Bacterial outer membrane vesicle nanorobot.

Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.

Proteus mirabilis UreR coordinates cellular functions required for urease activity.

A hallmark of Proteus mirabilis infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of ions to form stones. Transcriptional regulator UreR activates expression of urease genes in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant. Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from one of two nickel transporters and facilitate incorporation of nickel to form mature urease. In this study, we performed a comprehensive RNA-seq to define the P. mirabilis urea-induced transcriptome as well as the UreR regulon. We identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for the direct regulation of the Ynt nickel transporter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Our study demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis. IMPORTANCE: Catheter-associated urinary tract infections (CAUTIs) account for over 40% of acute nosocomial infections in the USA and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. Transcriptional regulation of urease genes by UreR is well established; here, we expand the UreR regulon to include regulation of nickel import, a function required to produce mature urease. Furthermore, we reflect on the role of urea catalysis in P. mirabilis metabolism and provide evidence for its importance.

Embedding of exogenous B cell epitopes on the surface of UreB structure generates a broadly reactive antibody response against Helicobacter pylori.

Since Helicobacter pylori (H. pylori) resistance to antibiotic regimens has increased, vaccination is becoming an increasingly important alternative therapy to control H. pylori infection. UreB, FlaA, AlpB, SabA, and HpaA proteins of H. pylori were previously proved to be used as candidate vaccine antigens. Here, we developed an engineered antigen based on a recombinant chimeric protein containing a structural scaffold from UreB and B cell epitopes from FlaA, AlpB, SabA, and HpaA. The multi-epitope chimeric antigen, named MECU, could generate a broadly reactive antibody response including antigen-specific antibodies and neutralising antibodies against H. pylori urease and adhesins. Moreover, therapeutic immunisation with MECU could reduce H. pylori colonisation in the stomach and protect the stomach in BALB/c mice. This study not only provides promising immunotherapy to control H. pylori infection but also offers a reference for antigen engineering against other pathogens.

Synthetic DNA-based Swimmers Driven by Enzyme Catalysis.

Here, we report DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA-enzyme swimmers) that self-propel by converting the enzymatic substrate to the product in solution. The DNA-enzyme swimmers are obtained from tubular DNA structures that self-assemble spontaneously by the hybridization of DNA tiles. We functionalize these DNA structures with two different enzymes, urease and catalase, and show that they exhibit concentration-dependent movement and enhanced diffusion upon addition of the enzymatic substrate (i.e., urea and H2O2). To demonstrate the programmability of such DNA-based swimmers, we also engineer DNA strands that displace the enzyme from the DNA scaffold, thus acting as molecular "brakes" on the DNA swimmers. These results serve as a first proof of principle for the development of synthetic DNA-based enzyme-powered swimmers that can self-propel in fluids.

Detection of Helicobacter pylori Infection in Human Gastric Fluid Through Surface-Enhanced Raman Spectroscopy Coupled With Machine Learning Algorithms.

Diagnostic methods for Helicobacter pylori infection include, but are not limited to, urea breath test, serum antibody test, fecal antigen test, and rapid urease test. However, these methods suffer drawbacks such as low accuracy, high false-positive rate, complex operations, invasiveness, etc. Therefore, there is a need to develop simple, rapid, and noninvasive detection methods for H. pylori diagnosis. In this study, we propose a novel technique for accurately detecting H. pylori infection through machine learning analysis of surface-enhanced Raman scattering (SERS) spectra of gastric fluid samples that were noninvasively collected from human stomachs via the string test. One hundred participants were recruited to collect gastric fluid samples noninvasively. Therefore, 12,000 SERS spectra (n = 120 spectra/participant) were generated for building machine learning models evaluated by standard metrics in model performance assessment. According to the results, the Light Gradient Boosting Machine algorithm exhibited the best prediction capacity and time efficiency (accuracy = 99.54% and time = 2.61 seconds). Moreover, the Light Gradient Boosting Machine model was blindly tested on 2,000 SERS spectra collected from 100 participants with unknown H. pylori infection status, achieving a prediction accuracy of 82.15% compared with qPCR results. This novel technique is simple and rapid in diagnosing H. pylori infection, potentially complementing current H. pylori diagnostic methods.

Self-Assembled Integrated Nanozyme Cascade Biosensor with Dual Catalytic Activity for Portable Urease Analysis.

In this work, a novel nanozyme (Cu@Zr) with all-in-one dual enzyme and fluorescence properties is designed by simple self-assembly. A nanozyme cascade sensor with disodium phenyl phosphate (PPDS) as substrate was first established by exploiting the dual enzymatic activities of phosphatase and laccase. Specifically, phosphatase cleaves the P-O bond of PPDS to produce colorless phenol, which is then oxidized by laccase and complexed with the chromogenic agent 4-aminoantipyrine (4-AP) to produce red quinoneimine (QI). Strikingly, the NH3 produced by the urease hydrolysis of urea can interact with Cu@Zr, accelerating the electron transfer rate and ultimately leading to a significantly improved performance of the cascade reaction. Moreover, the fluorescence at 440 nm of Cu@Zr is further quenched by the inner filter effect (IFE) of QI. Thus, the colorimetric and fluorescence dual-mode strategy for sensitive urease analysis with LODs of 3.56 and 1.83 U/L was established by the proposed cascade sensor. Notably, a portable swab loaded with Cu@Zr was also prepared for in situ urease detection with the aid of a smartphone RGB readout. It also provides a potentially viable analytical avenue for environmental and biological analysis.

Enzymatic Nanomotors Surviving Harsh Conditions Enabled by Metal Organic Frameworks Encapsulation.

Enzyme-driven micro/nanomotors (MNMs) have demonstrated potentials in the biomedical field because of their excellent biocompatibility, versatility, and fuel bioavailability. However, the fragility of enzymes limits their practical application, because of their susceptibility to denaturation and degradation in realistic scenarios. Herein, a simple yet versatile and effective approach is reported to preserve the enzymatic activity and propulsion capability of enzymatic MNMs under various harsh conditions using metal organic frameworks (MOFs) as a protective shell. Urease can be encapsulated within the exoskeleton of zeolitic imidazolate framework-8 (ZIF-8) via biomimetic mineralization to form ZIF-8@urease (ZU-I) nanomotors that exhibit self-propulsion in the presence of urea. When exposed to harsh conditions, including high temperature, presence of proteases, and organic solvents, the ZU-I nanomotors still maintained their activity and mobility, whereas ZIF-8 with externally modified urease (ZU-O) nanomotors with externally modified urease as a control rapidly lost their motion capabilities owing to the inactivation of urease. Furthermore, ZU-I nanomotors exhibit effectively enhanced diffusion within the small intestine fluid, achieving a fourfold higher mucus penetration than the ZU-O nanomotors. The results highlight the effectiveness of using MOFs as protective shells for enzyme nano-engines, which can greatly advance the practical applications of enzymatic MNMs under realistic conditions, especially for biomedical purpose.

Effect of nitrogen reduction by chemical fertilization with green manure (Vicia sativa L.) on soil microbial community, nitrogen metabolism and and yield of Uncaria rhynchophylla by metagenomics.

Uncaria rhynchophylla is an important herbal medicine, and the predominant issues affecting its cultivation include a single method of fertilizer application and inappropriate chemical fertilizer application. To reduce the use of inorganic nitrogen fertilization and increase the yield of Uncaria rhynchophylla, field experiments in 2020-2021 were conducted. The experimental treatments included the following categories: S1, no fertilization; S2, application of chemical NPK fertilizer; and S3-S6, application of chemical fertilizers and green manures, featuring nitrogen fertilizers reductions of 0%, 15%, 30%, and 45%, respectively. The results showed that a moderate application of nitrogen fertilizer when combined with green manure, can help alleviate soil acidification and increase urease activity. Specifically, the treatment with green manure provided in a 14.71-66.67% increase in urease activity compared to S2. Metagenomics sequencing results showed a decrease in diversity in S3, S4, S5, and S6 compared to S2, but the application of chemical fertilizer with green manure promoted an increase in the relative abundance of Acidobacteria and Chloroflexi. In addition, the nitrification pathway displayed a progressive augmentation in tandem with the reduction in nitrogen fertilizer and application of green manure, reaching its zenith at S5. Conversely, other nitrogen metabolism pathways showed a decline in correlation with diminishing nitrogen fertilizer dosages. The rest of the treatments showed an increase in yield in comparison to S1, S5 showing significant differences (p < 0.05). In summary, although S2 demonstrate the ability to enhance soil microbial diversity, it is important to consider the long-term ecological impacts, and S5 may be a better choice.

Recombinant L. lactis vaccine LL-plSAM-WAE targeting four virulence factors provides mucosal immunity against H. pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) causes chronic gastric disease. An efficient oral vaccine would be mucosa-targeted and offer defense against colonization of invasive infection in the digestive system. Proteolytic enzymes and acidic environment in the gastrointestinal tract (GT) can, however, reduce the effectiveness of oral vaccinations. For the creation of an edible vaccine, L. lactis has been proposed as a means of delivering vaccine antigens. RESULTS: We developed a plSAM (pNZ8148-SAM) that expresses a multiepitope vaccine antigen SAM-WAE containing Urease, HpaA, HSP60, and NAP extracellularly (named LL-plSAM-WAE) to increase the efficacy of oral vaccinations. We then investigated the immunogenicity of LL-plSAM-WAE in Balb/c mice. Mice that received LL-plSAM-WAE or SAM-WAE with adjuvant showed increased levels of antibodies against H. pylori, including IgG and sIgA, and resulted in significant reductions in H. pylori colonization. Furthermore, we show that SAM-WAE and LL-plSAM-WAE improved the capacity to target the vaccine to M cells. CONCLUSIONS: These findings suggest that recombinant L. lactis could be a promising oral mucosa vaccination for preventing H. pylori infection.

Accuracy of gastric nodule combined with rapid urease test prediction in diagnosing Helicobacter pylori infection in children.

BACKGROUND: The diagnosis of Helicobacter pylori (H. pylori) infection in children remains challenging with the lack of a rapid, cost-effective, and highly accurate diagnostic method. Consequently, this study aimed to investigate the accuracy of the combination of gastric nodule and rapid urease test (RUT) as a diagnostic method for H. pylori infection in children. METHODS: The study included participants who underwent a thorough examination, including gastroscopy, a 13C breath test, RUT, and pathological methylene blue staining, with the gold standard for diagnosing of H. pylori infection being a positive result from both pathological methylene blue staining and 13C breath test. The sensitivity, specificity, positive and negative predictive values, and accuracy of the diagnostic methods were calculated. RESULTS: The accuracy of the different tests for H. pylori infection was evaluated in 2202 participants. A total of 730 (33.2%) children were diagnosed with H. pylori infection (pathological methylene blue staining and 13C breath test, both positive). Gastric nodule had a sensitivity of 87.1% and a specificity of 93.1%, whereas combining gastric nodule and RUT in parallel had the higher accuracy of 95.4%. The accuracy of gastric nodule diagnosis was higher in younger age groups and increased after excluding patients with a history of anti-H. pylori treatment. CONCLUSIONS: The findings of this study suggest that gastric nodules, particularly when combined with RUT, can be a valuable predictor of H. pylori infection in children, offering a simple and feasible alternative to other invasive methods.

Advances in Helicobacter pylori vaccine research: From candidate antigens to adjuvants-A review.

BACKGROUND: Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that infects approximately 50% of the world's population and has been strongly associated with chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoma, and gastric cancer. The elimination of H. pylori is currently considered one of the most effective strategies for the treatment of gastric-related diseases, so antibiotic therapy is the most commonly used regimen for the treatment of H. pylori infection. Although this therapy has some positive effects, antibiotic resistance has become another clinically prominent problem. Therefore, the development of a safe and efficient vaccine has become an important measure to prevent H. pylori infection. METHODS: PubMed and ClinicalTrials.gov were systematically searched from January 1980 to March 2023 with search terms-H. pylori vaccine, adjuvants, immunization, pathogenesis, and H. pylori eradication in the title and/or abstract of literature. A total of 5182 documents were obtained. Based on the principles of academic reliability, authority, nearly publicated, and excluded the similar documents, finally, 75 documents were selected, organized, and analyzed. RESULTS: Most of the candidate antigens used as H. pylori vaccines in these literatures are whole-cell antigens and virulence antigens such as UreB, VacA, CagA, and HspA, and the main types of vaccines for H. pylori are whole bacteria vaccines, vector vaccines, subunit vaccines, nucleic acid vaccines, epitope vaccines, etc. Some vaccines have shown good immune protection in animal trials; however, few vaccines show good in clinical trials. The only H. pylori vaccine passed phase 3 clinical trial is a recombinant subunit vaccine using Urease subunit B (UreB) as the vaccine antigen, and it shows good prophylactic effects. Meanwhile, the adjuvant system for vaccines against this bacterium has been developed considerably. In addition to the traditional mucosal adjuvants such as cholera toxin (CT) and E. coli heat labile enterotoxin (LT), there are also promising safer and more effective mucosal adjuvants. All these advances made safe and effective H. pylori vaccines come into service as early as possible. CONCLUSIONS: This review briefly summarized the advances of H. pylori vaccines from two aspects, candidates of antigens and adjuvants, to provide references for the development of vaccine against this bacterium. We also present our prospects of exosomal vaccines in H. pylori vaccine research, in the hope of inspiring future researchers.

Diversity and ecological function of urease-producing bacteria in the cultivation environment of Gracilariopsis lemaneiformis.

Urease-producing bacteria (UPB) provide inorganic nitrogen for primary producers by hydrolyzing urea, and play an important role in marine nitrogen cycle. However, there is still an incomplete understanding of UPB and their ecological functions in the cultivation environment of the red macroalgae Gracilariopsis lemaneiformis. This study comprehensively analyzed the diversity of culturable UPB and explored their effects on urea uptake by G. lemaneiformis. A total of 34 isolates belonging to four main bacterial phyla i.e. (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) were identified through 16S rRNA sequencing and were screened for UPB by urea agar chromogenic medium assay and ureC gene cloning. Our data revealed that only 8 strains contained urease. All of these UPB exhibited different urease activities, which were determined by the Berthelot reaction colorimetry assay. Additionally, the UPB strain (G13) isolated from G. lemaneiformis with higher urease activity was selected for co-culture with G. lemaneiformis to explore its role in promoting or inhibiting nitrogen uptake by macroalgae. The results showed a significant increase in urea consumption in the culture medium and the total cellular nitrogen in G. lemaneiformis in the UPB-co culture group compared to the sterile group. This suggests that the selected UPB strain positively influences nitrogen uptake by G. lemaneiformis. Similarly, isotopic assays revealed that the δ15N content of G. lemaneiformis was significantly higher in the UPB-co culture than in the control group, where δ15N-urea was the only nitrogen source in the culture medium. This indicates that the UPB helped G. lemaneiformis to absorb more nitrogen from urea. Moreover, the highest content of δ15N was found in G. lemaneiformis with epiphytic bacteria compared to sterilized (i.e. control), showing that epiphytic bacteria, along with UPB, have a compound effect in helping G. lemaneiformis absorb more nitrogen from urea. Taken together, these results provide unique insight into the ecological role of UPB and suggest that urease from macroalgae environment-associated bacteria might be an important player in marine nitrogen cycling.

Uncovering the Dynamics of Urease and Carbonic Anhydrase Genes in Ureolysis, Carbon Dioxide Hydration, and Calcium Carbonate Precipitation.

The hydration of CO2 suffers from kinetic inefficiencies that make its natural trapping impractically sluggish. However, CO2-fixing carbonic anhydrases (CAs) remarkably accelerate its equilibration by 6 orders of magnitude and are, therefore, "ideal" catalysts. Notably, CA has been detected in ureolytic bacteria, suggesting its potential involvement in microbially induced carbonate precipitation (MICP), yet the dynamics of the urease (Ur) and CA genes remain poorly understood. Here, through the use of the ureolytic bacteriumSporosarcina pasteurii, we investigate the differing role of Ur and CA in ureolysis, CO2 hydration, and CaCO3 precipitation with increasing CO2(g) concentrations. We show that Ur gene up-regulation coincides with an increase in [HCO3-] following the hydration of CO2 to HCO3- by CA. Hence, CA physiologically promotes buffering, which enhances solubility trapping and affects the phase of the CaCO3 mineral formed. Understanding the role of CO2 hydration on the performance of ureolysis and CaCO3 precipitation provides essential new insights, required for the development of next-generation biocatalyzed CO2 trapping technologies.

Synthesis and biological evaluation of triazolones/oxadiazolones as novel urease inhibitors.

Urease is the main virulence factor of infectious gastritis and gastric ulcers. Urease inhibitors are regarded as the first choice for the treatment of such diseases. Based on the triazolone/oxadiazolone skeleton, a urea-like fragment being able to specifically bind the urease activity pocket and prevent urea from hydrolysis, we designed and synthesized 45 triazolones/oxadiazolones as urease inhibitors. Eight compounds were proved to show excellent inhibitory activity against Helicobacter pylori urease, being more potency than the clinically used urease inhibitor acetohydroxamic acid. The most active inhibitor with IC50 value of 1.2 µM was over 20-fold higher potent than the positive control. Enzymatic kinetic assays showed that these novel inhibitors reversibly inhibited urease with a mixed competitive mechanism. Molecular dockings provided evidence for the observations in enzyme assays. Furthermore, these novel inhibitors were proved as drug-like compounds with very low cytotoxicity to mammalian cells and favorable water solubility. These results suggested that triazolone and oxadiazolone were promising scaffolds for the design and discovery of novel urease inhibitors, and were expected as good candidates for further drug development.

Artificial neural networks in the modeling of the catalytic activity of a biosensor composed of conjugated polymers and urease.

Thin films of conjugated polymer and enzyme can be used to unravel the interaction between components in a biosensor. Using artificial neural networks (ANNs) improves data interpretability and helps construct models with great capacity for classifying and processing information. The present work used kinetic data from the catalytic activity of urease immobilized in different conjugated polymers to create ANN models using time, substrate concentration, and absorbance as input variables since the models had absorbance in a posterior instant as output value to explore the predictivity of the ANNs. The performance of the models was evaluated by Pearson's correlation coefficient (ρ) and mean squared error (MSE) values. After the learning process, a series of new experiments were performed to verify the generality of the models. As the main results, the best ANN model presented 0.9980 and 3.0736 × 10-5 for ρ and MSE, respectively. For the simulation step, intermediary values of substrate concentration were used. The mean absolute percentage error (MAPE) values were 3.34, 3.07, and 3.78 for 12 mM, 22 mM, and 32 mM concentrations, respectively. Overall, with the simulations, it was possible to ascertain the interpolatory capacity of the model, which has a learning mechanism based on absorbance and time as variables. Thus, the potential of ANNs would be in their use in pre-evaluations, helping to determine the substrate concentration at which there is higher catalytic activity or in determining the linear range of the sensor.

New strategies for Helicobacter pylori isolation and sequencing analysis for virulence genes contributing to its pathogenicity.

BACKGROUND: Helicobacter pylori is a fastidious pathogen that is required a complicated medium for growth. Invading epithelial cells of the stomach. H. pylori virulence factors are classified by function, acidic resistivity, adhesion, chemotaxis and motility, molecular mimicry, immunological invasion and modulation, and toxins formation such as cytotoxin-associated genes A (cagA) and vacuolating cytotoxin A (vacA). This study aims to determine a simple and innovative technique to isolate H. pylori from gastric biopsies and assess pathogenicity by virulence factor gene detection. METHODS: A total of 200 patients who were suspected of having H. pylori infection had two antral gastric biopsies undertaken. A rapid urease test (RUT) was used for one, and Brain Heart Infusion broth (BHI) was used to cultivate the other. The molecular study included diagnostics utilizing the 16sRNA housekeeping gene along with the identification of the virulence factors genes (cagA, cagT, and vacA) and sequencing, RESULT: Of the 200 antral gastric biopsies collected, 135 were positive rapid urease tests, and 17 H. pylori isolates were successfully obtained from 135 biopsies. The 16SrRNA as a housekeeping gene is confirmed, and about 53%, 70.5%, and 82.3% of the 17 isolates show carrying cagA, cagT, and vacA genes, respectively. All peptic ulcer isolates have the cagA gene, while Gastroesophageal Reflux Disease (GERD) and non-peptic ulcer disease (NPUD) isolates show the lack of the cagA gene. All bacteria, which were isolated from peptic ulcer, nodular gastritis, and gastritis patients, have a vacA gene. CONCLUSION: The effective method for isolating H. pylori is centrifuging the transport broth after 24 h of incubation. The cagA toxin causes peptic ulcer while vacA toxin induces several histopathological changes in the stomach. Three virulence genes were present in all peptic ulcer-causing bacteria, while only one or none were present in the GERD and NPUD biopsy isolates.

Synergizing structure and function: Cinnamoyl hydroxamic acids as potent urease inhibitors.

The current investigation encompasses the structural planning, synthesis, and evaluation of the urease inhibitory activity of a series of molecular hybrids of hydroxamic acids and Michael acceptors, delineated from the structure of cinnamic acids. The synthesized compounds exhibited potent urease inhibitory effects, with IC50 values ranging from 3.8 to 12.8 µM. Kinetic experiments unveiled that the majority of the synthesized hybrids display characteristics of mixed inhibitors. Generally, derivatives containing electron-withdrawing groups on the aromatic ring demonstrate heightened activity, indicating that the increased electrophilicity of the beta carbon in the Michael Acceptor moiety positively influences the antiureolytic properties of this compounds class. Biophysical and theoretical investigations further corroborated the findings obtained from kinetic assays. These studies suggest that the hydroxamic acid core interacts with the urease active site, while the Michael acceptor moiety binds to one or more allosteric sites adjacent to the active site.

Epiberberine: a potential rumen microbial urease inhibitor to reduce ammonia release screened by targeting UreG.

Rumen microbial urease inhibitors have been proposed for regulating nitrogen emission and improving nitrogen utilization efficiency in ruminant livestock industry. However, studies on plant-derived natural inhibitors of rumen microbial urease are limited. Urease accessory protein UreG, plays a crucial role in facilitating urease maturation, is a new target for design of urease inhibitor. The objective of this study was to select the potential effective inhibitor of rumen microbial urease from major protoberberine alkaloids in Rhizoma Coptidis by targeting UreG. Our results showed that berberine chloride and epiberberine exerted superior inhibition potential than other alkaloids based on GTPase activity study of UreG. Berberine chloride inhibition of UreG was mixed type, while inhibition kinetics type of epiberberine was uncompetitive. Furthermore, epiberberine was found to be more effective than berberine chloride in inhibiting the combination of nickel towards UreG and inducing changes in the second structure of UreG. Molecular modeling provided the rational structural basis for the higher inhibition potential of epiberberine, amino acid residues in G1 motif and G3 motif of UreG formed interactions with D ring of berberine chloride, while interacted with A ring and D ring of epiberberine. We further demonstrated the efficacy of epiberberine in the ruminal microbial fermentation with low ammonia release and urea degradation. In conclusion, our study clearly indicates that epiberberine is a promising candidate as a safe and effective inhibitor of rumen microbial urease and provides an optimal strategy and suitable feed additive for regulating nitrogen excretion in ruminants in the future. KEY POINTS: • Epiberberine is the most effective inhibitor of rumen urease from Rhizoma Coptidis. • Urease accessory protein UreG is an effective target for design of urease inhibitor. • Epiberberine may be used as natural feed additive to reducing NH3 release in ruminants.

Genome Sequence Analysis of Calcifying Bacteria Bacillus paranthracis CT5 and Its Biomineralization Efficacy to Improve the Strength and Durability Properties of Civil Structures.

Bacteria producing urea amidohydrolases (UA) and carbonic anhydrases (CA) are of great importance in civil engineering as these enzymes are responsible for microbially induced calcium carbonate precipitation (MICCP). In this investigation, genomic insights of Bacillus paranthracis CT5 and the expression of genes underlying in MICCP were studied. B. paranthracis produced a maximum level of UA (669.3 U/ml) and CA (125 U/ml) on 5th day of incubation and precipitated 197 mg/100 ml CaCO3 after 7 days of incubation. After 28 days of curing, compressive strength of bacterial admixed and bacterial cured (B-B) specimens was 13.7% higher compared to water-mixed and water-cured (W-W) specimens. A significant decrease in water absorption was observed in bacterial-cured specimens compared to water-cured specimens after 28 days of curing. For genome analysis, reads were assembled de novo producing 5,402,771 bp assembly with N50 of 273,050 bp. RAST annotation detected six amidohydrolase and three carbonic anhydrase genes. Among 5700 coding sequences found in genome, COG gene annotation grouped 4360 genes into COG categories with highest number of genes to transcription (435 genes), amino acid transport and metabolism (362 genes) along with cell wall/membrane/envelope biogenesis and ion transport and metabolism. KEGG functional classification predicted 223 pathways consisting of 1,960 genes and the highest number of genes belongs to two-component system (101 genes) and ABC transporter pathways (98 genes) enabling bacteria to sense and respond to environmental signals and actively transport various minerals and organic molecules, which facilitate the active transport of molecules required for MICCP.

Inoculation of cadmium-tolerant bacteria to regulate microbial activity and key bacterial population in cadmium-contaminated soils during bioremediation.

The perennial ryegrass Lolium perenne can be used in conjunction with cadmium (Cd)-tolerant bacteria such as Cdq4-2 (Enterococcus spp.) for bioremediation of Cd-contaminated soil. In this study, a theoretical basis was provided to increase the efficiency of L. perenne remediation of Cd-contaminated soil using microorganisms to maintain the stability of the soil microbiome. The experimental design involved three treatment groups: CK (soil without Cd addition) as the control, 20 mg·kg-1 Cd-contaminated soil, and 20 mg·kg-1 Cd-contaminated soil + Cdq4-2, all planted with L. perenne. The soil was collected on day 60 to determine the soil microbial activity and bacterial community structure and to analyze the correlation between soil variables, the bacterial community, available Cd content in the soil, Cd accumulation, and L. perenne growth. The soil microbial activity and bacterial community diversity decreased under Cd stress, and the soil microbial community composition was changed; while inoculation with Cdq4-2 significantly increased soil basal respiration and the activities of urease, invertase, and fluorescein diacetate (FDA) hydrolase by 83.65%, 79.72%, 19.88%, and 96.15% respectively; and the stability of the community structure was also enhanced. The Actinobacteriota biomass, the amount of available Cd, and the above- and belowground Cd content of L. perenne were significantly negatively correlated with the total phosphorus, total potassium, and pH. The activity of urease, invertase, and FDA hydrolase were significantly positively correlated with the biomasses of Acidobacteriota and L. perenne and significantly negatively correlated with the Chloroflexi biomass. Further, the available soil Cd content and the above- and belowground Cd levels of L. perenne were significantly positively correlated with the Actinobacteriota biomass and significantly negatively correlated with the Gemmatimonadetes biomass. Overall, inoculating Cd-tolerant bacteria improved the microbial activity, diversity, and abundance, and changed the microbial community composition, facilitating the remediation of Cd-contaminated soil by L. perenne.