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1.
PLoS One ; 15(4): e0231426, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271848

RESUMO

Demand for agricultural crop continues to escalate in response to increasing population and damage of prime cropland for cultivation. Research interest is diverted to utilize soils with marginal plant production. Moisture stress has negative impact on crop growth and productivity. The plant growth promoting rhizobacteria (PGPR) and plant growth regulators (PGR) are vital for plant developmental process under moisture stress. The current study was carried out to investigate the effect of PGPR and PGRs (Salicylic acid and Putrescine) on the physiological activities of chickpea grown in sandy soil. The bacterial isolates were characterized based on biochemical characters including Gram-staining, P-solubilisation, antibacterial and antifungal activities and catalases and oxidases activities and were also screened for the production of indole-3-acetic acid (IAA), hydrogen cyanide (HCN) and ammonia (NH3). The bacterial strains were identified as Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium based on the results of 16S-rRNA gene sequencing. Chickpea seeds of two varieties (Punjab Noor-2009 and 93127) differing in sensitivity to drought were soaked for 3 h before sowing in fresh grown cultures of isolates. Both the PGRs were applied (150 mg/L), as foliar spray on 20 days old seedlings of chickpea. Moisture stress significantly reduced the physiological parameters but the inoculation of PGPR and PGR treatment effectively ameliorated the adverse effects of moisture stress. The result showed that chickpea plants treated with PGPR and PGR significantly enhanced the chlorophyll, protein and sugar contents. Shoot and root fresh (81%) and dry weights (77%) were also enhanced significantly in the treated plants. Leaf proline content, lipid peroxidation and antioxidant enzymes (CAT, APOX, POD and SOD) were increased in reaction to drought stress but decreased due to PGPR. The plant height (61%), grain weight (41%), number of nodules (78%) and pod (88%), plant yield (76%), pod weight (53%) and total biomass (54%) were higher in PGPR and PGR treated chickpea plants grown in sandy soil. It is concluded from the present study that the integrative use of PGPR and PGRs is a promising method and eco-friendly strategy for increasing drought tolerance in crop plants.


Assuntos
Agricultura , Bacillaceae/fisiologia , Cicer/crescimento & desenvolvimento , Reguladores de Crescimento de Planta/farmacologia , Amônia/metabolismo , Bacillaceae/genética , Bacillaceae/isolamento & purificação , Bacillus megaterium/genética , Bacillus megaterium/isolamento & purificação , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Bacillus subtilis/fisiologia , Biomassa , Clorofila/análise , Cicer/efeitos dos fármacos , Cicer/metabolismo , Ácidos Indolacéticos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Putrescina/metabolismo , Putrescina/farmacologia , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Chuva , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Microbiologia do Solo
2.
J Sci Food Agric ; 100(3): 1164-1173, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31680258

RESUMO

BACKGROUND: This study was conducted to analyze the effects of endophytic Bacillus megaterium (BM 18-2) colonization on structure strengthening, microbial community, chemical composition and stabilization properties of Hybrid Pennisetum. RESULTS: The BM 18-2 had successfully colonized in the interior tissues in both leaf and stem of Hybrid Pennisetum. During ensiling, the levels of pH, acetic acid (AA), butyric acid (BA), propionic acid (PA), and the population of yeast and aerobic bacteria were significantly (P > 0.05) lower, while lactic acid bacteria (LAB) and lactic acid (LA) were significantly (P < 0.001) higher with the steps forward of ensiling in with BM 18-2 as compared to without BM 18-2 colonized of Hybrid Pennisetum. During the different ensiling days, at days 3, 6, 15, and 30, the genus Brevundimonas, Klebsiella, Lactococcus, Weissella, Enterobacter, Serratia, etc. population were significantly decreased, while genus Pediococcus acidilactici and Lactobacillus plantarum were significantly influenced in treated groups as compared to control. The genus Lactobacillus and Pediococcus were positively correlated with treatment groups. CONCLUSIONS: It is concluded that the endophytic bacteria strain BM 18-2 significantly promoted growth characteristics and biomass yield before ensiling and after ensiling inoculated with or without Lactobacillus plantarum could improve the distinct changes of the undesirable microbial diversity, chemical composition, and stabilization properties in with BM 18-2 as compared to without BM 18-2 colonized Hybrid Pennisetum. © 2019 Society of Chemical Industry.


Assuntos
Bacillus megaterium/crescimento & desenvolvimento , Endófitos/crescimento & desenvolvimento , Microbiota , Pennisetum/microbiologia , Ácido Acético/metabolismo , Bacillus megaterium/genética , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Ácido Butírico/metabolismo , Endófitos/genética , Ácido Láctico/metabolismo , Pennisetum/genética , Pennisetum/crescimento & desenvolvimento , Leveduras/classificação , Leveduras/genética , Leveduras/isolamento & purificação , Leveduras/metabolismo
3.
J Basic Microbiol ; 60(1): 22-26, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31692013

RESUMO

Metals are among the most prevalent pollutants released into the environment. For these reasons, the use of biomarkers for environmental monitoring of individuals and populations exposed to metal pollution has gained considerable attention, offering fast and sensitive detection of chemical stress in organisms. There are different metal resistance genes in bacteria that can be used as biomarkers, including cation diffusion facilitators carrying metal ions; the prototype is the cobalt-zinc-cadmium transporter (czcD). The present study reports the expression changes in the czcD gene in Bacillus megaterium and Microbacterium liquefaciens under nickel and vanadium exposure by real-time polymerase chain reaction. The nickel-vanadium-resistant strains of B. megaterium and M. liquefaciens used in this study were isolated from mine tailings in Guanajuato, Mexico. The czcD gene showed high expression under exposure to 200 ppm of Ni and 200 ppm of V during the logarithmic growth phase of M. liquefaciens in PHGII liquid media. In contrast, no changes were observed in B. megaterium during logarithmic and stationary growth, perhaps due to the gene having differential expression during the growth phases. The expression profiles obtained for czcD show the possibility of using this gene from M. liquefaciens as a biomarker of nickel and vanadium pollution in microorganisms.


Assuntos
Actinobacteria/genética , Bacillus megaterium/genética , Biomarcadores Ambientais/genética , Genes Bacterianos/genética , Actinobacteria/metabolismo , Bacillus megaterium/metabolismo , Expressão Gênica , México , Mineração , Níquel/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Vanádio/metabolismo
4.
Science ; 365(6460): 1469-1475, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31604277

RESUMO

The study of cellular processes occurring inside intact organisms requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling noninvasive imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce mammalian acoustic reporter genes. Starting with a gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of intracellular air-filled protein nanostructures called gas vesicles, which produce ultrasound contrast. Mammalian acoustic reporter genes allow cells to be visualized at volumetric densities below 0.5% and permit high-resolution imaging of gene expression in living animals.


Assuntos
Expressão Gênica , Genes Reporter , Proteínas/genética , Ultrassonografia , Acústica , Anabaena flos-aquae/genética , Animais , Bacillus megaterium/genética , Células CHO , Cricetulus , Células HEK293 , Halobacterium salinarum/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Família Multigênica , Nanoestruturas/química , Transfecção
5.
Microb Cell Fact ; 18(1): 132, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31405374

RESUMO

BACKGROUND: Heparosan is the unsulfated precursor of heparin and heparan sulfate and its synthesis is typically the first step in the production of bioengineered heparin. In addition to its utility as the starting material for this important anticoagulant and anti-inflammatory drug, heparosan is a versatile compound that possesses suitable chemical and physical properties for making a variety of high-quality tissue engineering biomaterials, gels and scaffolds, as well as serving as a drug delivery vehicle. The selected production host was the Gram-positive bacterium Bacillus megaterium, which represents an increasingly used choice for high-yield production of intra- and extracellular biomolecules for scientific and industrial applications. RESULTS: We have engineered the metabolism of B. megaterium to produce heparosan, using a T7 RNA polymerase (T7 RNAP) expression system. This system, which allows tightly regulated and efficient induction of genes of interest, has been co-opted for control of Pasteurella multocida heparosan synthase (PmHS2). Specifically, we show that B. megaterium MS941 cells co-transformed with pT7-RNAP and pPT7_PmHS2 plasmids are capable of producing heparosan upon induction with xylose, providing an alternate, safe source of heparosan. Productivities of ~ 250 mg/L of heparosan in shake flasks and ~ 2.74 g/L in fed-batch cultivation were reached. The polydisperse Pasteurella heparosan synthase products from B. megaterium primarily consisted of a relatively high molecular weight (MW) heparosan (~ 200-300 kD) that may be appropriate for producing certain biomaterials; while the less abundant lower MW heparosan fractions (~ 10-40 kD) can be a suitable starting material for heparin synthesis. CONCLUSION: We have successfully engineered an asporogenic and non-pathogenic B. megaterium host strain to produce heparosan for various applications, through a combination of genetic manipulation and growth optimization strategies. The heparosan products from B. megaterium display a different range of MW products than traditional E. coli K5 products, diversifying its potential applications and facilitating increased product utility.


Assuntos
Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Dissacarídeos/biossíntese , Glicosiltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , RNA Polimerases Dirigidas por DNA/genética , Engenharia Genética , Glicosiltransferases/genética , Engenharia Metabólica , Pasteurella multocida/enzimologia , Proteínas Virais/genética
6.
J Biosci Bioeng ; 128(6): 683-689, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31326332

RESUMO

Directed evolution methodologies have been used as promising strategies for improving the catalytic properties of many existing enzymes. In the presented work, this approach was applied to improve the enzyme activity of l-asparaginase I obtained from Bacillus megaterium H-1. After two rounds of error-prone polymerase chain reaction (epPCR) and two generations of sequential DNA shuffling, all of 5 different mutants showed a significant increase in the enzyme activity of l-asparaginase I, ranging from 6.27 to 22.78 IU/mL. Among these mutants, D-9B and DD-12G displayed the relatively high catalytic activity, which were 20.22-fold and 21.33-fold higher than the wild-type enzyme (WT), respectively. Furthermore, the catalytic efficiency (kcat/Km) of D-9B and DD-12G were also improved, which were 132.73 min-1mM-1 and 146.39 min-1mM-1, respectively, in comparison to that of WT (3.39 min-1mM-1). In addition, mutant DD-12G showed tolerance toward wider range of pH values and higher temperatures than its WT counterpart. Homology modeling of above two mutants reflected a reduction of hydrogen bonds and an introduction of flexible residues in the loops near the active catalytic site Thr15. These changes contributed to the flexibility of loops, which may lead to further enhancement in catalytic efficiency. Results also showed that approximately 88.5% (0.978 mg/kg) acrylamide could be removed from mutant DD-12G pre-treated fried potato chips. This study clearly shows that directional evolution methods can indeed be utilized to improve the activity of l-asparaginase, which could also provide research basis for future application in food industry.


Assuntos
Asparaginase/metabolismo , Bacillus megaterium/enzimologia , Asparaginase/química , Asparaginase/genética , Bacillus megaterium/genética , Biocatálise , Domínio Catalítico , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína
7.
Curr Microbiol ; 76(10): 1215-1224, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254008

RESUMO

Nejayote is an alkaline wastewater generated during the nixtamalization process. Nejayote contains high-value compounds such as ferulic acid (FA), which is widely employed as a substrate for the biotechnological production of flavors and aromas. In the present study, the isolation, identification, and characterization of a native strain of Bacillus megaterium were performed, and its capacity to produce 4-vinylguaiacol (4VG) from ferulic acid was evaluated by employing growing cell and resting cell systems. Growing cells of native B. megaterium biotransformed 6 mM crude FA in nejayote into 2.1 mM 4VG, reaching a productivity of 0.21 mM h-1 4VG, while nejayote enriched with FA at 10, 15, and 25 mM resulted in the formation of 2.4, 3.8, and 6.2 mM 4VG and productivities of 0.24, 0.38, and 0.51 mM h-1 4VG, respectively. In the resting cell system, from 6 and 25 mM pure FA, 3.5 mM 4VG was produced (0.18 mM h-1 4VG), while at 10 and 15 mM FA, 4.6 and 5.1 mM 4VG (average of 0.24 mM h-1 4VG) were obtained, respectively. The native B. megaterium strain, isolated from nejayote, showed great biotechnological potential to produce 4VG from crude FA contained in this wastewater, in which other Bacillus species, such as B. licheniformis and B. cereus, were unable to grow and biotransform FA into 4VG.


Assuntos
Bacillus megaterium/classificação , Bacillus megaterium/metabolismo , Ácidos Cumáricos/metabolismo , Águas Residuárias/microbiologia , Zea mays , Bacillus megaterium/genética , Bacillus megaterium/crescimento & desenvolvimento , Biomassa , Biotransformação , Ácidos Cumáricos/química , Guaiacol/análogos & derivados , Guaiacol/metabolismo , Filogenia , Águas Residuárias/química
8.
Appl Microbiol Biotechnol ; 103(18): 7537-7552, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31227867

RESUMO

Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G to 6-aminopenicillanic acid and phenylacetic acid, which provides the precursor for most semisynthetic penicillins. Most applications rely on PGAs from Gram-negative bacteria. Here we describe the first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability. PGAs from Bacillus megaterium (BmPGA, Tm = 56.0 °C), Bacillus thermotolerans (BtPGA, Tm = 64.5 °C), and Bacillus sp. FJAT-27231 (FJAT-PGA, Tm = 74.3 °C) were recombinantly produced with B. megaterium, secreted, purified to apparent heterogeneity, and crystallized. Structures with resolutions of 2.20 Å (BmPGA), 2.27 Å (BtPGA), and 1.36 Å (FJAT-PGA) were obtained. They revealed high overall similarity, reflecting the high identity of up to approx. 75%. Notably, the active center displays a deletion of more than ten residues with respect to PGAs from Gram-negatives. This enlarges the substrate binding site and may indicate a different substrate spectrum. Based on the structures, ten single-chain FJAT-PGAs carrying artificial linkers were produced. However, in all cases, complete linker cleavage was observed. While thermostability remained in the wild-type range, the enzymatic activity dropped between 30 and 60%. Furthermore, four hybrid PGAs carrying subunits from two different enzymes were successfully produced. Their thermostabilities mostly lay between the values of the two mother enzymes. For one PGA increased, enzyme activity was observed. Overall, the three novel PGA structures combined with initial protein engineering experiments provide the basis for establishment of new PGA-based biotechnological processes.


Assuntos
Bacillus megaterium/enzimologia , Penicilina Amidase/química , Engenharia de Proteínas/métodos , Bacillus megaterium/genética , Fenômenos Bioquímicos , Biotecnologia , Cristalização , Estabilidade Enzimática , Hidrólise , Penicilina Amidase/genética
9.
Metab Eng ; 55: 59-67, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31212031

RESUMO

Steroidal compounds are one of the most widely marketed pharmaceutical products. Chemical synthesis of steroidal compounds faces many challenges, including the requirement for multiple chemical steps, low yield and selectivity in several synthesis steps, low profitability and the production of environmental pollutants. Consequently, in recent decades there has been growing interest in the use of microbial systems to produce pharmaceutical compounds. Several microbial systems have recently been developed for the microbial synthesis of the glucocorticoid hydrocortisone, which serves as a key intermediate in the production of several other pharmaceutically important steroidal compounds. In this study, we sought to establish an efficient, microbial-based system, for the conversion of hydrocortisone into cortisone. To this end, we developed a strategy for high-yield cortisone production based on ectopic expression of the guinea-pig 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in Bacillus megaterium. We screened different constructs, containing a variety of promoters tailored for B. megaterium, and created modified versions of the enzyme by protein engineering to optimize cortisone yield. Furthermore, we utilized co-expression of an alcohol dehydrogenase to promote NADP+ regeneration, which significantly improved 11ß-HSD1 activity. The process thereby developed was found to show a remarkably high regioselectivity of >95% and to generate cortisone yields of up to 13.65 g L-1 d-1, which represents a ∼1000-fold improvement over the next-best reported system. In summary, we demonstrate the utility of B. megaterium MS941 as a suitable host for recombinant protein production and its high potential for industrial steroid production.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , Bacillus megaterium , Cortisona/biossíntese , Hidrocortisona/metabolismo , Microrganismos Geneticamente Modificados , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Animais , Bacillus megaterium/enzimologia , Bacillus megaterium/genética , Cortisona/genética , Cobaias , Hidrocortisona/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Oxirredução , Engenharia de Proteínas
10.
J Biotechnol ; 301: 52-55, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31150680

RESUMO

(+)-Nootkatone is a natural ingredient that occurs in grapefruit and certain other plants and is responsible for the characteristic smell of grapefruit. Due to its versatile applications in the flavor and fragrance industry as well as its application in some medical uses it recruits the interests of academic research along with industrial biotechnology. In the current work we present the application of a novel short chain dehydrogenase from Bacillus megaterium in an in vivo whole-cell biocatalyst system for the conversion of the intermediate nootkatol into the industrially valuable (+)-nootkatone. The newly identified dehydrogenase converted nootkatol selectively and efficiently into the final product. The conversion ratio of about 100% was achieved within 40 min yielding about 44 mg/L (+)-nootkatone. Furthermore, the herein identified dehydrogenase provides a new tool to overcome the limitation of the two-step enzymatic biotechnological process for the production of (+)-nootkatone.


Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Sesquiterpenos Policíclicos/metabolismo , Sesquiterpenos/metabolismo , Redutases-Desidrogenases de Cadeia Curta/metabolismo , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Engenharia Metabólica , Sesquiterpenos Policíclicos/análise , Sesquiterpenos/análise , Redutases-Desidrogenases de Cadeia Curta/genética
11.
Chemistry ; 25(26): 6533-6541, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30820987

RESUMO

Selective chemical modification of proteins plays a pivotal role for the rational design of enzymes with novel and specific functionalities. In this study, a strategic combination of genetic and chemical engineering paves the way for systematic construction of biocatalysts by tuning the product spectrum of a levansucrase from Bacillus megaterium (Bm-LS), which typically produces small levan-like oligosaccharides. The implementation of site-directed mutagenesis followed by a tyrosine-specific modification enabled control of the product synthesis: depending on the position, the modification provoked either enrichment of short oligosaccharides (up to 800 % in some cases) or triggered the formation of high molecular weight polymer. The chemical modification can recover polymerization ability in variants with defective oligosaccharide binding motifs. Molecular dynamic (MD) simulations provided insights into the effect of modifying non-native tyrosine residues on product specificity.


Assuntos
Bacillus megaterium/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Hexosiltransferases/química , Hexosiltransferases/genética , Oligossacarídeos/metabolismo , Tirosina/química , Bacillus megaterium/química , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Reação de Cicloadição , Frutanos/química , Frutanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismo
12.
J Chem Inf Model ; 59(2): 743-753, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30758202

RESUMO

Cytochrome P450 102A1 from Bacillus megaterium (BM3) is a fatty acid hydroxylase that has one of the highest turnover rates of any mono-oxygenase. Recent studies have shown how mutants of BM3 can produce metabolites of known drug compounds similar to those observed in humans. Single-point mutations in the binding pocket change the regioselective metabolism of fenamic acids from aromatic hydroxylation to aliphatic hydroxylation. This study is concerned with the individual contribution from accessibility and reactivity for drug metabolism with a future goal to develop fast methods for prediction. For a BM3 M11 mutant as well as the M11 V87F and M11 V87I mutants, we studied the metabolism of the nonsteroidal anti-inflammatory drugs (NSAIDs) mefenamic acid, meclofenamic acid, tolfenamic acid, and diclofenac. Density functional theory (DFT; B3LYP and B3LYP-D3) calculations for all possible reactions were performed using a porphyrin model reacting with the four substrates. Molecular dynamics (MD) simulations were used to determine the potential sites of metabolism that are accessible. Finally, we combine reactivity and accessibility for each potential site to interpret the experimentally determined metabolism. Generally, the 3 and 5 positions (on the ring containing the acidic substituent) and the 2', 3', and 4' positions are most reactive, whereas 4, 5, 3', and 4' are most accessible. Combining reactivity and accessibility show that the 5, 3', and 4' positions are predicted to be most prone to be metabolized, in agreement with experimentally observed data. Reactivity seems to be the dominant factor in the CYP-mediated metabolism of these NSAIDs, which is consistent with previously published methods based solely on reactivity.


Assuntos
Bacillus megaterium/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Teoria da Densidade Funcional , Simulação de Dinâmica Molecular , Mutação , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismo , Bacillus megaterium/genética , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/genética , Conformação Molecular , Estereoisomerismo , Especificidade por Substrato
13.
J Biotechnol ; 294: 38-48, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30771444

RESUMO

Over the past decades, Bacillus megaterium has gained significant interest in the biotechnological industry due to its high capacity for protein production. Although many proteins have been expressed efficiently using the optimized xylose inducible system so far, there is a considerable demand for novel promoters with varying activities, particularly for the adjustment of protein levels in multi-enzyme cascades. Genome-wide microarray analyses of the industrially important B. megaterium strain MS941 were applied to identify constitutive and growth phase dependent promoters for the expression of heterologous proteins from the early exponential to the early stationary phase of bacterial growth. Fifteen putative promoter elements were selected based on differential gene expression profiles and signal intensities of the generated microarray data. The corresponding promoter activities were evaluated in B. megaterium via ß-galactosidase screening. ß-Galactosidase expression levels ranged from 15% to 130% compared to the optimized xylose inducible promoter. Apart from these constitutive promoters we also identified and characterized novel inducible promoters, which were regulated by the addition of arabinose, galactose and the commonly used allolactose analog IPTG. The potential application of the identified promoters for biotechnologically relevant processes was demonstrated by overexpression of the cholesterol oxidase II from Brevibacterium sterolicum, thus obtaining product yields of up to 1.13 g/l/d. The provided toolbox of novel promoters offers versatile promoter strengths and will significantly contribute to harmonize protein expression in synthetic metabolic pathways, thereby pushing forward the engineering of B. megaterium as microbial cell factory for the biosynthesis and conversion of valuable compounds.


Assuntos
Bacillus megaterium/genética , Regiões Promotoras Genéticas , Bacillus megaterium/metabolismo , Colesterol Oxidase , Genoma Bacteriano , Engenharia Metabólica , Análise de Sequência com Séries de Oligonucleotídeos , Pregnenolona/metabolismo , Progesterona/metabolismo , beta-Galactosidase/metabolismo
14.
Comput Biol Chem ; 79: 1-5, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30684864

RESUMO

Bacillus megaterium strain JX285, isolated from rhizosphere red soil sample, can solubilize inorganic phosphorus, which increases the amount of available phosphorus and promotes plant growth. To investigate the mechanisms underlying phosphate solubilization, we sequenced the entire genome of B. megaterium strain JX285 (CGMCC 1.1621), which comprises a circular chromosome of 5,066,463 bp and seven plasmids of 167,030, 128,297, 60,905, 134,795, 9,598, 37,455, and 6332 bp, respectively. The whole genome sequence includes 5948 protein-coding genes, 124 tRNAs, and 29 rRNAs, and has been deposited at Genbank/EMBL/DDBJ with accession numbers CP018874-CP018881. We detected genes associated with organic acid production, which may be vital for phosphate conversion. Furthermore, phosphatase-encoding genes were also detected. The information embedded in the genome will assist in studying the mechanisms of phosphate solubilization. In conclusion, analysis of the JX285 genome will further our knowledge regarding this strain and may contribute to its biotechnological application.


Assuntos
Bacillus megaterium/genética , Bacillus megaterium/isolamento & purificação , Camellia/microbiologia , Camellia/crescimento & desenvolvimento
15.
J Gen Appl Microbiol ; 65(3): 137-144, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30568045

RESUMO

An extracellular L-asparaginase was isolated and purified from Bacillus megaterium MG1 to apparent homogeneity. The purification procedure involved a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration techniques, resulting in a purification factor of 31.52 fold with a specific activity of 215 U mg-1. The molecular mass of the purified enzyme was approximately 47 kDa on SDS-PAGE and 185 kDa on native PAGE gel as well as in gel filtration column chromatography, revealing that the enzyme was a homotetramer. The Km and Vmax values of the purified enzyme were calculated to be 2.0 ⅹ 10-4 M and 1.198 mM s-1. Maximum enzyme activity was observed over a wide range of temperature and pH values with an optimum temperature of 37°C and pH 8.5. SDS and metal ions such as Fe2+, Cu2+, Mg2+, Co2+, Mn2+, and Ca2+ decreased the enzyme activity remarkably, whereas the addition of Na+ and K+ led to an increase in activity. The insensitivity of the protein in the presence of EDTA suggested that the enzyme might not essentially be a metalloprotein. Its marked stability and activity in organic solvents and reducing agents suggest that this asparaginase is highly suitable as a biotechnological tool with industrial applications.


Assuntos
Asparaginase/isolamento & purificação , Asparaginase/metabolismo , Bacillus megaterium/enzimologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Microbiologia da Água , Asparaginase/química , Asparaginase/genética , Asparagina/metabolismo , Bacillus megaterium/classificação , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ativação Enzimática , Estabilidade Enzimática , Espaço Extracelular/metabolismo , Florestas , Concentração de Íons de Hidrogênio , Índia , Cinética , Peso Molecular , Filogenia , Especificidade por Substrato , Temperatura
16.
FEBS J ; 286(6): 1240-1249, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30537187

RESUMO

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4-108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4-108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4-108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein-protein docking, as well as conversion studies, using the mutants demonstrated that the iron-sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5-fold increase in 15ß-hydroxyprogesterone, the main product of progesterone conversion by CYP106A2.


Assuntos
Adrenodoxina/metabolismo , Bacillus megaterium/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Progesterona/metabolismo , Adrenodoxina/química , Adrenodoxina/genética , Bacillus megaterium/genética , Bacillus megaterium/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Hidroxilação , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Oxirredução , Conformação Proteica
17.
J Basic Microbiol ; 59(1): 111-119, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30318739

RESUMO

Biofertilizers are the eco-friendly bio-input being used to sustain the agriculture by reducing the chemical inputs and improving the soil health. Quality is the major concern of biofertilizer technology which often leads to poor performance in the field and thereby loses the farmers' faith. To authenticate the strain as well as its presumed cell load of a commercial product, sequence characterized amplified region (SCAR) markers were developed for three biofertilizer strains viz., Azospirillum brasilense (Sp7), Bacillus megaterium (Pb1) and Azotobacter chroococcum (Ac1). We evaluated the feasibility of multiplex-PCR and quantitative real-time PCR for SCAR marker-based quality assessment of the product as well as the persistence of the strains during crop growth. We showed that multiplex PCR can concurrently discriminate the strains based on the amplicons' size and detects up to 104 cells per g or per ml of carrier-based or liquid formulation of biofertilizer, respectively. The detection limit of quantitative PCR targeting SCAR markers is 103 cells per g or ml of biofertilizer. Both the PCR methods detected and quantified them in the maize rhizosphere. Hence SCAR marker-based quality assessment would be a sensitive tool to monitor the biofertilizer production as well as its persistence in the inoculated crop rhizosphere.


Assuntos
Azospirillum brasilense/isolamento & purificação , Azotobacter/isolamento & purificação , Bacillus megaterium/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Fertilizantes/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Microbiologia do Solo , Agricultura , Azospirillum brasilense/genética , Azotobacter/genética , Bacillus megaterium/genética , Sequência de Bases , Impressões Digitais de DNA , Primers do DNA/genética , DNA Bacteriano/análise , Marcadores Genéticos , Raízes de Plantas/microbiologia , Rizosfera , Sensibilidade e Especificidade , Zea mays/microbiologia
18.
Appl Microbiol Biotechnol ; 103(1): 303-313, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30392122

RESUMO

A novel inducible gene expression system using p-isopropyl benzoate (cumate) as an inducer was developed for the industrial production hosts, Bacillus subtilis and Bacillus megaterium. Cumate is non-toxic to the host, inexpensive, and carbon source-independent inducer which provides an economical option for large-scale production of valuable proteins and chemicals from Bacillus strains. The synthetic cumate-inducible system was constructed by combining the strong constitutive Bacillus promoter Pveg with regulatory elements of the Pseudomonas putida, CymR repressor, and its operator sequence CuO. The designed expression cassette containing a sfGFP reporter under the cumate-inducible promoter was assembled into a Bacillus-E. coli shuttle and gene expression investigated in the two Bacillus strains. Characterization of gene expression levels, expression kinetics, and dose-response to cumate inducer concentration confirmed high-level, but tightly controlled GFP reporter expression in tunable, cumate concentration-dependent manner. Unexpectedly, this expression system works equally well for Escherichia coli, resulting in a platform that can be used both in gram-positive and gram-negative expression host. Its tight regulation and controllable expression makes this system useful for metabolic engineering, synthetic biology studies as well industrial protein production.


Assuntos
Bacillus megaterium/genética , Bacillus subtilis/genética , Benzoatos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Engenharia Genética/métodos , Bacillus megaterium/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Benzoatos/administração & dosagem , Escherichia coli/genética , Perfilação da Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Microrganismos Geneticamente Modificados , Plasmídeos/genética , Regiões Promotoras Genéticas , Pseudomonas putida/genética , Sequências Reguladoras de Ácido Nucleico
19.
J Microbiol Biotechnol ; 30(5): 777-784, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32482945

RESUMO

Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named CYP102A15 and CYP102A170, from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724, respectively, were cloned and expressed in E. coli. The genes are homologues of CYP102A1 from Bacillus megaterium. They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C12-C20, with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with Km 15.3 µM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 µM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.


Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Paenibacillus/enzimologia , Bacillus megaterium/genética , Bacillus megaterium/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência , Especificidade por Substrato
20.
FEMS Microbiol Lett ; 366(22)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913456

RESUMO

In the present study, the taxonomic position of Bacillus aryabhattai and Bacillus megaterium was evaluated using morphological, biochemical, phylogenomic and genome analysis. The morphological and biochemical of these two species were almost similar with few exceptions. The major fatty acids in B. megaterium DSM 32T and B. aryabhattai 21047T were anteiso-C15:0 and iso-C15:0. In the phylogenomic tree, both species clade together and shared high 16S rRNA gene sequence similarity (99.6%). The average nucleotide identity values between Bacillus aryabhattai and Bacillus megaterium were above the threshold values for bacterial species delineation. Based upon morphological, biochemical, chemotaxonomic and comparative genome analysis, we propose to reclassify Bacillus aryabhattai Shivaji et al. 2009 as a later heterotypic synonym of Bacillus megaterium de Bary 1884 (Approved Lists 1980).


Assuntos
Bacillus megaterium/classificação , Bacillus megaterium/genética , Bacillus/classificação , Bacillus/genética , Bacillus/citologia , Bacillus/fisiologia , Bacillus megaterium/citologia , Bacillus megaterium/fisiologia , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Genômica , Técnicas de Tipagem Micológica , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
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