Protein Gas Vesicles of Bacillus megaterium as Enhancers of Ultrasound-Induced Transcriptional Regulation.

Gas vesicles (GVs) are large cylindrical gas-filled protein assemblies found in diverse aquatic bacteria that enable their adaptation of buoyancy. GVs have already been used as ultrasound contrasting agents. Here, we investigate GVs derived from Bacillus megaterium, aiming to minimize the number of accessory Gvps within the GV gene cluster and demonstrate the use of GVs as enhancers of acoustic radiation force administered by ultrasound. Three (GvpR, GvpT, and GvpU) out of 11 genes in the cluster were found to be dispensable for functional GV formation, and their omission resulted in narrower GVs. Two essential proteins GvpJ and GvpN were absent from recently determined GV structures, but GvpJ was nevertheless found to be tightly bound to the cylindrical part of GVs in this study. Additionally, the N-terminus of GvpN was observed to play an important role in the formation of mature GVs. The binding of engineered GvpC fromAnabaena flos-aquae to HEK293 cells via integrins enhanced the acoustic force delivered by ultrasound and resulted in an increased Ca2+ influx into cells. Coupling with a synthetic Ca2+-dependent signaling pathway GVs efficiently enhanced cell stimulation by ultrasound, which expands the potentials of noninvasive sonogenetics cell stimulation.

[Effects of Different Microbial Fertilizers on Soil Quality and Maize Yield in Coastal Saline Soil].

Microbial fertilizers have the characteristics of high efficiency and environmental protection in improving saline soils, and the application of functional microbial fertilizers is of great significance for the green abatement of saline barriers and the improvement of soil quality in coastal areas. The experiment was based on moderately saline soil in the coastal area of Hebei Province, with corn as the indicator crop, on the basis of conventional chemical fertilizer application. Different microbial fertilizer treatments, namely, T1 （conventional chemical fertilizer 750 kg·hm-2 + compound microbial agent 75 kg·hm-2）, T2 （conventional chemical fertilizer 750 kg·hm-2 + Bacillus megaterium 300 kg·hm-2）, T3 （conventional chemical fertilizer 750 kg·hm-2 + B. mucilaginosus 300 kg·hm-2）, T4 （conventional chemical fertilizer 750 kg·hm-2 + organic silicon fertilizer 600 kg·hm-2）, T5 （conventional chemical fertilizer 750 kg·hm-2 + bio-organic fertilizer 600 kg·hm-2）, T6 （conventional fertilizer 750 kg·hm-2 + active microalgae 15 kg·hm-2）, and CK （only fertilizer 750 kg·hm-2）, were used for these seven treatments, to study the effects of different microbial fertilizers on soil nutrients, salinity, bacterial community, and corn yield and economic efficiency during two critical periods （V12 stage and maturity stage） of corn. The results showed that compared with that in CK, T1 significantly increased soil total nitrogen （TN） and available phosphorus （AP） contents during the whole growth period. Over the whole reproductive period, soil organic matter （OM） at maturity increased by 10.35% over the V12 stage compared to that in CK, but there was no significant difference between treatments. Compared with that in CK, T5 and T6 significantly reduced soil total salinity and Ca2+ content during the whole growth period by an average of 14.51%-18.48% and 24.25%-25.51%. T1 significantly increased the bacterial diversity index over the whole growth period by 45.16% compared to that in CK. The dominant soil phyla were Actinobacteria, Proteobacteria, Acidobacteria, and Chloroflexi, and the dominant genera were Bacillus and Geminicoccaceae. The most abundant functions of the bacterial community in the study area were chemoheterotrophy and aerobic chemoheterotrophy, with average relative abundances of 28.89% and 27.11%, and T3 and T6 significantly improved soil N cycling function. The results of redundancy analysis （RDA） indicated that Na+, SO42-, pH, and EC were important factors driving the structure of the bacterial community, and correlation heatmaps showed that Na+, SO42-, pH, and EC were significantly and positively correlated mainly with the phylum Planctomycetota, whereas soil OM and TN were significantly and positively correlated with Cyanobacteria. Compared with that in CK, T6 increased the relative abundance of Cyanobacteria and optimized the bacterial community structure during the whole growth period. Using recommended dosages of bacterial fertilizers T1 and T6 increased maize yield by 7.31%-24.83% and economic efficiency by 9.05%-23.23%, respectively. The preliminary results of soil chemical properties and yield correlation analysis revealed that EC, AP, HCO3-, and Mg2+ were the obstacle factors limiting soil productivity in coastal areas. In conclusion, the use of the compound bacterial agent （T1） and active microalgae （T6） at the recommended dosage can significantly enhance soil nutrients, reduce salinity, and improve the structural diversity of soil bacterial communities, which not only ensures the increase in maize yield and efficiency but also realizes the efficient use of microbial fertilizers and the improvement of soil quality.

Microbially induced calcium carbonate precipitation through CO2 sequestration via an engineered Bacillus subtilis.

BACKGROUND: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase-an enzyme with the capability to hydrolyse CO2, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO3 biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry. RESULTS: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO2 and convert it to calcium carbonate. A significant decrease in CO2 was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO2 sequestration. CONCLUSION: Recombinantly produced carbonic anhydrase successfully sequestered CO2 and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Efficacy of DAP coated with bacterial strains and their metabolites for soil phosphorus availability and maize growth.

Phosphorus (P) use efficiency in alkaline/calcareous soils is only 20% due to precipitation of P2O5 with calcium and magnesium. However, coating Diammonium Phosphate (DAP) with phosphorus solubilizing bacteria (PSB) is more appropriate to increase fertilizer use efficiency. Therefore, with the aim to use inorganic fertilizers more effectively present study was conducted to investigate comparative effect of coated DAP with PSB strains Bacillus subtilis ZE15 (MN003400), Bacillus subtilis ZR3 (MN007185), Bacillus megaterium ZE32 (MN003401) and Bacillus megaterium ZR19 (MN007186) and their extracted metabolites with uncoated DAP under axenic conditions. Gene sequencing was done against various sources of phosphorus to analyze genes responsible for phosphatase activity. Alkaline phosphatase (ALP) gene amplicon of 380bp from all tested strains was showed in 1% w/v gel. Release pattern of P was also improved with coated fertilizer. The results showed that coated phosphatic fertilizer enhanced shoot dry weight by 43 and 46% under bacterial and metabolites coating respectively. Shoot and root length up to 44 and 42% with metabolites coated DAP and 41% with bacterial coated DAP. Physiological attributes also showed significant improvement with coated DAP over conventional. The results supported the application of coated DAP as a useful medium to raise crop yield even at lower application rates i.e., 50 and 75% DAP than non-coated 100% DAP application which advocated this coating technique a promising approach for advancing circular economy and sustainable development in modern agriculture.

The role and mechanism of Bacillus megaterium strain A14 in inhibiting the cadmium uptake by peanut plants in acidic red soil.

AIMS: This study explores the potential of cadmium (Cd)-resistant bacteria, specifically Bacillus megaterium A14, to decrease Cd accumulation in peanuts, a crop susceptible to metal uptake from contaminated soils, by understanding the bacterium's impact on plant Cd absorption mechanisms. METHODS AND RESULTS: Through pot experiments, we observed that A14 inoculation significantly increased peanut biomass under Cd stress conditions, primarily by immobilizing the metal and reducing its bioavailability. The bacterium effectively changed the distribution of Cd, with a notable 46.53% reduction in the exchangeable fraction, which in turn limited the expression of genes related to Cd transport in peanuts. Additionally, A14 enhanced the plant's antioxidant response, improving its tolerance to stress. Microbial analysis through 16S sequencing demonstrated that A14 inoculation altered the peanut rhizosphere, particularly by increasing populations of Firmicutes and Proteobacteria, which play crucial roles in soil remediation from heavy metals. CONCLUSION: The A14 strain effectively counters Cd toxicity in peanuts, promoting growth through soil Cd sequestration, root barrier biofilm formation, antioxidant system enhancement, suppression of Cd transport genes, and facilitation of Cd-remediating microorganisms.

High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Production and secretion of recombinant spider silk in Bacillus megaterium.

BACKGROUND: Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. RESULTS: In this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. CONCLUSION: It is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

Bioconversion of glycerol into polyhydroxyalkanoates through an atypical metabolism shift using Priestia megaterium during fermentation processes: A statistical analysis of carbon and nitrogen source concentrations.

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g-x. H-1, where the rate of PHA production was 0.28 g L-1. H-1 and PHA concentration was 4.94 g. L-1. This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown.

Biosynthesis of resveratrol by an endophytic Priestia megaterium PH3 via the phenylpropane pathway.

Resveratrol (RES) is a secondary metabolite synthesized by plants in response to environmental stress and pathogen infection, which is of great significance for the industrial production of RES by fermentation culture. In this study, we aimed to explore the biosynthesis pathway of RES and its key enzymes in the Priestia megaterium PH3, which was isolated and screened from peanut fruit. Through Liquid Chromatography-Mass Spectrometry (LC-MS) analysis, we quantified the RES content and distribution in the culture medium and determined that Priestia megaterium PH3 mainly secreted RES extracellularly. Furthermore, the highest production of RES was observed in YPD, yielding an impressive 127.46 ± 6.11 µg/L. By optimizing the fermentation conditions, we achieved a remarkable RES yield of 946.82 ± 24.74 µg/L within just 2 days, which represents the highest reported yield for a natural isolate produced in such a short time frame. Our investigation revealed that the phenylpropane pathway is responsible for RES synthesis in this bacterium, with cinnamate 4-hydroxylase (C4H) identified as the main rate-limiting enzyme. Overall, our findings highlight the robust RES production capabilities of Priestia megaterium PH3, offering novel insights and potential applications for bacterial fermentation in RES production. KEY POINTS: • RES synthesized by the bacterium was confirmed through the phenylpropane pathway. • The key rate-limiting enzyme for biosynthesis-RES is C4H. • RES reached 946.82 ± 24.74 µg/L after fermentation for 2 days.

The Versatile Biocatalyst of Cytochrome P450 CYP102A1: Structure, Function, and Engineering.

Wild-type cytochrome P450 CYP102A1 from Bacillus megaterium is a highly efficient monooxygenase for the oxidation of long-chain fatty acids. The unique features of CYP102A1, such as high catalytic activity, expression yield, regio- and stereoselectivity, and self-sufficiency in electron transfer as a fusion protein, afford the requirements for an ideal biocatalyst. In the past three decades, remarkable progress has been made in engineering CYP102A1 for applications in drug discovery, biosynthesis, and biotechnology. The repertoire of engineered CYP102A1 variants has grown tremendously, whereas the substrate repertoire is avalanched to encompass alkanes, alkenes, aromatics, organic solvents, pharmaceuticals, drugs, and many more. In this article, we highlight the major advances in the past five years in our understanding of the structure and function of CYP102A1 and the methodologies used to engineer CYP102A1 for novel applications. The objective is to provide a succinct review of the latest developments with reference to the body of CYP102A1-related literature.

Regio- and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X-ray crystallography and computational modeling.

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25-hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio- and stereoselective manner to 16ß-hydroxytestosterone. To reveal the structural determinants governing the regio- and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme-iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon-hydrogen bond was taken into account. Overall, the first-time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site-directed mutagenesis toward improved enzymatic properties of this enzyme.

Enhanced metabolism of 2,3',4,4',5-pentachlorobiphenyl (CB118) by bacterial cytochrome P450 monooxygenase mutants of Bacillus megaterium.

Bacterial cytochrome P450 monooxygenase P450BM3 is a promising enzyme to provide novel substrate specificity and enhanced enzymatic activity. The wild type (WT) has been shown to metabolize the widely distributed polychlorinated biphenyl (PCB) 2,3',4,4',5-pentachlorobiphenyl (CB118) to hydroxylated metabolites. However, this reaction requires the coexistence of perfluoroalkyl carboxylic acids (PFCAs). To locate P450BM3 mutants metabolizing CB118 without PFCAs, mutations were selected from amino acids comprising the substrate-binding cavity and the substrate entrance. The mutant A264G showed enhanced hydroxylation activities compared to the WT for the production of five hydroxylated metabolites. Perfluorooctanoic acid addition provided the highest activity, as found in the WT. The docking model of A264G and CB118 indicated that the enlargement of the space above the heme brought CB118 close to the heme, resulting in high activity. In contrast, the mutants L188Q, QG, LVQ, and GVQ, which contain the L188Q mutation, showed higher activity than WT even without PFCAs. Docking models revealed that the closed form found in substrate binding was induced by the L188Q mutation in the substrate non-binding state of the mutants. These mutants are promising for bioremediation of PCBs using enhanced metabolizing activities.

Integrative transcriptome and metabolome revealed the molecular mechanism of Bacillus megaterium BT22-mediated growth promotion in Arabidopsis thaliana.

Plant growth-promoting rhizobacteria (PGPR) can promote plant growth and protect plants from pathogens, which contributes to sustainable agricultural development. Several studies have reported their beneficial characteristics in facilitating plant growth and development and enhancing plant stress resistance through different mechanisms. However, there is still a challenge to study the molecular mechanism of plant response to PGPR. We integrated the transcriptome and metabolome of Arabidopsis thaliana (Arabidopsis) to understand its responses to the inoculation with an isolated PGPR strain (BT22) of Bacillus megaterium. Fresh shoot weight, dry shoot weight and leaf number of Arabidopsis were increased by BT22 treatment, showing a positive growth-promoting effect. According multi-omics analysis, 878 differentially expressed genes (296 up-regulated, 582 down-regulated) and 139 differentially expressed metabolites (66 up-regulated, 73 down-regulated) response to BT22 inoculation. GO enrichment results indicate that the up-regulated genes mainly enriched in the regulation of growth and auxin response pathways. In contrast, the down-regulated genes mainly enriched in wounding response, jasmonic acid and ethylene pathways. BT22 inoculation regulated plant hormone signal transduction of Arabidopsis, including auxin and cytokinin response genes AUX/IAA, SAUR, and A-ARR related to cell enlargement and cell division. The contents of nine flavonoids and seven phenylpropanoid metabolites were increased, which help to induce systemic resistance in plants. These results suggest that BT22 promoted Arabidopsis growth by regulating plant hormone homeostasis and inducing metabolome reprogramming.

Bacterial spore germination receptors are nutrient-gated ion channels.

Bacterial spores resist antibiotics and sterilization and can remain metabolically inactive for decades, but they can rapidly germinate and resume growth in response to nutrients. Broadly conserved receptors embedded in the spore membrane detect nutrients, but how spores transduce these signals remains unclear. Here, we found that these receptors form oligomeric membrane channels. Mutations predicted to widen the channel initiated germination in the absence of nutrients, whereas those that narrow it prevented ion release and germination in response to nutrients. Expressing receptors with widened channels during vegetative growth caused loss of membrane potential and cell death, whereas the addition of germinants to cells expressing wild-type receptors triggered membrane depolarization. Therefore, germinant receptors act as nutrient-gated ion channels such that ion release initiates exit from dormancy.

Study on the mechanism of biochar affecting the effectiveness of phosphate solubilizing bacteria.

Low phosphorus utilization and phosphorus fertilizer pollution are serious issues primarily affecting soil health. To investigate the effects of biochar on the growth, phosphorus solubilization, and metabolites of phosphorus-solubilizing bacteria (PSB), rice husk biochar (RH) and rice straw biochar (RS) were incubated with Bacillus megatherium (BM1) and Bacillus mucilaginosus (BM2), respectively. The highest phosphorus solubilization was observed in BM2 following the addition of RS. The dissolved amount of phosphorus was 244.99 mg/L, which was 43.86% higher than that of the control group. Hence, biochar can improve the phosphorus solubilization capacity of PSB by affecting the organic acid and polysaccharide contents, and phosphatase activity secreted by the PSB, as the porous structure and surface characteristics of biochar ensured the adsorption of PSB. This study can help improve the functional activity of PSB and provide basis for improving the utilization of soil phosphorus, which in turn, aid in the development of biochar-based microbial fertilizers.

A non-K+-solubilizing PGPB (Bacillus megaterium) increased K+ deprivation tolerance in Oryza sativa seedlings by up-regulating root K+ transporters.

Potassium is one of the principal macronutrients required by all plants, but its mobility is restricted between soil compartments. Numerous studies have shown that Plant Growth Promoting Bacteria (PGPB) can facilitate nutrient uptake. The present work examined the effects of the PGPB (Bacillus megaterium) on rice plants subjected to potassium deprivation. To study only direct effects of B. megaterium, we first checked its lack of capacity to solubilize soil K. Rice plants were provided with 1.5 mM K (100%) or 0.015 mM K (1%) and growth related parameters, nutrient concentrations and gene expression of K+ transporters were determined. After two weeks, the 1% K treatment reduced growth of non-inoculated plants by about 50% compared with the 100% K treatment. However, there was no effect of reduced K nutrition on growth of inoculated plants. The reduction in growth in non-inoculated plants was accompanied by a similar reduction in K+ concentration in both roots and leaves and an overall 80% reduction of the plant potassium concentrations. In inoculated plants a 50% reduction occurred only in leaves. The expression of the K+ transporters HKT1;1, 1;2, 1;5, 2;2, 2;3 and 2;4 was up-regulated by the inoculation of B. megaterium under K deprivation conditions, explaining their higher K tissue concentrations and growth. Thus, the bacterial strain improved plant potassium nutrition without affecting K+ availability in the soil. The results demonstrate the potential of this bacteria for using as a biofertilizer to reduce the amount of potassium fertilizers to be applied in the field.

Degradation of alkane hydrocarbons by Priestia megaterium ZS16 and sediments consortia with special reference to toxicity and oxidative stress induced by the sediments in the vicinity of an oil refinery.

Petroleum hydrocarbon is a critical ecological issue with impact on ecosystems through bioaccumulation. It poses significant risks to human health. Due to the extent of alkane hydrocarbon pollution in some environments, biosurfactants are considered as a new multifunctional technology for the efficient removal of petroleum-based contaminants. To this end, Yamuna river sediments were collected at different sites in the vicinity of Mathura oil refinery, UP (India). They were analysed by atomic absorption spectrophotometry and gas chromatography-mass spectrometry (GC-MS) for heavy metals and organic pollutants. Heptadecane, nonadecane, oleic acid ester and phthalic acid were detected. In total 107 bacteria were isolated from the sediments and screened for biosurfactant production. The most efficient biosurfactant producing strain was tested for its capability to degrade hexadecane efficiently at different time intervals (0 h, 7 d, 14 d and 21 d). FT-IR analysis defined the biosurfactant as lipopeptide. 16S rRNA gene sequencing identified the bacterium as Priestia megaterium. The strain lacks resistance to common antibiotics thus making it an important candidate for remediation. The microbial consortia present in the sediments were also investigated for their capability to degrade C16, C17 and C18 alkane hydrocarbons. By using gas chromatography-mass spectrophotometry the metabolites were identified as 1-docosanol, dodecanoic acid, 7-hexadecenal, (Z)-, hexadecanoic acid, docosanoic acid, 1-hexacosanal, 9-octadecenoic acid, 3-octanone, Z,Z-6,28-heptatriactontadien-2-one, heptacosyl pentafluoropropionate, 1,30-triacontanediol and decyl octadecyl ester. Oxidative stress in Vigna radiata L. roots was observed by using Confocal Laser Scanning Microscopy. A strong reduction in seed germination and radicle and plumule length was observed when Vigna radiata L. was treated with different concentrations of sediment extracts, possibly due to the toxic effects of the pollutants in the river sediments. Thus, this study is significant since it considers the toxicological effects of hydrocarbons and to degrade them in an environmentally friendly manner.

Characterization of a novel detergent-resistant type I pullulanase from Bacillus megaterium Y103 and its application in laundry detergent.

This study aims to find a moderate pullulanase for detergent industry. The pulY103B gene (2217 bp) from Bacillus megaterium Y103 was cloned and expressed in Escherichia coli. PulY103B contained four conserved regions of glycoside hydrolase family (GH) 13 and the typical sequence of type I pullulanase. The optimal reaction conditions of PulY103B were pH 6.5 and 40 °C. In addition, it remained stable below 40 °C and over 80% of activity was retained at pH ranging from 6.0 to 8.5. The best substrate for the enzyme was pullulan. Furthermore, it exhibited activity toward wheat starch (36.5%) and soluble starch (33.4%) but had no activity toward amylose and glycogen. Maltotriose and maltohexaose were major pullulan hydrolysis products. Soluble starch and amylopectin were mainly hydrolyzed into maltotetraose. These results indicated that PulY103B is a novel type I pullulanase with transglycosylation activity via formation of α-1,4-glucosidic linkages. Moreover, PulY103B was strongly stimulated by nonionic detergents [viz, Tween 20 (10%), Tween 80 (1%), Triton X-100 (20%)] and commercial liquid detergents (3.0 g/L). Wash performance tests demonstrated that the mixture of PulY103B and detergent removed starch-based stains better than using detergent alone (p < 0.05). Therefore, this pullulanase has big potential as a detergent additive.

Tryptophan-96 in cytochrome P450 BM3 plays a key role in enzyme survival.

Flavocytochrome P450 from Bacillus megaterium (P450BM3 ) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long-chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

The antibacterial mechanism of phenylacetic acid isolated from Bacillus megaterium L2 against Agrobacterium tumefaciens.

Background: Agrobacterium tumefaciens T-37 can infect grapes and other fruit trees and cause root cancer. Given the pollution and damage of chemical agents to the environment, the use of biological control has become an important area of focus. Bacillus megaterium L2 is a beneficial biocontrol strain isolated and identified in the laboratory, which has a good antibacterial effect on a variety of plant pathogens. The antibacterial metabolites of L2 were separated and purified to obtain a bioactive compound phenylacetic acid (PAA). Methods: The potential antibacterial mechanism of PAA against A. tumefaciens T-37 strain was determined by relative conductivity, leakage of nucleic acids, proteins, and soluble total sugars, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reactive oxygen species (ROS). Results: PAA showed good antibacterial activity against strain A. tumefaciens T-37 with IC50 of 0.8038 mg/mL. Our data suggested that after treatment with PAA, the relative conductivity, nucleic acid, protein, and total soluble sugar of T-37 were increased significantly compared with the chloramphenicol treatment group and the negative treatment group. The total protein synthesis of T-37 cells was inhibited, the consumption of phosphorus decreased with the increase of incubation time, and the content of ROS was significantly higher than that in the negative treatment group. Meanwhile, the activity of two key enzymes (MDH and SDH) involved in the tricarboxylic acid cycle (TCA cycle) decreased. In addition, T-37 cells were found to be damaged by scanning electron microscopy observation. Our results showed that PAA can destroy cell membrane integrity, damage cell structures, affect cell metabolism, and inhibit protein synthesis to exert an antibacterial effect. Conclusions: We concluded that the mechanism of action of the PAA against strain T-37 might be described as PAA exerting antibacterial activity by affecting cell metabolism, inhibiting protein synthesis, and destroying cell membrane integrity and cell ultrastructure. Therefore, PAA has a promising application prospect in the prevention and treatment of root cancer disease caused by A. tumefaciens.