Characterization of three novel stem rot pathogens and their antagonistic endophytic bacteria associated with Cistanche deserticola.

Cistanche deserticola is a precious Chinese medicinal material with extremely high health care and medicinal value. In recent years, the frequent occurrence of stem rot has led to reduced or even no harvests of C. deserticola. The unstandardized use of farm chemicals in the prevention and control processes has resulted in excessive chemical residues, threatening the fragile desert ecological environment. Therefore, it is urgent to explore safe and efficient prevention and control technologies. Biocontrol agents, with the advantages of safety and environment-friendliness, would be an important idea. The isolation, screening and identification of pathogens and antagonistic endophytic bacteria are always the primary basis. In this study, three novel pathogens causing C. deserticola stem rot were isolated, identified and pathogenicity tested, namely Fusarium solani CPF1, F. proliferatum CPF2, and F. oxysporum CPF3. For the first time, the endophytic bacteria in C. deserticola were isolated and identified, of which 37 strains were obtained. Through dual culture assay, evaluation experiment and tissue culture verification, a biocontrol candidate strain Bacillus atrophaeus CE6 with outstanding control effect on the stem rot was screened out. In the tissue culture system, CE6 showed excellent control effect against F. solani and F. oxysporum, with the control efficacies reaching 97.2% and 95.8%, respectively, indicating its great potential for application in the production. This study is of great significance for the biocontrol of plant stem rot and improvement of the yield and quality of C. deserticola.

Proteome of spores from biological indicators in sterilization processes: Bacillus pumilus and Bacillus atrophaeus.

Bacillus atrophaeus and Bacillus pumilus spores are widely used as biological indicators to assess the effectiveness of decontamination procedures. Spores are intricate, multi-layered cellular structures primarily composed of proteins, which significantly contribute to their extreme resistance. Therefore, conducting a comprehensive proteome analysis of spores is crucial to identify the specific proteins conferring spore resistance. Here, we employed a high-throughput shotgun proteomic approach to compare the spore proteomes of B. atrophaeus DSM675 and B. pumilus DSM492, identifying 1312 and 1264 proteins, respectively. While the overall number of proteins found in both strains is roughly equivalent, a closer examination of a subset of 54 spore-specific proteins revealed noteworthy distinctions. Among these 54 proteins, 23 were exclusively detected in one strain, while others were shared between both. Notably, of the 31 proteins detected in both strains, 10 exhibited differential abundance levels, including key coat layer morphogenetic proteins. The exploration of these 54 proteins, considering their presence, absence, and differential abundance, provides a unique molecular signature that may elucidate the differences in sensitivity/resistance profiles between the two strains.

Impact of filter material and holding time on spore sampling efficiency in water.

Bacillus anthracis and other environmentally persistent pathogens pose a significant threat to human and environmental health. If contamination is spread over a wide area (e.g. resulting from a bioterrorism or biowarfare incident), readily deployable and scalable sample collection methods will be necessary for rapidly developing and implementing effective remediation strategies. A recent surge in environmental (eDNA) sampling technologies could prove useful for quantifying the extent and levels of contamination from biological agents in environmental and drinking water. In this study, three commonly used membrane filtration materials (cellulose acetate, cellulose nitrate, and nylon) were evaluated for spore filtration efficiency, yielding recoveries from 17%-68% to 25%-117% for high and low titer samples, respectively, where cellulose nitrate filters generated the highest recoveries. A holding time test revealed no statistically significant differences between spore recoveries when analyzed at the specified timepoints, suggesting that eDNA filter sampling techniques can yield and maintain a relatively high recovery of spores for an extended period of time between filtration and analysis without a detrimental impact on spore recoveries. The results shown here indicate that emerging eDNA technologies could be leveraged for sampling following a wide-area contamination incident and for other microbiological water sampling applications.

Exploration of the Biocontrol Activity of Bacillus atrophaeus Strain HF1 against Pear Valsa Canker Caused by Valsa pyri.

Valsa pyri-induced pear Valsa canker is among the most prevalent diseases to impact pear quality and yields. Biocontrol strategies to control plant disease represent an attractive alternative to the application of fungicides. In this study, the potential utility of Bacillus atrophaeus strain HF1 was assessed as a biocontrol agent against pear Valsa canker. Strain HF1 suppressed V. pyri mycelium growth by 61.20% and induced the development of malformed hyphae. Both culture filtrate and volatile organic compounds (VOCs) derived from strain HF1 were able to antagonize V. pyri growth. Treatment with strain HF1-derived culture filtrate or VOCs also induced the destruction of hyphal cell membranes. Headspace mixtures prepared from strain HF1 were analyzed, leading to the identification of 27 potential VOCs. Of the thirteen pure chemicals tested, iberverin, hexanoic acid, and 2-methylvaleraldehyde exhibited the strongest antifungal effects on V. pyri, with respective EC50 values of 0.30, 6.65, and 74.07 µL L-1. Fumigation treatment of pear twigs with each of these three compounds was also sufficient to prevent the development of pear Valsa canker. As such, these results demonstrate that B. atrophaeus strain HF1 and the volatile compounds iberverin, hexanoic acid, and 2-methylvaleraldehyde exhibit promise as novel candidate biocontrol agents against pear Valsa canker.

Pharmacological potential of seaweed-associated heterotrophic bacterium Bacillus atrophaeus.

Extremities in marine environmental conditions led the marine macroalga-associated bacteria to adapt and biosynthesize potential bioactive agents. The myriad of marine macroalgae and the bacterial flora they are associated with constitute a potential source of bioactive components with significant biotechnological and pharmacological applications. Heterotrophic bacteria associated with the intertidal macroalgae were isolated and assessed for their pharmacological properties. Subsequently, Firmicutes dominated more than half of the 152 cultivable isolates from macroalgae-associated bacteria collected from the Gulf of Mannar (9°17'0'' N, 79°7'0'' E), on Peninsular India's southern coast. A total of 43 of those demonstrated steady antibacterial activities against a wide range of nosocomial pathogens. Among the bacteria isolated from marine macroalgae, Bacillus atrophaeus SHB2097 (MW821482) exhibited significant antimicrobial activities against clinically important pathogens. Organic extract of B. atrophaeus SHB2097 showed potential antimicrobial activities against test pathogens (minimum inhibitory concentration 6.25 µg/mL). Organic extract of B. atrophaeus SHB2097 revealed promising inhibition potential against cyclooxygenase-2 (IC90 53.26 µg/mL) and 5-lipoxygenase (IC90 9.74 µg/mL). The carbolytic enzyme α-glucosidase inhibition potential of the organic extract of the studied heterotrophic bacterium was significantly greater than (IC90 118 µg/mL) than that displayed by acarbose (IC90 645 µg/mL, p < 0.05). The significance of nuclear magnetic resonance-centered analyses of distinguishing signals in the organic extract and correlating those with bioactive potential was accentuated. The utilities of nuclear magnetic resonance-based fingerprinting emphasized the assessment of the distinctive signals in the solvent extracts and their correlation with the pharmacological properties. Thus, the heterotrophic B. atrophaeus SHB2097 could be used to develop potential therapeutic and biomedical agents.

Antibiotic-active heterotrophic Firmicutes sheltered in seaweeds: can they add new dimensions to future antimicrobial agents?

Appearance of drug-resistant microorganisms prompted researchers to unravel new environments for development of novel antimicrobial agents. Culture-supported analysis of heterotrophic bacteria associated with seaweeds yielded 152 strains, in that larger share of the isolates was embodied by Bacillus atrophaeus SHB2097 (54%), B. velezensis SHB2098 (24%), B. subtilis SHB2099 (12%), and B. amyloliquefaciens SHB20910 (10%). One of the most active strains characterized as B. atrophaeus SHB2097 (MW821482) with an inhibition zone more than 30 mm on spot-over-lawn experiment, was isolated from a seaweed Sargassum wightii, was selected for bioprospecting studies. Significant antibacterial potential was displayed by bacterial organic extract against vancomycin-resistant Enterococcus faecalis, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Klebsiella pneumonia with minimum inhibitory concentration 6.25 µg/mL and comparable to the antibiotics ampicillin and chloramphenicol. The genes of type 1 pks (MZ222383, 700 bp) and hybrid nrps/pks (MZ222389, 1000-1400 bp) of B. atrophaeus MW821482 could be amplified. The bacterium displayed susceptibility to the commercially available antibiotic agents, and was negative for the pore-forming non-hemolytic hemolysin BL (hbl) and enterotoxin (nhe) genes, and therefore, was not pathogenic. The bacterium was found to possess genes (1000-1400 bp) involved in the biosynthesis of siderophore-class of compounds (MZ222387 and MZ222388) that showed 99% of similarity in BLAST search, and showed production of siderophore. Noteworthy antibacterial activities against clinically important pathogenic bacteria in conjunction with occurrence of genes coding for antimicrobial metabolites inferred that the marine heterotrophic bacterium B. atrophaeus SHB2097 could be used for the development of antibacterial agents against the emerging antibiotic resistance.

Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores.

The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

Deciphering the Biomolecules from Bacillus atrophaeus NMB01 Untangles the Anti-Oomycetes Action of Trioxsalen and Corynan-17-ol, Against Phytophthora infestans Inciting Late Blight of Potato.

The antagonistic Bacillus spp. is known well for the production of versatile antimicrobial biomolecules with broad spectrum of action against different types of plant pathogens. Considering the significance of metabolically active biomolecules, attempts were made to decipher the anti-oomycete nature of biomolecules produced by Bacillus atrophaeus NMB01 during di-trophic interaction with Phytophthora infestans. Ten biomolecules produced by B. atrophaeus NMB01 during di-trophic interaction with P. infestans were docked against the twelve target proteins of P. infestans. Molecular docking of biomolecules reported trioxsalen and corynan-17-ol,18,19-didehydro-10-methoxy-acetate(ester) as best hits with highest binding energy in the range of - 7.5 to - 5 kcal/mol against target proteins of P. infestans. Comparatively less binding energy was observed for commercially available fungicides mandipropamid and metalaxyl on docking against the target proteins of P. infestans. We also confirmed the direct impact of trioxsalen andcorynan-17-ol, on P. infestans under in vitro with 66% and 50% inhibition of mycelial growth of P. infestans, respectively. This is the first study attempted to untangle the role of bioactive anti-oomycete compounds produced by B. atrophaeus strain NMB01 during di-trophic interaction with P. infestans against late blight pathogen P. infestans infecting potato. From the present study, we conclude that the biomolecules, trioxsalen and corynan-17-ol, can be explored for the management of P. infestans, the incitant of late blight of potato. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01044-7.

Lipopeptide production by Bacillus atrophaeus strain B44 and its biocontrol efficacy against cotton rhizoctoniosis.

OBJECTIVES: An assay was conducted to show the comparisons the effects of nine metal ions on antagonistic metabolites (lipopeptides, siderophores and gibberellins) by Bacillus atrophaeus strain B44 using well-diffusion assays, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, chrome azurol S plus mannitol salt agar (CAS-MSA) tests, and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. This assay is also designed to demonstrate the biocontrol efficacy of B44 against cotton rhizoctoniosis using pot culture tests. RESULTS: Both the lipopeptide yield and the antimicrobial activity of B44 increase with the MnSO4, MgSO4, CaCO3, and CuSO4 treatments and either have no effect or decreased lipopeptide yield and antimicrobial activity with the FeSO4, K2HPO4, KCl, KH2PO4 and ZnSO4 treatments. The medium containing MgSO4 has no significant effect on either the lipopeptide yield or antimicrobial activity. MALDI-TOF-MS analysis shows a broad range of m/z peaks, indicating that strain B44 produces a complex mixture of iturin, surfactin, and fengycin lipopeptides. Gibberellin production by strain B44 varies greatly depending on the culture medium, and the siderophore production is not significantly affected by the culture medium. Pot tests show that lipopeptide production affects the disease control efficacy of strain B44. CONCLUSION: The biocontrol efficacy of B. atrophaeus strain B44 is related to the lipopeptide yield. Moreover, B. atrophaeus strain B44 significantly increases the size of cotton seedlings, which is related to the GA3 concentration.

Decontamination of soil contaminated at the surface with Bacillus anthracis spores using dry thermal treatment.

In the event of a large, aerosol release of Bacillus anthracis spores in a major metropolitan area, soils and other outdoor materials may become contaminated with the biological agent. A study was conducted to assess the in-situ remediation of soil using a dry thermal treatment approach to inactivate a B. anthracis spore surrogate inoculated into soil samples. The study was conducted in two phases, using loam, clay and sand-based soils, as well as biological indicators and spore-inoculated stainless-steel coupons. Initial experiments were performed in an environmental test chamber with temperatures controlled between 80 and 110 °C, with and without added humidity, and with contact times ranging from 4 h to 7 weeks. Tests were then scaled up to assess the thermal inactivation of spores in small soil columns, in which a heating plate set to 141 °C was applied to the soil surface. These column tests were conducted to assess time requirements to inactivate spores as a function of soil depth and soil type. Results from the initial phase of testing showed that increasing the temperature and relative humidity reduced the time requirements to achieve samples in which no surrogate spores were detected. For the test at 80 °C with no added humidity, 49 days were required to achieve soil samples with no spores detected in clay and loam. At 110 °C, 24 h were required to achieve samples in which no spores were detected. In the column tests, no spores were detected at the 2.5 cm depth at four days and at the 5.1 cm depth at 21 days, for two of the three soils. The experiments described in the study demonstrate the feasibility of using dry thermal techniques to decontaminate soils that have been surficially contaminated with B. anthracis spores.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus.

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.

Effect of Sporulation Conditions Following Submerged Cultivation on the Resistance of Bacillus atrophaeus Spores against Inactivation by H2O2.

The resistance formation of spores in general and of Bacillus atrophaeus in particular has long been the focus of science in the bio-defense, pharmaceutical and food industries. In the food industry, it is used as a biological indicator (BI) for the evaluation of the inactivation effects of hydrogen peroxide in processing and end packaging lines' sterilization. Defined BI resistances are critical to avoid false positive and negative tests, which are salient problems due to the variable resistance of currently available commercial BIs. Although spores for use as BIs have been produced for years, little is known about the influence of sporulation conditions on the resistance as a potential source of random variability. This study therefore examines the dependence of spore resistance on the temperature, pH and partial oxygen saturation during submerged production in a bioreactor. For this purpose, spores were produced under different sporulation conditions and their resistance, defined by the D-value, was determined using a count reduction test in tempered 35% liquid hydrogen peroxide. The statistical analysis of the test results shows a quadratic dependence of the resistance on the pH, with the highest D­values at neutral pH. The sporulation temperature has a linear influence on the resistance. The higher the temperature, the higher the D­value. However, these factors interact with each other, which means that the temperature only influences the resistance when the pH is within a certain range. The oxygen partial pressure during sporulation has no significant influence. Based on the data obtained, a model could be developed enabling the resistance of BIs to be calculated, predicted and standardized depending on the sporulation conditions. BI manufacturers could thus produce BIs with defined resistances for the validation of sterilization effects in aseptic packaging/filling lines for the reliable manufacture of shelf-stable and safe food products.

Comparison of surface sampling methods for an extended duration outdoor biological contamination study.

Bacillus anthracis, the causative agent for anthrax, is a dangerous pathogen to humans and has a history as a bioterrorism agent. While sampling methods have been developed and evaluated for characterizing and clearing contaminated indoor sites, the performance of these sampling methods is unknown for use in outdoor environments. This paper presents surface sampling data for Bacillus atrophaeus spores, a surrogate for B. anthracis, from a 210-day outdoor study that evaluated the detection and recovery of spores using five different sampling methods as follows: sponge sticks, 37-mm vacuum filter cassettes, residential wet vacuums, robotic floor cleaners, and grab samples of soil, leaves, and grass. The spores were applied by spraying a liquid suspension onto the surfaces. Both asphalt and concrete surfaces were sampled by all the surface sampling methods, excluding grab sampling. Stainless steel coupons placed outdoors were additionally sampled using sponge sticks. Sampling methods differed in their ability to collect detectable spores over the duration of the study. The 37-mm vacuums and sponge sticks consistently detected spores on asphalt through day 37 and robots through day 99. The wet vacuums detected spores on asphalt for days 1 and 4, but not again until day 210. On concrete, all samplers detected spores until day 210 except for sponge stick samplers that detected spores only up until the day 99 time point. For all sampling methods, spore recoveries were higher from concrete than from asphalt surfaces. There was no statistically significant difference in recoveries of sponge sticks and 37-mm vacuums from either asphalt or concrete surfaces. Processing of grab samples was challenging due to non-target background microorganisms resulting in high detection limits for the samples.

Two sporulated Bacillus enhance immunity in Galleria mellonella protecting against Candida albicans.

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.

ytiB and ythA Genes Reduce the Uranium Removal Capacity of Bacillus atrophaeus.

Two Bacillus atrophaeus strains, the first being a highly stress-resistant ATCC 9372 strain and the Ua strain identified from a chromium mine by our lab, differ in their abilities to tolerate and remove Uranium (VI) from contaminated water. An increase in U(VI) concentration in growth media led to a decrease in the tolerance and bio-remedial capacity of both strains. However, under high concentrations of U(VI) in the growth media, the ATCC 9372 strain demonstrated a higher tolerance and a higher removal capacity than the Ua strain. Two approaches, transcriptome sequencing and transgenic technology, were used to elucidate the relationship between particular genes within these two strains and their U(VI) removal capacity. Sequencing confirmed the expression of two genes unique to the Ua strain, previously designated ytiB and ythA. They encode putative proteins that show the highest levels of identity to carbonic anhydrase and cytochrome bd terminal oxidase I, respectively. Using the pBE-S DNA vector, ytiB and ythA were transformed into the ATCC 9372 strain of Bacillus atrophaeus. Under a U(VI) concentration of 120 mg/L, the removal rates of the transgenic ATCC 9372-ytiB and ATCC 9372-ythA strains decreased by 7.55% and 7.43%, respectively, compared to the removal rate of the control strain transformed with empty plasmid. The results suggest that both ythA and ytiB genes have a negative influence on the uranium removing capacity of Bacillus atrophaeus. This finding will help to elucidate the molecular mechanisms of uranium removal by bacteria.

Label-Free Proteomics of a Defined, Binary Co-culture Reveals Diversity of Competitive Responses Between Members of a Model Soil Microbial System.

Interactions among members of microbial consortia drive the complex dynamics in soil, gut, and biotechnology microbiomes. Proteomic analysis of defined co-cultures of well-characterized species provides valuable information about microbial interactions. We used a label-free approach to quantify the responses to co-culture of two model bacterial species relevant to soil and rhizosphere ecology, Bacillus atrophaeus and Pseudomonas putida. Experiments determined the ratio of species in co-culture that would result in the greatest number of high-confidence protein identifications for both species. The 281 and 256 proteins with significant shifts in abundance for B. atrophaeus and P. putida, respectively, indicated responses to co-culture in overall metabolism, cell motility, and response to antagonistic compounds. Proteins associated with a virulent phenotype during surface-associated growth were significantly more abundant for P. putida in co-culture. Co-culture on agar plates triggered a filamentous phenotype in P. putida and avoidance of P. putida by B. atrophaeus colonies, corroborating antagonistic interactions between these species. Additional experiments showing increased relative abundance of P. putida under conditions of iron or zinc limitation and increased relative abundance of B. atrophaeus under magnesium limitation were consistent with patterns of changes in abundance of metal-binding proteins during co-culture. These results provide details on the nature of interactions between two species with antagonistic capabilities. Significant challenges remaining for the development of proteomics as a tool in microbial ecology include accurate quantification of low-abundance peptides, especially from rare species present at low relative abundance in a consortium.

Substrate selection of adenylation domains for nonribosomal peptide synthetase (NRPS) in bacillamide C biosynthesis by marine Bacillus atrophaeus C89.

Nonribosomal peptide synthetases (NRPSs) are multi-modular enzymes involved in the biosynthesis of natural products. Bacillamide C was synthesized by Bacillus atrophaeus C89. A nonribosomal peptide synthetase (NRPS) cluster found in the genome of B. atrophaeus C89 was hypothesized to be responsible for the biosynthesis of bacillamide C using alanine and cysteine as substrates. Here, the structure analysis of adenylation domains based on homologous proteins with known crystal structures indicated locations of the substrate-binding pockets. Molecular docking suggested alanine and cysteine as the potential substrates for the two adenylation domains in the NRPS cluster. Furthermore, biochemical characterization of the purified recombinant adenylation domains proved that alanine and cysteine were the optimum substrates for the two adenylation domains. The results provided the in vitro evidence for the hypothesis that the two adenylation domains in the NRPS of B. atrophaeus C89 preferentially select alanine and cysteine, respectively, as a substrate to synthesize bacillamide C. Furthermore, this study on substrates selectivity of adenylation domains provided basis for rational design of bacillamide analogs.

Antifungal activity of the bioactive substance from Bacillus atrophaeus strain HAB-5 and its toxicity assessment on Danio rerio.

The prevalence of pathogen inhibitors bacteria has motivate the study for antimicrobial compounds. Bioactive fungicide have always received considerable attention. A bacterial isolated strain HAB-5 showed antifungal activity against plant fungi. Based on morphological, physiological, biochemical and 16SrDNA sequence analysis, the strain was identified to be a Bacillus atrophaeus. This strain possessed a broad spectrum antifungal activity against various plant pathogenic fungi. Extraction of antifungal substance was performed and the crude extract had potent antifungal ability and showed great potential for swelling and inhibiting spore germination. This antifungal displayed heat stability and active in a wide pH range 5.0-10.0. Moreover no reduction was found in its activity after enzyme treatment. The toxicity test was evaluated in Danio rerio. The acute toxicity test indicated that the 24, 48, 72, 96h LC50 values of UMTLS to the zebrafish were 14.4, 13.8, 13.4, and 12.9%, respectively. Based on the results obtained in this study, antifungal substance was not toxic to zebra. Analyses of disease suppression showed that HAB-5 was effective to reduce the incidence of anthracnose symptoms on mango fruits, also prevent disease infection and protect tobacco seedling from Phytophtora nicotianae. The bioactive substance from Bacillus atrophaeus HAB-5 could be a candidate in the generation of new antifungal agents in crop.

Screening of novel bacteria for the 2,3-butanediol production.

Biotechnologically produced 2,3-butanediol (2,3-BDO) is a potential starting material for industrial bulk chemicals such as butadiene or methyl ethyl ketone which are currently produced from fossil feedstocks. So far, the highest 2,3-BDO concentrations have been obtained with risk group 2 microorganisms. In this study, three risk group 1 microorganisms are presented that are so far unknown for an efficient production of 2,3-BDO. The strains Bacillus atrophaeus NRS-213, Bacillus mojavensis B-14698, and Bacillus vallismortis B-14891 were evaluated regarding their ability to produce high 2,3-BDO concentrations with a broad range of different carbon sources. A maximum 2,3-BDO concentration of 60.4 g/L was reached with the strain B. vallismortis B-14891 with an initial glucose concentration of 200 g/L within 55 h in a batch cultivation. Besides glucose, B. vallismortis B-14891 converts 14 different substrates that can be obtained from residual biomass sources to 2,3-BDO. Therefore B. vallismortis B-14891 is a promising candidate for the large-scale production of 2,3-BDO with low-cost substrates.

Effect of static magnetic field on endospore germination.

This work investigated the effect of static magnetic field (SMF) on Bacillus atrophaeus endospore germination. Germination was triggered by L-alanine in 1.3-T SMF and characterized by ion release, Ca2+ -dipicolinic acid release, and water influx. These events were monitored by electrical conductivity, Tb-DPA fluorescence, and optical density, respectively. Culturability of endospore germinated in SMF exposure was evaluated by CFU enumeration. Results indicated that 1.3-T SMF failed to significantly affect endospore germination and culturability, suggesting that the three aforementioned processes were not sensitive to SMF. Bioelectromagnetics. 38:121-127, 2017. © 2016 Wiley Periodicals, Inc.