Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319

Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B(12), penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B(12) through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.     

Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C

An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis.     

Effects of Mitomycin C and Thymidine Deprivation on Lysogenic and Sensitive Strains of Bacillus megaterium

A triple auxotroph of Bacillus megaterium strain KM was lysogenized with a phage suspension from B. megaterium 899a. The lysogenic and phage-sensitive derivatives of KM were found to die at the same exponential rate during thymineless incubation, despite the fact that the lysogenic strain became induced. The lysogenic strain was also induced by mitomycin C, and died at an exponential rate which was approximately twice that of the sensitive strain. With both strains, the lethality of mitomycin C was the same in the presence and absence of thymidine; thymidine was required for maximal phage production. Mitomycin C preferentially inhibited deoxyribonucleic acid (DNA) synthesis of both strains for the first 60 min. The (DNA) synthetic ability of the lysogenic strain was subsequently restored, due to phage production. Since there was no evidence that sensitive strains of KM contained other inducible elements (prophage or probacteriocins), it is concluded that both thymineless death and mitomycin C death can occur via mechanisms not involving induction.     

混合培养中巨大芽孢杆菌对氧化葡萄糖酸杆菌的作用

为查明维生素Ｃ二步发酵混合培养中巨大芽孢杆菌与氧化葡萄糖酸杆菌间的关系,通过生长曲线测定、静息细胞实验及摇瓶发酵实验研究了巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产生２－酮基－Ｌ－古龙酸作用的影响;采用超滤分离、凝胶层析及聚丙烯酰胺凝胶电泳技术对巨大芽孢杆菌胞外液中具有促进氧化葡萄糖酸杆菌产酸作用的活性物质进行了分离和纯化。结果表明,大菌胞内液和胞外液均可促进小菌生长,大菌胞外液中具有该作用的组分分子     

响应面法优化Vc二步发酵新菌系产2-酮基-L-古龙酸的培养基

以短小芽胞杆菌(Bacillus pumilus)HJ-04作为维生素C二步发酵第2步中的伴生菌,促进产酸菌产维生素C(Vitamin C,Vc)前体2-酮基-L-古龙酸(2-keto-L-gulonic acid,2-KGA)的能力强于工业生产用菌株巨大芽胞杆菌(Bacillus megaterium) B2980.采用单因素试验、Plackett-Burman(PB)试验及Box-Behnken试验对影响新菌系发酵产2-KGA的6个因素进行分析优化.结果表明,L-山梨糖、尿素、玉米浆为显著影响因子.最佳产酸条件为L-山梨糖94.95 g/L,尿素11.99 g/L,玉米浆14.13g/L.优化后产酸量提高12.31 mg/mL,产酸周期缩短6h.     

SYNTHESIS AND DEGRADATION OF POLY-beta-HYDROXYBUTYRIC ACID IN CONNECTION WITH SPORULATION OF BACILLUS MEGATERIUM.

Slepecky, Ralph A. (Northwestern University, Evanston, Ill.), and John H. Law. Synthesis and degradation of poly-beta-hydroxybutyric acid in connection with sporulation of Bacillus megaterium. J. Bacteriol. 82:37-42. 1961.-The production of poly-beta-hydroxybutyrate has been followed in Bacillus megaterium, a sporulating strain, and B. megaterium strain KM, a nonsporulating strain, by an improved assay procedure and by the use of C(14)-acetate.The production of polymer in the KM strain follows the growth curve very slowly and reaches a peak at the time the cells are entering the stationary phase of growth. Slow utilization of polymer follows.When the sporulating strain is grown under conditions favorable for polymer production, no spores are formed; polymer production and utilization follow kinetics similar to those observed with asporogenous strains.When the sporulating strain is grown under conditions unfavorable for polymer production but favorable for sporulation, less polymer is produced and peak production occurs during the log phase of growth. Rapid utilization of the polymer precedes sporulation.If the medium is made favorable for polymer production by the addition of glucose and acetate and vigorous aeration conditions are used, sporulation can be obtained after good polymer production and subsequent utilization.     

Complete genome sequence of the industrial strain Ketogulonicigenium vulgare WSH-001

Ketogulonicigenium vulgare is an industrial organism commonly used in the vitamin C industry. Here, we report the finished, annotated, and compared 3.28-Mbp high-quality genome sequence of Ketogulonicigenium vulgare WSH-001, a 2-keto-l-gulonic acid-producing industrial strain stocked in our laboratory.     

Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25

Ketogulonicigenium vulgare is characterized by the efficient production of 2KGA from L-sorbose. Ketogulonicigenium vulgare Y25 is known as a 2-keto-L-gulonic acid-producing strain in the vitamin C industry. Here we report the finished, annotated genome sequence of Ketogulonicigenium vulgare Y25.     

Spore pool glutamic acid as a metabolite in germination

Spore glutamic acid pools were examined in dormant and germinating spores using colorimetric and (14)C analytical procedures. Germination of spores of Bacillus megaterium (parent strain), initiated by d-glucose, was accompanied by a rapid drop in the level of spore pool glutamate, from 12.0 mug/mg of dry spores to 7.7 mug/mg of dry spores after 30 sec of germination. Similar decreases in extractable spore pool glutamate were observed with l-alanine-initiated germination of B. licheniformis spores. On the other hand, glutamate pools of mutant spores of B. megaterium, with a requirement of gamma-aminobutyric acid for spore germination, remained unchanged for 9 min of germination, at which time more than 50% of the spore population had germinated. Evidence for conversion of spore pool glutamate to gamma-aminobutyric acid during germination of spores of B. megaterium (parent strain) was obtained.     

Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.     

A short story about a big magic bug

Bacillus megaterium, the "big beast," is a Gram-positive bacterium with a size of 4 × 1.5 μm. During the last years, it became more and more popular in the field of biotechnology for its recombinant protein production capacity. For the purpose of intra- as well as extracellular protein synthesis several vectors were constructed and commercialized (MoBiTec GmbH, Germany). On the basis of two compatible vectors, a T7 RNA polymerase driven protein production system was established. Vectors for chromosomal integration enable the direct manipulation of the genome. The vitamin B(12) biosynthesis of B. megaterium served as a model for the systematic development of a production strain using these tools. For this purpose, the overexpression of chromosomal and plasmid encoded genes and operons, the synthesis of anti-sense RNA for gene silencing, the removal of inhibitory regulatory elements in combination with the utilization of strong promoters, directed protein design, and the recombinant production of B(12) binding proteins to overcome feedback inhibition were successfully employed. For further system biotechnology based optimization strategies the genome sequence will provide a closer look into genomic capacities of B. megaterium. DNA arrays are available. Proteome, fluxome and metabolome analyses are possible. All data can be integrated by using a novel bioinformatics platform. Finally, the size of the "big beast" B. megaterium invites for cell biology research projects. All these features provide a solid basis for challenging biotechnological approaches.     

Methylation of basic proteins in ribosomes from wild-type and thiostrepton-resistant strains of Bacillus megaterium and their electrophoretic analysis.

Ribosomes, radioactively labelled in vivo with both [1-14C]methionine and [methyl-3H]methionine, have been isolated from both wild-type and thiostrepton-resistant strains of Bacillus megaterium and their constituent proteins separated by two-dimensional gel electrophoresis. Ribosomes from the wild-type strain possess one basic protein that is extensively methylated. In contrast no such protein can be detected in ribosomes from the thiostrepton-resistant strain.     

巨大芽孢杆菌表达外源蛋白的特点及其研究进展

巨大芽孢杆菌是一种很有潜力的分泌型基因工程宿主菌,现已广泛应用于工业和学术研究领域。它具有表达率高、遗传稳定、产物可分泌、发酵工艺成熟等优点。本文综述了其自身的优点,表达载体的特点,巨大芽孢杆菌的转化方法,外源蛋白的表达,以及高效表达的策略,并对未来进行展望。     

一株番茄青枯菌拮抗菌的鉴定及抗病效果初探

从形态、生理生化、16S rDNA3个方面确定了番茄青枯菌拮抗菌株3-1-16的分类地位。光学显微镜下观察到菌体为杆状细胞,革兰氏染色均匀,并可见菌体染成蓝紫色。透射电镜进一步观察到细胞内有许多颗粒状物质,无伴胞晶体。Biolog鉴定,3-1-16与巨大芽孢杆菌(Bacillus megaterium)具有最高相似率为98%。16S rRNA分析,3-1-16与巨大芽孢杆菌MO31同源性最高为99.4%。聚类分析显示3-1-16与3株巨大芽孢杆菌聚成一支,支持度为100%。生理生化特征及培养特征测定结果表明,菌株3-1-16鉴定为巨大芽孢杆菌(Bacillus megaterium)。盆栽试验表明该菌株对番茄青枯病防病效果达到81.3%。     

巨大芽孢杆菌B．m2980产孢对氧化葡萄糖酸杆菌产酸的影响

为确定维生素C二步发酵中巨大芽孢杆菌（伴生菌）芽孢形成对氧化葡萄酸杆菌（产酸菌）产酸的影响,本研究通过对巨大芽孢杆菌生长特性分析,选取培养12h（未形成芽孢）和36h（芽孢大量形成）巨大芽孢杆菌B．m2980,检测其胞外液、胞内液以及混合液对产酸菌生成2-酮基-L-古龙酸的影响。结果表明,在未开始形成芽孢时,伴生菌胞外液、胞内液及混合液对产酸菌的生长和产酸有较低的促进作用,其中胞内液的促进能力大于胞外液;在芽孢生成后,胞外液以及混合液对产酸菌生长和产酸的促进能力显著提高。     

Aflatoxin B1 Induction of Lysogenic Bacteria

A technique for biological verification of aflatoxin B(1) was developed based on toxin-mediated induction of lysis in a lysogenic strain of Bacillus megaterium NNRL B-3695. Reduction of culture turbidity was determined at various concentrations of toxin. Incubation of 1.1 x 10(-4) g (dry weight) of cells/ml of growth medium containing 25 mug of B(1) per ml at 37 C reduced initial turbidity 0.20 absorbance units in 4 hr. If the bacterial lysate of the lysogenic strain, after a 2-hr incubation with 25 mug of B(1) per ml, was plated with a sensitive B. megaterium strain (NRRL B-3694), plaque-forming units increased approximately 150 times relative to the control. Comparable testing of the effects of aflatoxin on the nonlysogenic, sensitive strain demonstrated that 75 mug of B(1) per ml neither induced lysis nor plaque-forming units. Although induction is not an exclusive property of aflatoxin B(1), the differential response of the lysogenic and sensitive Bacillus strains to B(1) offers a unique and rapid technique for biological verification of the toxin.     

Isolation of recombination-defective and UV-sensitive mutants of Bacillus megaterium.

Mutants of Bacillus megaterium QMB1551 sensitive to mitomycin C or methyl methanesulfonate were isolated and characterized phenotypically. Cell survival after UV-light and gamma-ray exposure was determined, as was transductional recombination. Of the mutants tested, three were sensitive to UV but remained recombination proficient. The UV-sensitive mutants were also reduced in host cell reactivation. At least three mutants had undetectable transduction frequencies, i.e., less than 0.3 to 1.3% of the parental strain frequencies, and so appear to be recombination deficient. Sensitivities of these mutant strains to UV light and gamma radiation were compared with those of parental B. megaterium as well as parental, recE4, recA1, uvrA19, and uvrB109 strains of Bacillus subtilis. In each case, the strains of B. megaterium, including the parental strains, showed a higher percentage of cell survival than B. subtilis.     

Identification of germination gene of Bacillus megaterium

Glucose, KNO3, proline and leucine initiate the spore germination of B. megaterium ATCC 12872, but not of B. megaterium ATCC 19213. In order to isolate the gene concerning germination of B. megaterium ATCC 12872, we constructed its gene library in plasmid vector, and introduced into B. megaterium ATCC 19213. We obtained a transformant whose spores differed from those of the wild type strain with respect to germinability. Spores of this transformant could be germinated by glucose, proline or leucine. The recombinant plasmid prepared from this transformant was found to carry 2 kilobase pairs fragment of B. megaterium ATCC 12872 DNA. This fragment may contain the gene encoding the protein which plays an important role in germination.     

Antibacterial and antifungal lysozyme-type activity in Cameraria ohridella pupae

Lysozyme-type antibacterial and antifungal activity in pupae of Cameraria ohridella was studied. Activity against Micrococcus luteus and Bacillus megaterium was detected in pupae extract. Also antifungal activity from C. ohridella pupae extract directed against Saccharomyces cerevisiae strain W 303 was shown. During immunoblotting two bands in pupae extract, with molecular mass of about 15 and 28 kDa were recognized by antibodies directed against HEWL. After acid electrophoresis followed by bioautography of the extract, two lytic zones showing lysozyme-type activity against M. luteus were observed. Two bacteria: Gram-positive Aerococcus viridans and Gram-negative Aeromonas salmonicida ssp. masoucida were isolated from pupae of C. ohridella. Their activity against M. luteus, B. megaterium, and S. cerevisiae W303 was detected. After immunoblotting with antibodies against HEWL, also two proteins from bacterial suspensions of A. viridans and A. salmonicida were detected, about 15 and 28 kDa.     

Purification and characterization of keratinase from recombinant Pichia and Bacillus strains

The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 degrees C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K(m) and V(max) of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.