Bacillus Atrophaeus Research Articles

Synergizing Bacillus halotolerans, Pseudomonas sihuiensis and Bacillus atrophaeus with folic acid for enhanced drought resistance in wheat by metabolites and antioxidants

Drought stress imposes a serious challenge to cultivate wheat, restricting its growth. Drought reduces the capability of plant to uptake essential nutrients. This causes stunted growth, development and yield. Traditional ways to increase wheat growth under drought stress have shortcomings. Using plant-growth-promoting rhizobacteria (PGPR) has proved feasible and eco-friendly way to enhance wheat growth even under the drought stress. Combining PGPR in consortiums further boosts up their effects. In this study, we have checked the efficacy of drought-tolerant Bacillus halotolerans, Pseudomonas sihuiensis and Bacillus atrophaeus in combination. These strains were allowed to grow on PEG 6000 with concentrations (-0.15, -0.49, -0.73 and − 1.2) Mega Pascal (MPa) alone and in combination. Furthermore, Fourier transmission infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used. Their biochemical traits such as solubilization of K, P and Zn and the synthesis of siderophore, indole acetic acid (IAA), protease, amylase, hydrogen cyanide (HCN) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase were done. In addition to this, we investigated the optimum folic acid concentration i.e 150 ppm for wheat against drought stress. We conducted a pot experiment to check the growth-enhancing and drought-mitigating effects of consortium and folic acid alone and in combination. As a result, we found a significantly increased wheat biomass, relative water content (RWC), chlorophyll content, antioxidants including glutathione reductase and total soluble sugars and protein content under all treatments. However, the combined treatment of bacterial consortium and folic acid showed maximum potential to boost wheat growth and survival even under drought. We also investigated the minerals uptake by wheat after the treatments and found maximum nutrient uptake under the co-effect of folic acid and bacterial consortium We believe this is the first study that has investigated the optimal dose of folic acid for wheat. Our research is also novel in that we seek to investigate the effects of folic acid along with a bacterial consortium comprising Bacillus halotolerans, Pseudomonas sihuiensis and Bacillus atrophaeus on wheat grown under the drought stress.

Exploring the aroma profile and biomedical applications of Scutellaria nuristanica Rech. F.: A new insight as a natural remedy

BackgroundThe Scutellaria genus has promising therapeutic capabilities as an aromatherapy. Based on that and local practices of S. nuristanica Rech. F. The essential oil was studied for the first time for its diverse biomedical applications. PurposeThis study aimed to evaluate and validate their therapeutic capabilities by screening the essential oil ingredients and examining their antimicrobial, antioxidant, carbonic anhydrase, and antidiabetic using further In silico assessment and In vivo anti-inflammatory and analgesic capabilities to devise novel sources as natural remedies alternative to the synthetic drugs. MethodsEssential oil was obtained through hydrodistillation, and the constituents were profiled using GC–MS. The antimicrobial assessment was conducted using an agar well diffusion assay. Free radical scavenging capabilities were determined by employing DPPH and ABTS assay. The carbonic anhydrase-II was examined using colorimetric assay, while the antidiabetic significance was performed using α-Glucosidase assay. The anti-inflammatory significance was examined through carrageenan-induced paw edema, and the analgesic features of the essential oil were determined using an acetic acid-induced writhing assay. ResultsFifty constituents were detected in S. nuristanica essential oil (SNEO), contributing 95.93 % of the total EO, with the predominant constituents being 24-norursa-3,12-diene (10.12 %), 3-oxomanoyl oxide (9.94 %), methyl 7-abieten-18-oate (8.85 %). SNEO presented significance resistance against the Gram-positive bacterial strains (GPBSs), Bacillus atrophaeus and Bacillus subtilis, as compared to the Salmonella typhi and Klebsiella pneumoniae, Gram-negative bacterial strains (GNBSs) as well as two fungal strains Aspergillus parasiticus and Aspergillus niger associated with their respective standards. Considerable free radical scavenging capacity was observed in DPPH compared to the ABTS assay when correlated with ascorbic acid. In addition, when equated with their standards, SNEO offered considerable in vitro carbonic anhydrase II and antidiabetic capabilities. Additionally, the antidiabetic behavior of the 9 dominant compounds of SNEO was tested via In silico techniques, such as molecular docking, which assisted in the assessment of the significance of binding contacts of protein with each chemical compound and pharmacokinetic evaluations to examine the drug-like characteristics. Molecular dynamic simulations at 100 ns and binding free energy evaluations such as PBSA and GBSA models explain the molecular mechanics and stability of molecular complexes. It was also observed that SNEO depicted substantial anti-inflammatory and analgesic capabilities. ConclusionHence, it was concluded that the SNEO comprises bioactive ingredients with biomedical significance, such as anti-microbial, antioxidant, CA-II, antidiabetic, anti-inflammatory, and analgesic agents. The computational validation also depicted that SNEO could be a potent source for the discovery of anti-diabetic drugs.

Inhibition of Microbial Growth and Biofilm Formation in Pure and Mixed Bacterial Samples.

Hydraulic fracturing, or fracking, requires large amounts of water to extract fossil fuel from rock formations. As a result of hydraulic fracturing, the briny wastewater, often termed back-produced fracturing or fracking water (FW), is pumped into holding ponds. One of the biggest challenges with produced water management is controlling microbial activity that could reduce the pond water's reusable layer and pose a significant environmental hazard. This study focuses on the characterization of back-produced water that has been hydraulically fractured using chemical and biological analysis and the development of a high-throughput screening method to evaluate and predict the antimicrobial effect of four naturally and commercially available acidic inhibitors (edetic acid, boric acid, tannic acid, and lactic acid) on the growth of the FW microbiome. Liquid cultures and biofilms of two laboratory model strains, the vegetative Escherichia coli MG1655, and the spore-forming Bacillus atrophaeus (also known as Bacillus globigii, BG) bacteria, were used as reference microorganisms. Planktonic bacteria in FW were more sensitive to antimicrobials than sessile bacteria in biofilms. Spore-forming BG bacteria exhibited more sensitivity to acidic inhibitors than the vegetative E. coli cells. Organic acids were the most effective bacterial growth inhibitors in liquid culture and biofilm.

Purification and characterization of glutaminase and urease-free L-asparaginase from Bacillus atrophaeus with acrylamide reduction potential

L-asparaginase (L-asnase) is a versatile enzyme with uses in food industry and medicine. Current study aimed to isolate L-asnase producing microorganism/s without urease and glutaminase and optimize L-asnase production. First, screening and isolation of L-asnase-producing bacterial strains that did not produce glutaminase and urease from chicken gizzards were performed. For this purpose, the enzyme producing bacteria were screened on the agar medium supplied with substrate and phenol red indicator dye. Among the isolated bacteria, 1 isolate showed L-asnase free of glutaminase and urease. The selected strain was identified by biochemical, morphological and 16s rRNA sequencing. The selected strain was identified as Bacillus atrophaeus by 16S rRNA sequencing. The effects of incubation temperature (30°C) and time (72 hours), medium pH (8.0) and nutritional sources (glucose and NaNO3) on L-asnase production were determined. L-asnase was purified with acetone, and its molecular weight was determined to be 42 kDa by SDS-Page. Enzyme kinetics were also calculated, and it was determined that Vmax was 43 μmol/mL/min and Km was 2.7 mM. L-asnase activity was highest at 40 ℃ and the optimal pH was 8.0. L-asnase activity was stimulated by Mn2+, Mg2+, and Ca2+ but inhibited by Co2+, Na+, Zn2+, and Hg2+. L-asnase was utilized to treat potato chips before they were fried in order to assess its capacity to mitigate acrylamide. The result was an 80% reduction in acrylamide concentration when compared to the untreated control. Based on these findings, it appears that L-asnase could have potential use in the food industry.

Fate and transport of viable Bacillus anthracis simulant spores in ambient air during a large outdoor decontamination field exercise

ABSTRACT The Wide Area Demonstration (WAD) was a field exercise conducted under the U.S. EPA’s Analysis of Coastal Operational Resiliency program, in conjunction with the U.S. Department of Homeland Security and the U.S. Coast Guard. The purpose of the WAD was to operationalize at field scale aspects of remediation activities that would occur following an outdoor release of Bacillus anthracis spores, including sampling and analysis, decontamination, data management, and waste management. The WAD was conducted in May 2022 at Fort Walker (formerly known as Fort A.P. Hill) and utilized Bacillus atrophaeus as a benign simulant for B. anthracis. B. atrophaeus spores were inoculated onto the study area at the beginning of the study, and air samples were collected daily during each of the different phases of the WAD using Dry Filter Units (DFUs). Ten DFU air samplers were placed at the perimeter of the study area to collect bioaerosols onto two parallel 47-mm diameter polyester felt filters, which were then subsequently analyzed in a microbiological laboratory for the quantification of B. atrophaeus. The study demonstrated the use of DFUs as a rugged and robust bioaerosol collection device. The results indicated that the highest B. atrophaeus spore air concentrations (up to ~ 5 colony forming units/m3) occurred at the beginning of the demonstration (e.g. during inoculation and characterization sampling phases) and generally downwind from the test site, suggesting transport of the spores was occurring from the study area. Very few B. atrophaeus spores were detected in the air after several weeks and following decontamination of exterior surfaces, thus providing an indication of the site decontamination procedures’ effectiveness. No B. atrophaeus spores were detected in any of the blank or background samples. Implications: Following an incident involving a release of Bacillus anthracis spores or other biological threat agent into the outdoor environment, understanding the factors that may affect the bioagent’s fate and transport can help predict viable contaminant spread via the ambient air. This paper provides scientific data for the first time on ambient air concentrations of bacterial spores over time and location during different phases of a field test in which Bacillus atrophaeus (surrogate for B. anthracis) spores were released outdoors as part of a full-scale study on sampling and decontamination in an urban environment. This study advances the knowledge related to the fate and transport of bacterial spores (such as those causing anthrax disease) as an aerosol in the outdoor environment over the course of three weeks in a mock urban environment and has exposure and health risk implications. The highest spore air concentrations occurred at the beginning of the study (e.g. during inoculation of surfaces and characterization sampling), and in the downwind direction, but diminished over time; few B. atrophaeus spores were detected in the air after several weeks and following decontamination. Therefore, in an actual incident, potential reaerosolization of the microorganism and subsequent transport in the air during surface sampling and remediation efforts should be considered for determining exclusion zone locations and estimating potential risk to neighboring communities. The data also provide evidence suggesting that the large-scale decontamination of outdoor surfaces may reduce air concentrations of the bioagent, which is important since exposure of B. anthracis via inhalation is a primary concern.

The Effect of Two Siderophore-Producing Bacillus Strains on the Growth Promotion of Perennial Ryegrass under Cadmium Stress.

Cadmium (Cd) is a highly toxic and cumulative environmental pollutant. Siderophores are heavy metal chelators with high affinity to heavy metals, such as Cd. Ryegrass (Lolium perenne L.) has a potential remediation capacity for soils contaminated by heavy metals. Consequently, using ryegrass alongside beneficial soil microorganisms that produce siderophores may be an effective means to remediate soils contaminated with Cd. In this study, the Bacillus strains WL1210 and CD303, which were previously isolated from the rhizospheres of Nitraria tangutorum in Wulan and Peganum harmala L. in Dachaidan, Qinghai, China, respectively, both arid and sandy environments, were evaluated for heavy metal pollution mitigation. Our quantitative analyses have discerned that the two bacterial strains possess commendable attributes of phosphorus (P) solubilization and potassium (K) dissolution, coupled with the capacity to produce phytohormones. To assess the heavy metal stress resilience of these strains, they were subjected to a cadmium concentration gradient, revealing their incremental growth despite cadmium presence, indicative of a pronounced tolerance threshold. The subsequent phylogenetic analysis, bolstered by robust genomic data from conserved housekeeping genes, including 16S rDNA, gyr B gene sequencing, as well as dnaK and recA, delineated a species-level phylogenetic tree, thereby confirming the strains as Bacillus atrophaeus. Additionally, we identified the types of iron-carrier-producing strains as catechol (WL1210) and carboxylic acid ferrophilin (CD303). A genomic analysis uncovered functional genes in strain CD303 associated with plant growth and iron carrier biosynthesis, such as fnr and iscA. Ryegrass seed germination assays, alongside morphological and physiological evaluations under diverse heavy metal stress, underscored the strains' potential to enhance ryegrass growth under high cadmium stress when treated with bacterial suspensions. This insight probes the strains' utility in leveraging alpine microbial resources and promoting ryegrass proliferation.

Optimization of Culture Conditions and Batch Process Control for the Augmented Production of Bacteriocin by Bacillus Species.

The emergence of antibiotic-resistant microorganisms poses a significant threat to human health worldwide. Recent advances have led to the discovery of molecules with potent antimicrobial activity from environmental sources. In this study, fifteen bacterial isolates were obtained from agricultural and polluted soil samples collected from different areas of the cities of Jizan and Jeddah. These isolates were screened for antagonistic activity against a set of human pathogenic bacterial strains. The results showed that two Bacillus strains, identified as Bacillus atrophaeus and Bacillus amyloliquefaciens based on 16S rDNA, synthesized bacteriocin with strong antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimum ATCC 14028, carbapenem-resistant E. coli, and MRSA 2. To optimize bacteriocin production, the effects of medium composition, incubation period, temperature, and pH were investigated. Nutrient broth and Mueller-Hinton broth were chosen as the optimal original media for bacteriocin production. The optimal incubation period, temperature, and pH were found to be 48 h at 37 °C and 7 pH in Bacillus atrophaeus and 72 h at 37 °C and 8 pH in Bacillus amyloliquefaciens. Batch cultures of Bacillus atrophaeus and Bacillus amyloliquefaciens were grown in a 10 L benchtop bioreactor, and pH control was found to significantly increase the production of bacteriocin by two-fold compared to uncontrolled conditions. The time course of growth, substrate consumption, pH, and enzyme production were investigated. This study demonstrates the potential of optimizing culture conditions and batch process control to enhance bacteriocin production by Bacillus spp.

Precursor-Directed Biosynthesis of Antialgal Fluorinated Bacillamide Derivatives in Bacillus atrophaeus.

The bacillamides are a class of indole alkaloids produced by the Bacillus genus that possess significant antialgal activity. Incorporation of fluorine into the bacillamides was carried out using a precursor-directed biosynthesis approach, with 4-, 5-, and 6-fluorotryptophan added to growing cultures of Bacillus atrophaeus IMG-11. This yielded the corresponding fluorinated analogues of bacillamides A and C, in addition to new derivatives of the related metabolite N-acetyltryptamine, thus demonstrating a degree of plasticity in the bacillamide biosynthetic pathway. The bacillamide derivatives were tested for activity against bloom-forming algae, which revealed that fluorination could improve the antialgal activity of these compounds in a site-specific manner, with fluorination at the 6-position consistently resulting in improved activity.

Bacterial crude oil and polyaromatic hydrocarbon degraders from Kazakh oil fields as barley growth support

Bacterial strains of the genera Arthrobacter, Bacillus, Dietzia, Kocuria, and Micrococcus were isolated from oil-contaminated soils of the Balgimbaev, Dossor, and Zaburunye oil fields in Kazakhstan. They were selected from 1376 isolated strains based on their unique ability to use crude oil and polyaromatic hydrocarbons (PAHs) as sole source of carbon and energy in growth experiments. The isolated strains degraded a wide range of aliphatic and aromatic components from crude oil to generate a total of 170 acid metabolites. Eight metabolites were detected during the degradation of anthracene and of phenanthrene, two of which led to the description of a new degradation pathway. The selected bacterial strains Arthrobacter bussei/agilis SBUG 2290, Bacillus atrophaeus SBUG 2291, Bacillus subtilis SBUG 2285, Dietzia kunjamensis SBUG 2289, Kocuria rosea SBUG 2287, Kocuria polaris SBUG 2288, and Micrococcus luteus SBUG 2286 promoted the growth of barley shoots and roots in oil-contaminated soil, demonstrating the enormous potential of isolatable and cultivable soil bacteria in soil remediation.Key points• Special powerful bacterial strains as potential crude oil and PAH degraders.• Growth on crude oil or PAHs as sole source of carbon and energy.• Bacterial support of barley growth as resource for soil remediation.

Influence of Temperature, Light, and H2O2 Concentration on Microbial Spore Inactivation: In‐Situ Raman Spectroscopy Combined with Optical Trapping

To gain insight on chemical sterilization processes, the influence of temperature (up to 70 °C), intense green light, and hydrogen peroxide (H2O2) concentration (up to 30% in aqueous solution) on microbial spore inactivation is evaluated by in‐situ Raman spectroscopy with an optical trap. Bacillus atrophaeus is utilized as a model organism. Individual spores are isolated and their chemical makeup is monitored under dynamically changing conditions (temperature, light, and H2O2 concentration) to mimic industrially relevant process parameters for sterilization in the field of aseptic food processing. While isolated spores in water are highly stable, even at elevated temperatures of 70 °C, exposure to H2O2 leads to a loss of spore integrity characterized by the release of the key spore biomarker dipicolinic acid (DPA) in a concentration‐dependent manner, which indicates damage to the inner membrane of the spore. Intensive light or heat, both of which accelerate the decomposition of H2O2 into reactive oxygen species (ROS), drastically shorten the spore lifetime, suggesting the formation of ROS as a rate‐limiting step during sterilization. It is concluded that Raman spectroscopy can deliver mechanistic insight into the mode of action of H2O2‐based sterilization and reveal the individual contributions of different sterilization methods acting in tandem.

In silico analysis of Bacillus stearothermophilus as a bioindicator for sterilizationco analysis of Bacillus stearothermophilus as a bioindicator for sterilization

To kill every kind of viable bacterial cells, sterilization in food and pharmaceutical industries is essential. In this study, bio indicators are used to validate accurate sterility. Bacillus stearothermophilus is used as a reference standard heat stable bio indicator. A 16S ribosomal RNA ribotyping sequence of Bacillus stearothermophilus is compared with different bacterial bioindicators including Bacillus pumilus, Bacillus atrophaeus, Clostridium sporogenes, and Brevundimonas diminuta. Results showed that Bacillus pumilus has the highest percentage (90.88%) similarity with the Geobacillus stearothermophilus followed by the Bacillus atrophaeus (90.84%), and Clostridium sporogenes (85.35%). The Brevundimonas diminuta was the least similar organism having a percentage similarity of 81.71% with the Geobacillus stearothermophilus. Bacterial spores are used as biological indicators for sterilization because they exhibit resistance to chemical and physical sterilization techniques. The protein common in all bacteria under study is Spo0A that make the bacteria heat resistant. The eradication of all these kind of heat resistant spores needs a high temperature which will also kill other viable cells. 
 Keywords: Bacillus stearothermophilus; Bacillus pumilus; Bacillus atrophaeus; Clostridium sporogenes and Brevundimonas diminuta; Spo0A protein.

Bacterial and fungal bioburden reduction on material surfaces using various sterilization techniques suitable for spacecraft decontamination.

Planetary protection is a guiding principle aiming to prevent microbial contamination of the solar system by spacecraft (forward contamination) and extraterrestrial contamination of the Earth (backward contamination). Bioburden reduction on spacecraft, including cruise and landing systems, is required to prevent microbial contamination from Earth during space exploration missions. Several sterilization methods are available; however, selecting appropriate methods is essential to eliminate a broad spectrum of microorganisms without damaging spacecraft components during manufacturing and assembly. Here, we compared the effects of different bioburden reduction techniques, including dry heat, UV light, isopropyl alcohol (IPA), hydrogen peroxide (H2O2), vaporized hydrogen peroxide (VHP), and oxygen and argon plasma on microorganisms with different resistance capacities. These microorganisms included Bacillus atrophaeus spores and Aspergillus niger spores, Deinococcus radiodurans, and Brevundimonas diminuta, all important microorganisms for considering planetary protection. Bacillus atrophaeus spores showed the highest resistance to dry heat but could be reliably sterilized (i.e., under detection limit) through extended time or increased temperature. Aspergillus niger spores and D. radiodurans were highly resistant to UV light. Seventy percent of IPA and 7.5% of H2O2 treatments effectively sterilized D. radiodurans and B. diminuta but showed no immediate bactericidal effect against B. atrophaeus spores. IPA immediately sterilized A. niger spores, but H2O2 did not. During VHP treatment under reduced pressure, viable B. atrophaeus spores and A. niger spores were quickly reduced by approximately two log orders. Oxygen plasma sterilized D. radiodurans but did not eliminate B. atrophaeus spores. In contrast, argon plasma sterilized B. atrophaeus but not D. radiodurans. Therefore, dry heat could be used for heat-resistant component bioburden reduction, and VHP or plasma for non-heat-resistant components in bulk bioburden reduction. Furthermore, IPA, H2O2, or UV could be used for additional surface bioburden reduction during assembly and testing. The systemic comparison of sterilization efficiencies under identical experimental conditions in this study provides basic criteria for determining which sterilization techniques should be selected during bioburden reduction for forward planetary protection.

Application of rhizobacteria to improve microbial community structure and maize (Zea mays L.) growth in saline soil.

The utilization of plant growth-promoting rhizobacteria (PGPR) has emerged as a prominent focus in contemporary research on soil microbiology, microecology, and plant stress tolerance. However, how PGPR influence the soil bacterial community and related ecological functions remains unclear. The aim of this study was to investigate the effects of three natural PGPR inoculations (YL07, Planococcus soli WZYH02; YL10, Bacillus atrophaeus WZYH01; YL0710, Planococcus soli WZYH02 and Bacillus atrophaeus WZYH01) on maize (Zea mays L.) growth under two salt stress conditions (S1, ECe = 2.1 ~ 2.5 dS/m; S2, ECe = 5.5 ~ 5.9 dS/m). The results revealed that compared to the control (CK), the average plant height of maize seedlings significantly increased by 27%, 23%, and 29% with YL07, YL10, and YL0710 inoculation under S1 conditions, respectively, and increased by 30%, 20%, and 18% under S2 conditions, respectively. Moreover, PGPR inoculation positively influenced the content of superoxide dismutase, catalase, soluble sugar, and proline in maize under salt stress. Subsequent analysis of alpha diversity indices, relative microbial abundance, principal coordinate analysis, cladograms, and linear discriminant analysis effect size histograms indicated significant alterations in the rhizosphere microbial community due to PGPR inoculation. FAPROTAX analysis demonstrated that YL10 inoculation in S2 rhizosphere soil had a notable impact on carbon cycle functions, specifically chemoheterotrophy, fermentation, and phototrophy. Thus, this study provides evidence that PGPR inoculation improves soil microbial communities and plant indices under salt stress. These findings shed light on the potential of PGPR as a viable approach for enhancing plant stress tolerance and fostering sustainable agricultural practices.

An integrated approach for the assessment of the electrochemical oxidation of diclofenac: By-product identification, microbiological and eco-genotoxicological evaluation

Diclofenac (DCF), a contaminant of emerging concern, is a non-steroidal anti-inflammatory drug widely detected in water bodies, which demonstrated harmful acute and chronic toxicity toward algae, zooplankton and aquatic invertebrates, therefore its removal from impacted water is necessary. DCF is recalcitrant toward traditional treatment technologies, thus, innovative approaches are required. Among them, electrochemical oxidation (EO) has shown promising results. In this research, an innovative multidisciplinary approach is proposed to assess the electrochemical oxidation (EO) of diclofenac from wastewater by integrating the investigations on the removal efficiency and by-product identification with the disinfection capacity and the assessment of the effect on environmental geno-toxicity of by-products generated through the oxidation.The electrochemical treatment successfully degraded DCF by achieving >98 % removal efficiency, operating with NaCl 0.02 M at 50 A m−2. By-product identification analyses showed the formation of five DCF parental compounds generated by decarboxylic and CN cleavage reactions.The disinfection capacity of the EO technique was evaluated by carrying out microbiological tests on pathogens generally found in aquatic environments, including two rod-shaped Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), one rod-shaped Gram-positive bacterium (Bacillus atrophaeus), and one Gram-positive coccus (Enterococcus hirae).Eco-toxicity was evaluated in freshwater organisms (algae, rotifers and crustaceans) belonging to two trophic levels through acute and chronic tests. Genotoxicity tests were carried out by Comet assay, and relative expression levels of catalase, manganese and copper superoxide dismutase genes in crustaceans. Results highlight the effectiveness of EO for the degradation of diclofenac and the inactivation of pathogens; however, the downstream mixture results in being harmful to the aquatic ecosystem.

Genome-wide transformation reveals extensive exchange across closely related Bacillus species.

Bacterial transformation is an important mode of horizontal gene transfer that helps spread genetic material across species boundaries. Yet, the factors that pose barriers to genome-wide cross-species gene transfer are poorly characterized. Here, we develop a replacement accumulation assay to study the effects of genomic distance on transfer dynamics. Using Bacillus subtilis as recipient and various species of the genus Bacillus as donors, we find that the rate of orthologous replacement decreases exponentially with the divergence of their core genomes. We reveal that at least 96% of the B. subtilis core genes are accessible to replacement by alleles from Bacillus spizizenii. For the more distantly related Bacillus atrophaeus, gene replacement events cluster at genomic locations with high sequence identity and preferentially replace ribosomal genes. Orthologous replacement also creates mosaic patterns between donor and recipient genomes, rearranges the genome architecture, and governs gain and loss of accessory genes. We conclude that cross-species gene transfer is dominated by orthologous replacement of core genes which occurs nearly unrestricted between closely related species. At a lower rate, the exchange of accessory genes gives rise to more complex genome dynamics.

The combined application of Debaryomyces hansenii and Bacillus atrophaeus inhibits disease development and enhances postharvest quality in litchi fruit by activating flavonoid metabolism

Our previous study demonstrated that the postharvest application of Debaryomyces hansenii (Y-1) and Bacillus atrophaeus (TE-7) significantly reduced decay in litchi fruit, however, the molecular mechanism was not elucidated. Results of the present study indicated that a combined treatment (Y-TE) using both antagonistic strains significantly suppresses an increase in the disease index and inhibits a reduction in the antioxidant properties of litchi fruit during storage at 25 °C. Transcriptomic analysis revealed that genes enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis and plant-pathogen interaction pathways were involved in the response to theY-TE treatment. RT-qPCR analysis indicated that LcMYB102, LcMYB73, and LcMYB33 transcription factors (TFs), and flavonoid synthesis genes, including LcANS and LcF3H, were significantly up-regulated in litchi fruit by the Y-TE treatment after two days of storage. Moreover, an analysis of the metabolome identified eight differentially expressed metabolites associated with flavonoid synthesis, especially formononetin. Furthermore, the regulation of the major pathway and network for flavonoid biosynthesis in litchi fruit by Y-TE was also observed. In summary, our results indicate that the combined application of Y-1 and TE-7 promotes the accumulation of flavonoids in litchi fruit that enhance disease resistance and contributes to the maintenance of quality during storage. The use of these microbial antagonists represents a potential biological approach for the preservation of litchi fruit.

Совместное культивирование азотфиксирующего штамма Azomonas macrocytogenes и штамма-антагониста Bacillus atrophaeus

The possibility of co-cultivation of the nitrogen-fixing strain Azomonas macrocytogenes OSV-2 and the antagonist strain of phytopathogenic fungi Bacillus atrophaeus B-13893 for the purpose of using it in a complex biological preparation with nitrogen-fixing and antimycotic activity was studied. Both strains were isolated by the authors from agricultural soils and identified by the nucleotide sequence of the 16S rRNA gene. It has been established that the strains do not show mutual antagonism and grow well in a joint culture on an artificial nutrient medium. When measuring microcolonies, no statistically significant growth inhibition of the nitrogen-fixing strain A. macrocytogenes OSV-2 was found in the presence of the antagonist strain B. atrophaeus B-13893. Thus, both strains can be used together to improve the nitrogen nutrition of plants and the biological protection of plants from fungal diseases. Keywords: BIOLOGICAL PROTECTION OF PLANTS FROM DISEASES, ANTAGONIST STRAINS, NITROGEN-FIXING STRAINS, BACILLUS ATROPHAEUS, AZOMONAS MACROCYTOGENES, CO-CULTURATION

Beneficial microbial consortium improves winter rye performance by modulating bacterial communities in the rhizosphere and enhancing plant nutrient acquisition.

The beneficial effect of microbial consortium application on plants is strongly affected by soil conditions, which are influenced by farming practices. The establishment of microbial inoculants in the rhizosphere is a prerequisite for successful plant-microorganism interactions. This study investigated whether a consortium of beneficial microorganisms establishes in the rhizosphere of a winter crop during the vegetation period, including the winter growing season. In addition, we aimed for a better understanding of its effect on plant performance under different farming practices. Winter rye plants grown in a long-time field trial under conventional or organic farming practices were inoculated after plant emergence in autumn with a microbial consortium containing Pseudomonas sp. (RU47), Bacillus atrophaeus (ABi03) and Trichoderma harzianum (OMG16). The density of the microbial inoculants in the rhizosphere and root-associated soil was quantified in autumn and the following spring. Furthermore, the influence of the consortium on plant performance and on the rhizosphere bacterial community assembly was investigated using a multidisciplinary approach. Selective plating showed a high colonization density of individual microorganisms of the consortium in the rhizosphere and root-associated soil of winter rye throughout its early growth cycle. 16S rRNA gene amplicon sequencing showed that the farming practice affected mainly the rhizosphere bacterial communities in autumn and spring. However, the microbial consortium inoculated altered also the bacterial community composition at each sampling time point, especially at the beginning of the new growing season in spring. Inoculation of winter rye with the microbial consortium significantly improved the plant nutrient status and performance especially under organic farming. In summary, the microbial consortium showed sufficient efficacy throughout vegetation dormancy when inoculated in autumn and contributed to better plant performance, indicating the potential of microbe-based solutions in organic farming where nutrient availability is limited.

Staphylococcus aureus Bacteriophage 52 Endolysin Exhibits Anti-Biofilm and Broad Antibacterial Activity Against Gram-Positive Bacteria.

Bacteriophage endolysins have been shown to hold great promise as new antibacterial agents for animal and human health in food preservation. In the present study, endolysin from Staphylococcus aureus subsp. aureus ATCC 27692-B1 bacteriophage 52 (LysSA52) was cloned, expressed, and characterized for its antimicrobial properties. Following DNA extraction from bacteriophage 52, a 1446-bp DNA fragment containing the endolysin gene (lysSA52) was obtained by PCR amplification and cloned into pET SUMO expression vector. The positive clone was validated by sequencing and open-reading frame analysis. The LysSA52 sequence shared high homology with staphylococcal phage endolysins of the SA12, SA13, and DSW2 phages and others. The cloned lysSA52 gene encoding 481 amino acids endolysin was expressed in Escherichia coli BL21 with a calculated molecular mass of 66kDa (LysSA52). This recombinant endolysin LysSA52 exhibited lytic activity against 8 of 10 Gram-positive bacteria via agar spot-on lawn antimicrobial assay, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus atrophaeus. In addition, the 0.50mg/mL, LysSA52 endolysins reduced about 60% of the biofilms of S. aureus and S. epidermidis established on a microtiter plate in 12h treatment. The data from this study indicate that LysSA52 endolysin could be used as an antibacterial protein to prevent and treat infections caused by staphylococci and several other Gram-positive pathogenic bacteria irrespective of their antibiotic resistance.

Real-Time Monitoring of H2O2 Sterilization on Individual Bacillus atrophaeus Spores by Optical Sensing with Trapping Raman Spectroscopy

Hydrogen peroxide (H2O2), a strong oxidizer, is a commonly used sterilization agent employed during aseptic food processing and medical applications. To assess the sterilization efficiency with H2O2, bacterial spores are common microbial systems due to their remarkable robustness against a wide variety of decontamination strategies. Despite their widespread use, there is, however, only little information about the detailed time-resolved mechanism underlying the oxidative spore death by H2O2. In this work, we investigate chemical and morphological changes of individual Bacillus atrophaeus spores undergoing oxidative damage using optical sensing with trapping Raman microscopy in real-time. The time-resolved experiments reveal that spore death involves two distinct phases: (i) an initial phase dominated by the fast release of dipicolinic acid (DPA), a major spore biomarker, which indicates the rupture of the spore’s core; and (ii) the oxidation of the remaining spore material resulting in the subsequent fragmentation of the spores’ coat. Simultaneous observation of the spore morphology by optical microscopy corroborates these mechanisms. The dependence of the onset of DPA release and the time constant of spore fragmentation on H2O2 shows that the formation of reactive oxygen species from H2O2 is the rate-limiting factor of oxidative spore death.