental conjugation system in the Brachypodium distachyon rhizosphere in an EcoFAB using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor containing a mobilizable and non-self-transmissible plasmid. We observed that the frequency of plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage and the composition and amount of root exudates. The frequency of plasmid transfer also increased with higher numbers of donor cells. We demonstrate the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. OAS925, showing HGT within a rhizosphere microbial community. Environmental stresses also influenced the rate and efficiency of HGT in the rhizosphere between different species and genera. This study provides a robust workflow to evaluate transfer of engineered plasmids in the rhizosphere when such plasmids are potentially introduced in a field or other plant-associated environments.IMPORTANCEWe report the use of EcoFABs to investigate the HGT process in a rhizosphere environment. It highlights the potential of EcoFABs in recapitulating the dynamic rhizosphere conditions as well as their versatility in studying plant-microbe interactions. This study also emphasizes the importance of studying the parameters impacting the HGT frequency. Several factors such as plant developmental stages, nutrient conditions, number of donor cells, and environmental stresses influence gene transfer within the rhizosphere microbial community. This study paves the way for future investigations into understanding the fate and movement of engineered plasmids in a field environment.

Mammalian alkaline phosphatase (AP) is widely used in diagnostics and molecular biology but its widespread use is impaired because it is difficult to express in Escherichia coli and has low thermostability. To overcome these challenges, we employed sequence-based protein redesign methods, specifically full consensus design (FCD) and ancestral sequence reconstruction (ASR), to create APs with enhanced properties. Biochemical analyses revealed that these newly designed APs exhibited improved levels of expression in their active form and increased thermostability compared to bovine intestinal AP isozyme II (bIAPII), without impeding enzymatic activity. Notably, the activity in culture broth of the designed APs was an order of magnitude higher than that of bIAPII, and their thermal stability increased by 13°C-17°C (measured as T₅₀). We also assessed 28 single-point mutants of bIAPII to identify regions influencing thermostability and expression level; these mutations were common in the engineered APs but not in bIAPII. Specific mutations, such as T413E and G402S, enhanced thermostability, whereas increasing the expression level required multiple mutations. This suggests that a synergistic effect is required to enhance the expression level. Mutations enhancing thermostability were located in the crown domain, while those improving expression were close to the dimer interface, indicating distinct mechanisms underpinning these enhancements.

Importance: Sequence-based protein redesign methods, such as full consensus design (FCD) and ancestral sequence reconstruction (ASR), have the potential to construct new enzymes utilizing protein sequence data registered in a rapidly expanding sequence database. The high thermostability of these enzymes would expand their application in diagnostics and molecular biology. These enzymes have also demonstrated a high level of active expression by Escherichia coli. These characteristics make these APs attractive candidates for industrial application. In addition, different amino acid residues are primarily responsible for thermal stability and active expression, suggesting important implications for strategies for designing enzymes by FCD and ASR.

As a response regulator of the EsrA-EsrB two-component system, EsrB is conserved in Hafniaceae and plays a crucial role in virulence and pathogenicity. EsrB possesses DNA binding abilities, enabling it to regulate the transcription of virulence genes to confront different stresses and achieve systematic infections. Here, ChIP-seq analysis of EsrB in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments) revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7'' pseudopalindromic DNA motif and interact with metabolic-related promoters with a high AT DNA motif. The crystal structure of the C-terminal of EsrB (EsrB_C) was solved at 2.20-Å resolution. Specifically, Lys¹⁸¹ enabled the DNA-binding affinity of EsrB and promoted the in vitro and in vivo pathogenicity of Edwardsiella piscicida. Moreover, EsrB directly regulated the expression of genes associated with basal metabolism, including iron and tricarboxylic acid (TCA) cycles. Furthermore, EsrB enhanced iron transport capability and the enzyme activity of succinate dehydrogenase and pyruvate dehydrogenase in DMEM. Collectively, our structural and ChIP-seq analysis provides valuable insights into the DNA binding mechanism of EsrB which will facilitate our understanding of EsrB coordinating virulence and basal metabolism gene expression.

Importance: As a crucial virulence regulator, EsrB possesses a LuxR-like superfamily domain at the C-terminal, which is conserved within the canonical NarL family regulators. Due to its critically important role in virulence and pathogenicity in fish hosts, the DNA binding ability has been believed to allow EsrB to regulate genes associated with the invasion process of host cells and basal metabolism in response to environmental stimuli. The lack of EsrB's crystal structure has been a major obstacle in understanding the molecular mechanisms of EsrB-DNA interaction which choreographs EsrB-mediated pathogenic behavior. Here, we conducted ChIP-seq and solved the crystal structure of the C-terminal of EsrB (EsrBC) at 2.20-Å resolution, which revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7' pseudopalindromic DNA motif and interacted with metabolic-related promoters with a high AT DNA motif in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments). Our results facilitate a detailed understanding of EsrB's regulatory role in Edwardsiella piscicida pathogenesis and expand our knowledge of virulence regulators in the family Hafniaceae.

The Atacama Desert in Chile is one of the driest and most inhospitable places on Earth. To analyze the diversity and distribution of microbial communities in such an environment, one of the most important and challenging steps is DNA extraction. Using commercial environmental DNA extraction protocols, a mixture of living, dormant, and dead cells of microorganisms is extracted, but separation of the different DNA pools is almost impossible. To overcome this problem, we applied a novel method on soils across a west-east moisture transect in the Atacama Desert to distinguish between extracellular DNA (eDNA) and intracellular DNA (iDNA) at the cell extraction level. Here, we show that a large number of living and potentially active microorganisms, such as Acidimicrobiia, Geodermatophilaceae, Frankiales, and Burkholderiaceae, occur in the hyperarid areas. We observed viable microorganisms involved as pioneers in initial soil formation processes, such as carbon and nitrogen fixation, as well as mineral-weathering processes. In response to various environmental stressors, microbes coexist as generalists or specialists in the desert soil environment. Our results show that specialists compete in a limited range of niches, while generalists tolerate a wider range of environmental conditions. Use of the DNA separation approach can provide new insights into different roles within viable microbial communities, especially in low-biomass environments where RNA-based analyses often fail.IMPORTANCEThe novel e- and iDNA separation technique offers insights into the living community at the cell extraction level in the hyperarid Atacama Desert. This approach provides a new framework for analyzing the composition and structure of the potentially active part of the microbial communities as well as their specialization, ecological network and community assembly process. Our findings underscore the significance of utilizing alternative genomic techniques in low-biomass environments where traditional DNA- and RNA-based analyses may not be feasible. The results demonstrate the viability of the proposed study framework and show that specialized microorganisms are important in initial soil formation processes, including microbial-driven mineral weathering, as well as the fixation of carbon and nitrogen.

Acetic acid is a byproduct of lignocellulose pretreatment and a potent inhibitor of yeast-based fermentation processes. A thicker yeast plasma membrane (PM) is expected to retard the passive diffusion of undissociated acetic acid into the cell. Molecular dynamic simulations suggest that membrane thickness can be increased by elongating glycerophospholipids (GPL) fatty acyl chains. Previously, we successfully engineered Saccharomyces cerevisiae to increase GPL fatty acyl chain length but failed to lower acetic acid net uptake. Here, we tested whether altering the relative abundance of diacylglycerol (DAG) might affect PM permeability to acetic acid in cells with longer GPL acyl chains (DAG^EN). To this end, we expressed diacylglycerol kinase α (DGKα) in DAG^EN. The resulting DAG^EN_Dgkα strain exhibited restored DAG levels, grew in medium containing 13 g/L acetic acid, and accumulated less acetic acid. Acetic acid stress and energy burden were accompanied by increased glucose uptake in DAG^EN_Dgkα cells. Compared to DAG^EN, the relative abundance of several membrane lipids changed in DAG^EN_Dgkα in response to acetic acid stress. We propose that the ability to increase the energy supply and alter membrane lipid composition could compensate for the negative effect of high net acetic acid uptake in DAG^EN_Dgkα under stressful conditions.

Importance: In the present study, we successfully engineered a yeast strain that could grow under high acetic acid stress by regulating its diacylglycerol metabolism. We compared how the plasma membrane and total cell membranes responded to acetic acid by adjusting their lipid content. By combining physiological and lipidomics analyses in cells cultivated in the absence or presence of acetic acid, we found that the capacity of the membrane to adapt lipid composition together with sufficient energy supply influenced membrane properties in response to stress. We suggest that potentiating the intracellular energy system or enhancing lipid transport to destination membranes should be taken into account when designing membrane engineering strategies. The findings highlight new directions for future yeast cell factory engineering.

Wastewater surveillance for infectious agents has proved useful in identifying the circulation of viruses within populations. We investigated the presence and concentration of human immunodeficiency virus (HIV)-1 total nucleic acids (including both viral RNA and proviral DNA) in wastewater solids. We retrospectively measured HIV-1 nucleic acids in two samples per week for 26 months at two wastewater treatment plants serving populations with different prevalences of HIV infections in San Francisco and Santa Clara County, California, USA. We detected HIV nucleic acids in a majority of samples with concentrations ranging from non-detect to 3.9 × 10⁵ cp/g (N = 459 samples total). Concentrations of HIV-1 were significantly higher in samples from the wastewater treatment plant serving a population with a higher prevalence of people living with HIV than in the plant serving a population with a lower prevalence. The HIV-1 nucleic acids amplified were primarily DNA and thus represented proviral DNA shedding into wastewater. Additionally, we found that HIV-1 nucleic acid concentrations in wastewater solids were orders of magnitude higher than those in liquid wastewater indicating that the HIV-1 target preferentially sorbs to solids. Whether concentrations of HIV-1 in wastewater solids can be used to identify the number of incident cases remains unknown. Additional work on HIV-1 shedding from individuals with viremia and people living with HIV is needed to translate wastewater measurements into quantitative information on infections. Additional work may also be needed to document non-human sources of HIV-1 nucleic acids in wastewater.

Importance: Human immunodeficiency virus (HIV)-1 has infected nearly 100 million people since it emerged in the 1980s. Antiretroviral therapy prevents transmission of HIV and also allows infected individuals to live healthy lives with normal life expectancy. Consequently, identifying unrecognized cases of HIV is of paramount importance. Since wastewater represents a composite biological sample from a community, it may provide valuable information on HIV-1 prevalence that can be used to inform HIV testing and outreach.

Biological nitrogen fixation (BNF) is an essential source of new nitrogen (N) for terrestrial ecosystems. The abiotic factors regulating BNF have been extensively studied in various ecosystems and laboratory settings. Despite this, our understanding of the impact of neighboring bacteria on N₂ fixer activity remains limited. Here, we explored this question using a co-culture of the two model species: the free-living diazotroph Azotobacter vinelandii and the non-fixing plant growth-promoting rhizobacteria Bacillus subtilis. We observed that the interaction between the two bacteria was modulated by N availability. Under N-replete conditions, B. subtilis outcompeted A. vinelandii in the co-culture. Under N-limiting conditions, BNF activity by A. vinelandii was enhanced in the presence of B. subtilis. Reciprocally, the presence of A. vinelandii repressed sporulation by B. subtilis and supported its growth likely through N transfer. N inputs by A. vinelandii were doubled in the presence of B. subtilis compared to the monoculture, primarily due to the retention of a robust N₂ fixation activity in the stationary phase. A proteomic analysis revealed that A. vinelandii N metabolism, particularly the molybdenum nitrogenase isoform protein levels (NifK and NifD), was upregulated during the stationary growth phase in the presence of B. subtilis. This study revealed that N stress drives bacterial interactions and activity in a two-species community, especially in the stationary phase.

Importance: Reducing inputs of chemical N fertilizers is essential to develop a more sustainable agriculture. The stimulation of biological nitrogen fixation by N2 fixers in multispecies cultures, here the plant growth-promoting rhizobacteria Azotobacter vinelandii and Bacillus subtilis, opens opportunities for the formulation of biofertilizers consortia. While most research on N2 fixation historically focussed on the exponential growth phase of microorganisms, we observed that Bacillus subtilis stimulated Azotobacter vinelandii N2 fixation mostly during the stationary phase. This result highlights that more research on the factors controlling N2 fixation repression during the stationary growth phase, especially bacteria-bacteria interactions, is eagerly needed.