Applied and Environmental Microbiology最新文献

Lignin contains a variety of interunit linkages, leading to a range of potential decomposition products that can be used as carbon and energy sources by microbes. β-O-4 linkages are the most common in native lignin, and associated catabolic pathways have been well characterized. However, the fate of the mono-aromatic intermediates that result from β-O-4 dimer cleavage has not been fully elucidated. Here, we used experimental evolution to identify mutant strains of Novosphingobium aromaticivorans with improved catabolism of a model aromatic dimer containing a β-O-4 linkage, guaiacylglycerol-β-guaiacyl ether (GGE). We identified several parallel causal mutations, including a single nucleotide polymorphism in the promoter of an uncharacterized gene that roughly doubled the growth yield with GGE. We characterized the associated enzyme and demonstrated that it oxidizes an intermediate in GGE catabolism, β-hydroxypropiovanillone, to vanilloyl acetaldehyde. Identification of this enzyme and its key role in GGE catabolism furthers our understanding of catabolic pathways for lignin-derived aromatic compounds.IMPORTANCELignin degradation is a key step for both carbon cycling in nature and biomass conversion to fuels and chemicals. Bacteria can catabolize lignin-derived aromatic compounds, but the complexity of lignin means that full mineralization requires numerous catabolic pathways and often results in slow growth. Using experimental evolution, we identified an uncharacterized enzyme for the catabolism of a lignin-derived aromatic monomer, β-hydroxypropiovanillone. A single nucleotide polymorphism in the promoter of the associated gene significantly increased bacterial growth with either β-hydroxypropiovanillone or a related lignin-derived aromatic dimer. This work expands the repertoire of known aromatic catabolic genes and demonstrates that slow catabolism of lignin-derived aromatic compounds may be due to misregulation under laboratory conditions rather than inherent catabolic challenges.

Vitamin B₁₂ (cobalamin, herein B₁₂) is a key cofactor for most organisms being involved in essential metabolic processes. In microbial communities, B₁₂ is often scarce, largely because only few prokaryotes can synthesize B₁₂ de novo and are thus considered B₁₂-prototrophs. B₁₂-auxotrophy is mostly manifested by the absence of the B₁₂-independent methionine synthase, MetE. Here, we focus on bacteria that we classified as facultative B₁₂ consumers as they encode both B₁₂-independent and -dependent (MetH) methionine synthases yet largely cannot synthesize B₁₂ de novo. The genus Vibrio belongs to this group, and our work shows that upon B₁₂ supply growth of Vibrio campbellii is accelerated and autoinducer-2 (AI-2) concentrations are enhanced. We speculate that methionine synthesis efficiency, dependent on B₁₂ availability, is linked to AI-2 synthesis. The precursor for AI-2 synthesis is S-adenosyl-L-homocysteine (SAH), which in turn requires methionine as a precursor. In almost all Vibrio species studied, btuF (B₁₂-binding protein), which is required for B₁₂ uptake, and cobD (Adenosylcobinamide-phosphate synthase), which enables remodeling of B₁₂-like compounds, are encoded on the same operon as pfs (or mtnN, Adenosylhomocysteine nucleosidase), the first enzyme in the two-step AI-2 synthesis reaction. Transcriptomic analyses show that virulence factors, such as toxin synthesis, fimbriae formation, and activation of the type-6 secretion system, which have been shown to be regulated by quorum sensing via AI-2, are significantly upregulated in V. campbellii when B₁₂ is available. Our results demonstrate that V. campbellii is a facultative B₁₂ consumer and indicate that B₁₂ availability affects AI-2 levels and thus potentially virulence factor regulation.IMPORTANCEMetabolites play a key role in microbial metabolism and communication. While vitamin B₁₂ is an essential cofactor for important enzymatic reactions, autoinducer-2 mediates interspecies signaling and can regulate the expression of genes that are crucial for virulence and survival. In our study, we hypothesize and present findings how these two important metabolites are linked in Vibrio species. Vibrio campbellii grows without B₁₂ but at an accelerated rate when B₁₂ is present, and we detect higher AI-2 values in cultures with B₁₂ amendment. Transcriptome analyses show how vitamin B₁₂ availability significantly upregulates gene expression of virulence factors such as toxin synthesis, fimbrial formation, and activation of the type-6 secretion system in V. campbellii.

Fish gut microbial communities are important for the breakdown and energy harvesting of the host diet. Microbes within the fish gut are selected by environmental and evolutionary factors. To understand how fish gut microbial communities are shaped by diet, three tropical fish species (hawkfish, Paracirrhites arcatus; yellow tang, Zebrasoma flavescens; and triggerfish, Rhinecanthus aculeatus) were fed piscivorous (fish meal pellets), herbivorous (seaweed), and invertivorous (shrimp) diets, respectively. From fecal samples, a total of 43 metagenome assembled genomes (MAGs) were recovered from all fish diet treatments. Each host-diet treatment harbored distinct microbial communities based on taxonomy, with Proteobacteria, Bacteroidota, and Firmicutes being the most represented. Based on their metagenomes, MAGs from all three host-diet treatments demonstrated a baseline ability to degrade proteinaceous, fatty acid, and simple carbohydrate inputs and carry out central carbon metabolism, lactate and formate fermentation, acetogenesis, nitrate respiration, and B vitamin synthesis. The herbivorous yellow tang harbored more functionally diverse MAGs with some complex polysaccharide degradation specialists, while the piscivorous hawkfish's MAGs were more specialized for the degradation of proteins. The invertivorous triggerfish's gut MAGs lacked many carbohydrate-degrading capabilities, resulting in them being more specialized and functionally uniform. Across all treatments, several MAGs were able to participate in only individual steps of the degradation of complex polysaccharides, suggestive of microbial community networks that degrade complex inputs.

Importance: The benefits of healthy microbiomes for vertebrate hosts include the breakdown of food into more readily usable forms and production of essential vitamins from their host's diet. Compositions of microbial communities in the guts of fish in response to diet have been studied, but there is a lack of a comprehensive understanding of the genome-based metabolic capabilities of specific microbes and how they support their hosts. Therefore, we assembled genomes of several gut microbes collected from the feces of three fish species that were being fed different diets to illustrate how individual microbes can carry out specific steps in the degradation and energy utilization of various food inputs and support their host. We found evidence that fish gut microbial communities share several core functions despite differences in microbial taxonomy. Herbivorous fish harbored a functionally diverse microbial community with plant matter degraders, while the piscivorous and invertivorous fish had microbiomes more specialized in protein degradation.

As a diverse and complex food matrix, the animal food microbiota and repertoire of antimicrobial resistance (AMR) genes remain to be better understood. In this study, 16S rRNA gene amplicon sequencing and shotgun metagenomics were applied to three types of animal food samples (cattle feed, dry dog food, and poultry feed). ZymoBIOMICS mock microbial community was used for workflow optimization including DNA extraction kits and bead-beating conditions. The four DNA extraction kits (AllPrep PowerViral DNA/RNA Kit, DNeasy Blood & Tissue Kit, DNeasy PowerSoil Kit, and ZymoBIOMICS DNA Miniprep Kit) were compared in animal food as well as the use of peptide nucleic acid blockers for 16S rRNA gene amplicon sequencing. Distinct microbial community profiles were generated, which varied by animal food type and DNA extraction kit. Employing peptide nucleic acid blockers prior to 16S rRNA gene amplicon sequencing was comparable with post-sequencing in silico filtering at removing chloroplast and mitochondrial sequences. There was a good agreement between 16S rRNA gene amplicon sequencing and shotgun metagenomics on community profiles in animal feed data sets; however, they differed in taxonomic resolution, with the latter superior at resolving at the species level. Although the overall prevalence of AMR genes was low, resistome analysis of animal feed data sets by shotgun metagenomics revealed 10 AMR gene/protein families, including beta-lactamases, erythromycin/lincomycin/pristinamycin/tylosin, fosfomycin, phenicol, and quinolone. Future expansion of microbiome and resistome profiling in animal food will help better understand the bacterial and AMR gene diversity in these commodities and help guide pathogen control and AMR prevention efforts.IMPORTANCEWith the growing interest and application of metagenomics in understanding the structure/composition and function of diverse microbial communities along the One Health continuum, this study represents one of the first attempts to employ these advanced sequencing technologies to characterize the microbiota and AMR genes in animal food. We unraveled the effects of DNA extraction kits on sample analysis by 16S rRNA gene amplicon sequencing and showed similar efficacies of two strategies at removing chloroplast and mitochondrial reads. The in-depth analysis using shotgun metagenomics shed light on the community compositions and the presence of an array of AMR genes in animal food. This exploration of microbiomes and resistomes in representative animal food samples by both sequencing approaches laid important groundwork for future metagenomic investigations to gain a better understanding of the baseline/core microbiomes and associated AMR functions in these diverse commodities and help guide pathogen control and AMR prevention efforts.

The bacterium Wolbachia pipientis is increasingly studied for its potential use in controlling insect vectors or pests due to its ability to induce Cytoplasmic Incompatibility (CI). CI can be exploited by establishing an opportunistic Wolbachia infection in a targeted insect species through trans-infection and then releasing the infected males into the environment as sterilizing agents. Several host life history traits (LHT) have been reported to be negatively affected by artificial Wolbachia infection. Wolbachia is often considered the causative agent of these detrimental effects, and the importance of the host's genetic origins in the outcome of trans-infection is generally overlooked. In this study, we investigated the impact of host genetic background using an Aedes albopictus line recently trans-infected with wPip from the Culex pipiens mosquito, which exhibited some fitness costs. We measured several LHTs including fecundity, egg hatch rate, and male mating competitiveness in the incompatible line after four rounds of introgression aiming at restoring genetic diversity in the nuclear genome. Our results show that introgression with a wild genetic background restored most fitness traits and conferred mating competitiveness comparable to that of wild males. Finally, we show that introgression leads to faster and stronger population suppression under laboratory conditions. Overall, our data support that the host genome plays a decisive role in determining the fitness of Wolbachia-infected incompatible males.IMPORTANCEThe bacterium Wolbachia pipientis is increasingly used to control insect vectors and pests through the Incompatible Insect Technique (IIT) inducing a form of conditional sterility when a Wolbachia-infected male mates with an uninfected or differently infected female. Wolbachia artificial trans-infection has been repeatedly reported to affect mosquitoes LHTs, which may in turn compromise the efficiency of IIT. Using a tiger mosquito (Aedes albopictus) line recently trans-infected with a Wolbachia strain from Culex pipiens and displaying reduced fitness, we show that restoring genetic diversity through introgression significantly mitigated the fitness costs associated with Wolbachia trans-infection. This was further demonstrated through experimental population suppression, showing that introgression is required to achieve mosquito population suppression under laboratory conditions. These findings are significant for the implementation of IIT programs, as an increase in female fecundity and male performance improves mass rearing productivity as well as the sterilizing capacity of released males.

Streptomyces fradiae is an important bioresource to produce various antibacterial natural products, however, the time-consuming and labor-intensive genome editing toolkits hindered the construction and application of engineered strains, and this study aimed to establish an efficient CRISPR/Cas9n genome editing system in S. fradiae. Initially, the CRISPR/Cas9-mediated editing tool was employed to replace those awkward genome editing tools that relied on homologous recombination, while the off-target Cas9 exhibited high toxicity to S. fradiae Sf01. Therefore, the nickase mutation D10A, high-fidelity mutations including N497A, R661A, Q695A, and Q926A, and thiostrepton-induced promotor P_tipA were incorporated into the Cas9 expression cassette, which reduced its toxicity. The deletion of single gene neoI and long fragment sequence (13.3 kb) were achieved with efficiencies of 77.8% and 44%, respectively. Additionally, the established tool was applied to facilitate the rapid deletion of nagB, replacement of P_frr with P_ermE*, and integration of exogenous vgbS, with respective efficiencies of 77.8%, 100%, and 67.8%, and all of the above modification strategies benefited neomycin synthesis in S. fradiae. Taken together, this research established an efficient CRISPR/Cas9n-mediated genome editing toolkit in S. fradiae, paving the way for developing high-performance neomycin-producing strains and facilitating the genetic modification of Streptomyces.IMPORTANCEThis study describes the development and application of a genome editing system mediated by CRISPR/Cas9n in Streptomyces fradiae for the first time, which overcomes the challenges associated with genome editing caused by high GC content (74.5%) coupling with complex genome structure, and reduces the negative impact of "off-target effect." Our work not only provides a facile editing tool for constructing S. fradiae strains of high-yield neomycin but also offers the technical guidance for the design of a CRISPR/Cas9n mediated genome editing tool in those creatures with high GC content genomes.

Microorganisms adapted to high hydrostatic pressures at depth in the oceans and within the subsurface of Earth's crust represent a phylogenetically diverse community thriving under extreme pressure, temperature, and nutrient availability conditions. To better understand the microbial function, physiological responses, and metabolic strategies at in-situ conditions requires high-pressure (HP) continuous culturing techniques that, although commonly used in bioengineering and biotechnology applications, remain relatively rare in the study of the Earth's microbiomes. Here, we focus on recent developments in the design of HP chemostats, with particular emphasis on adaptations for delivery and sampling of dissolved gases. We present protocols for sterilization, inoculation, agitation, and sampling strategies that minimize cell lysis, applicable to a wide range of chemostat designs.

The SpoVAF/FigP complex, a newly identified dormant spore ion channel, has been shown to amplify the response of germinant receptors (GRs) to nutrient germinants. However, its contribution to high-pressure-induced germination remains unexplored. In this study, we discovered that the 5AF/FigP complex played an important role in the GR-dependent germination of Bacillus subtilis spores under moderate high pressure (MHP) by facilitating the release of ions, such as potassium (K⁺), a mechanism in parallel with its role in nutrient-induced germination. Despite its predicted function as an ion channel, the 5AF/FigP complex failed to be activated by MHP in the absence of GerA-type GRs. We quantitatively examined the factors that influence the 5AF/FigP complex's function in MHP-induced germination using modeling and fitting techniques. Our results indicated that the complex's amplification effect was both enhanced and accelerated as pressure levels increase from 50 to 200 MPa. However, raising the MHP treatment temperature from 22°C to 30°C only speeded up the complex's function without enhancing its effectiveness. Moreover, extreme conditions of higher pressure (300 MPa) and temperature (34°C-37°C) could diminish the complex's functionality. Additionally, the amplification effect was weakened in spores produced at both elevated and reduced sporulation temperatures. Taken together, our findings highlight the essential role of the 5AF/FigP complex in boosting the efficiency of MHP-induced germination. This revelation has enriched our understanding of the intricate mechanisms underlying GR-dependent germination in Bacillus spores, offering valuable insights that can be utilized to refine the germination-inactivation strategies within the food industry.

Importance: High-pressure-induced spore germination has been discovered for more than half a century, but the signal transduction pathway of the process still needs to be refined. In this study, for the first time, we revealed the role of the newly identified SpoVAF/FigP complex in high-pressure-induced spore germination, as well as the factors influencing its function in this process. The new findings in this work not only enhance the theoretical understanding of spore germination mechanisms under high pressure but also pave the way for developing novel strategies to inactivate spores during high-pressure food processing, a technology that is gaining popularity in the food industry as a promising non-thermal preservation method.

Human enteric viruses can remain infective in surface waters for extended periods of time, posing a public health risk. Microbial activity contributes to the inactivation of waterborne enteric viruses, but while individual bacteria-virus interactions have been characterized, the importance of microbial diversity remains unknown. Here, we experimentally manipulated the diversity of bacterial communities from Lake Geneva across three seasons using a dilution-to-extinction approach and monitored the inactivation and genome decay of echovirus 11, a member of the Enterovirus genus. Long-read sequencing of the 16S rRNA gene revealed diversity gradients ranging between 373 and 2,722 bacterial species. Compared to sterile controls, echovirus 11 inactivation was enhanced by the presence of active bacteria and depended both on season and sample dilution. Throughout all seasons, the highest inactivation (between 3.0 and 7.9 log₁₀ fold reduction in infectivity over 96 h) was observed in the least diluted incubations (i.e., the highest bacterial richness). Genome decay exhibited a 24-h lag and was less pronounced than the corresponding infectivity loss (ranging between 2.3 and 3.8 log₁₀ fold over 96 h), indicating that microbial inactivation primarily targets the echovirus 11 capsid. We found a positive-saturating relationship between bacterial species richness and viral inactivation, suggesting functional redundancy and pointing toward the importance of rare species for viral inactivation. Biomarker analysis revealed several clades of bacteria, particularly members of Chitinophagaceae, to be significantly associated with echovirus 11 inactivation. Overall, these findings suggest that high microbial diversity enhances the capacity of surface waters to rid themselves of contamination by enteric viruses and hence protects public health.IMPORTANCEHuman enteric viruses in natural waterbodies pose a public health risk. Microorganisms, particularly bacteria, contribute to the inactivation of enteroviruses, thereby mitigating this risk. We use experimental manipulations of lake water bacterial diversity to unravel the importance of diversity for the inactivation of echovirus 11, a model human pathogen. Our findings suggest that bacterial diversity is important for echovirus 11 inactivation and that specific, but numerically rare, bacteria present in the surface water of Lake Geneva across different seasons contribute to viral inactivation. These findings contribute to our understanding of the inactivation of human enteric viruses in natural waterbodies-a hitherto understudied ecosystem service.

Cattle and other domestic ruminants are the primary reservoirs of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC). Living in areas with high ruminant density has been associated with excess risk of infection, which could be due to both direct ruminant contact and residual environmental risk, but the role of each is unclear. We investigated whether there is any meaningful risk to individuals living in ruminant-dense areas if they do not have direct contact with ruminants. Using a Bayesian spatial framework, we investigated the association between the density of ruminants on feedlots and STEC incidence in Minnesota from 2010 to 2019, stratified by serogroup and season, and adjusting for direct ruminant contact. For every additional head of cattle or sheep per 10 acres, the incidence of O157 STEC infection increased by 30% (incidence rate ratio [IRR] 1.30; 95% credible interval [CrI] 1.18, 1.42) or 135% (IRR 2.35; 95% CrI 1.14, 4.20), respectively, during the summer months. Sheep density was also associated with O157 STEC risk during winter (IRR 4.28; 95% CrI 1.40, 8.92). The risk of non-O157 STEC infection was only elevated in areas with goat operations during summer (IRR 19.6; 95% CrI 1.69, 78.8). STEC risk associated with ruminant density was independent of direct ruminant contact across serogroups and seasons. Our findings demonstrate that living in a ruminant-dense area increases an individual's risk of O157 and non-O157 STEC infection even without direct ruminant contact, indicating that prevention efforts need to extend to community strategies for averting indirect transmission from local ruminant populations.IMPORTANCEShiga toxin-producing Escherichia coli (STEC) are zoonotic enteric bacteria responsible for 2.5 million illnesses each year. Infections in young children can be especially devastating, causing hemolytic uremic syndrome (HUS), a debilitating and sometimes fatal form of acute kidney injury. STEC's primary reservoirs are cattle and other domestic ruminants, and transmission can occur through food, water, animal contact, and person-to-person. Living near ruminants poses a significant risk of STEC infection; however, the proportion of that risk due to direct ruminant contact or other routes of transmission is unknown. Our research demonstrates that direct ruminant contact is a substantial risk irrespective of location, and that individuals living in ruminant-rich regions are at high risk of STEC infection regardless of whether they come into contact with ruminants. These findings indicate a need for multi-pronged prevention efforts that emphasize control of contamination in the environments surrounding ruminant populations, in addition to biosafety precautions when contacting ruminants directly.