Determination of the strength of the cell walls of vegetative cells ofBacillus megaterium andBacillus stearothermophilus

The tensile strength of the cell walls ofBacillus megaterium andBacillus stearothermophilus was found to be about 2.4×10⁷ N/m². The internal pressure and water activity of the cells were 14 atm, 0.99 a_w forB. megaterium and 28 atm, 0.98 a_w forB. stearothermophilus. The greater strength ofB. stearothermophilus cells, considered as pressure vessels, restricts absorption of water by the protoplasm so that the water content on a dry weight basis is 3.4 g/g forB. megaterium cells in water but only 1.8 g/g forB. stearothermophilus.     

Bacillus megaterium, KM beta-galactosidase: purification by affinity chromatography and characterization of the active species

The enzyme β-galactosidase from Bacillus megaterium, strain KM has been purified by affinity chromatography. The enzyme was found to have a dimeric subunit structure, with the monomer having a molecular weight of 120,000. The K_eq of the monomer-dimer equilibrium was strongly shifted towards dissociation in the isolated state. Inclusion of 5% sucrose in the buffer (and maintenance of the temperature at 5 °) minimized this dissociation. Molecularly homogeneous monomer and dimer could be prepared on sucrose gradients. The dimer was determined to have an S_20,w of 8, while the monomer had an S_20,w of 3. The amino acid composition was found to be similar to that of the E. coli β-galactosidase although significant differences occur. The activity of the monomer was studied by both urea-denaturation experiments and by immobilization of the monomer on Sepharose-4B. The monomer, bound to Sepharose-4B, was found to be inactive but still capable of binding the inhibitor thio-methyl galactoside. Activity was reconstituted by adding free monomer, in 8 M urea, to the Sepharose-bound monomer, followed by removal of the urea by dialysis. In addition, free monomers from E. coli β-galactosidase were found to form active hybrids with Sepharose-bound B. megaterium β-galactosidase monomers. We conclude on the basis of these studies that the free monomer is inactive, and that the dimer is the active species, in marked contrast to E. coli β-galactosidase where only the tetrameric form is active.     

Chlorxanthomycin, a Fluorescent, Chlorinated, Pentacyclic Pyrene from a Bacillus sp.

A gram-positive Bacillus sp. that fluoresces yellow under long-wavelength UV light on several common culture media was isolated from soil samples. On the basis of carbon source utilization studies, fatty acid methyl ester analysis, and 16S ribosomal DNA analysis, this bacterium was most similar to Bacillus megaterium. Chemical extraction yielded a yellow-orange fluorescent pigment, which was characterized by X-ray crystallography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The fluorescent compound, chlorxanthomycin, is a pentacyclic, chlorinated molecule with the molecular formula C₂₂H₁₅O₆Cl and a molecular weight of 409.7865. Chlorxanthomycin appears to be located in the cytoplasm, does not diffuse out of the cells into the culture medium, and has selective antibiotic activity.     

Biochemical Alternations in Bacillus megaterium as Produced by Aflatoxin B1

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 μg of aflatoxin B₁ per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 μg of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.     

Chemotactic attraction between hemocytes of the oyster,Crassostrea virginica,and bacteria

Experiments were conducted to ascertain whether there is chemotactic attraction by Bacillus megaterium and Micrococcus varians, both Gram-positive species, and Escherichia coli and Vibrio parahaemolyticus, both Gram-negative species, for hemocytes of the American oyster, Crassostrea virginica. It was ascertained quantitatively that oyster hemocytes are attracted to live E. coli, B. megaterium, and M. varians but not to heat-killed bacteria. Furthermore, oyster cells are not attracted to either live or heat-killed V. parahaemolyticus. It is concluded that the chemoattractant is some molecule emitted by living vegetative cells of certain Gram-positive as well as Gramnegative bacteria.     

Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium

Poly(3-hydroxybutyrate)—P(3HB)—is a natural biodegradable polyester synthesized by several bacteria, produced from renewable resources. The effects of oxygen transfer rate on the intracellular accumulation of P(3HB) was evaluated, aiming at increasing P(3HB) synthesized by Bacillus megaterium DSM 32^T in bioreactor batch cultures. Bench-scale bioreactor cultivations were performed under different volumetric oxygen mass transfer coefficients, k_La, setting stirrer speed on specified values. The results of this work show that oxygen transfer is a key factor on P(3HB) accumulation by B. megaterium, increasing the P(3HB) intracellular mass fraction from 39% to 62% of CDW at k_La condition of 0.006 s^?1.     

Production of single chain Fab (scFab) fragments in Bacillus megaterium

Background

The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.

Results

The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.

Conclusion

B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

The anaerobic pathway for the biosynthesis of cobalamin (vitamin B₁₂) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH₆₀, that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH₆₀ was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH₆₀ demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.     

Phosphate Solubilizing Bacillus megaterium mj1212 Regulates Endogenous Plant Carbohydrates and Amino Acids Contents to Promote Mustard Plant Growth

The current study was conducted to explore the potential of a phosphate solubilizing soil bacterium, Bacillus megaterium mj1212 for enhancing the growth of mustard plants. The newly isolated bacterial strain mj1212 was identified as B. megaterium using phylogenetic analysis and, its phosphate solubilization ability was shown by the clear zone formation on National Botanical Research Institute’s Phosphate medium. Moreover, the phosphate solubilization ability of B. megaterium mj1212 was enhanced by optimal culture conditions at pH 7.0 and 35 °C which might be due to the presence of malic and quinic acid in the culture medium. The beneficial effect of B. megaterium mj1212 in mustard plants was determined by an increasing shoot length, root length and fresh weight of plants. In the biochemical analysis revealed that chlorophyll, sucrose, glucose, fructose and amino acids (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ilu, Leu, Tyr, Phe, Lys, His, Arg and Pro) were higher in B. megaterium mj1212 treated plants, when compared to their control. The result of present study suggests that B. megaterium mj1212 treatment could be act as phosphate biofertilizer to improve the plant growth.     

Manipulation of B. megaterium growth for efficient 2-KLG production by K. vulgare

In the two-step Vitamin C fermentative production, its precursor 2-keto-l-gulonic acid (2-KLG) was synthesized by Ketogulonicigenium vulgare through co-culture with Bacillus megaterium. The rates of K. vulgare cell growth and 2-KLG production were closely related with B. megaterium concentration in the co-culture system. To enhance the 2-KLG production efficiency, a strategy of manipulating B. megaterium growth in the co-culture system and properly releasing its intracellular components was introduced. Lysozyme was used specifically to damage B. megaterium cell wall structure and subsequently inhibit its cell growth. When 10,000 U mL⁻¹ lysozyme was fed to the co-culture system at 12 h, the growth rate of K. vulgare, sorbose consumption rate, and 2-KLG productivity could increase 27.4%, 37.1%, and 28.2%, respectively.     

Phosphotransbutyrylase Expression in Bacillus megaterium

The molecular analysis of a genomic region of B. megaterium revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). The enzyme activity was measured throughout the different phases of growth in B. megaterium, and its activity was found to be maximal in the late exponential growth phase. The branched amino acids isoleucine and valine activated Ptb expression. Ptb_Bm was capable of using butyryl-CoA and 2-methyl-propionyl CoA as substrates. Act_Bm, a sigma⁵⁴ regulator from B. megaterium whose gene is situated upstream from the ptb gene, activated its expression. Received: 14 September 2000 / Accepted: 13 October 2000     

Recovery and partial purification of penicillin G acylase from E. coli homogenate and B. megaterium culture medium using an expanded bed adsorption column

The adsorption of penicillin G acylase (PGA) from B. megaterium and from Escherichia coli on a cationic resin, Streamline SP XL, was studied using both packed and expanded beds. Stability assays showed that penicillin acylases from the two sources presented high irreversible deactivation at pH 4.0 and 4.5, but remained stable at pH 4.8. Adsorption experiments performed in a packed bed (PB), in the pH range 4.8–5.8, showed highest adsorption yields at pH 4.8, for both enzymes. Using small expanded bed adsorption (EBA) columns, PGA was directly recovered and partially purified from E. coli crude extracts, E. coli homogenates, and from B. megaterium centrifuged broth in a single unit operation. Global recovery yields of 91.0, 55.0 and 7.4% and purification factors of 4.5-, 7.5- and 12.7-fold were achieved, respectively. The elution yields of penicillin acylase obtained with these cationic EBA processes when working with E. coli homogenate and B. megaterium centrifuged medium were of 100 and 52%, respectively. The comparison of adsorption capacities of E. coli penicillin acylase from crude extracts onto Streamline SP XL showed similar results for packed-bed and for expanded-bed modes. However, PGA adsorption yields for E. coli (homogenate) and B. megaterium (centrifuged medium) were substantially lower than the values obtained for E. coli crude extract, due to the competition of cell debris and other components present in the B. megaterium medium.     

The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus

Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.     

Characterization of phosphate-solubilizing bacteria exhibiting the potential for growth promotion and phosphorus nutrition improvement in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) in calcareous soils of Sinaloa,Mexico

Greenhouse bioassays were used to examine the ability of selected strains of the rhizobacteria Sinorhizobium meliloti, Bacillus flexus and B. megaterium to solubilize phosphorus (P) and to affect growth promotion and phosphorus nutrition in maize. These bacterial strains were found to decrease the pH and solubilize some forms of insoluble P, such as tricalcium phosphate and hydroxyapatite, as well as to exhibit acid and alkaline phosphatase enzymatic activities in culture medium, properties that are possibly involved in P solubilization. Inoculation of the strains separately and as a consortium of the three bacteria (S. meliloti, B. flexus and B. megaterium) in P-deficient soil (4.33 w/v P) fertilized without P improved plant height, shoot and root dry weight, as well as P nutrition in the maize plants. Use of the B. flexus and B. megaterium strains separately and in a consortium positively affected several growth parameters and P nutrition in plants supplemented with insoluble P. No effect was observed when pots in which the seedlings were growing were supplied with soluble fertilizer. A second assay using a P-deficient soil (6.64 w/v P) showed that inoculation with the consortium of B. flexus and B. megaterium significantly increased growth and total P content in maize plants. A dose–response P fertilization experiment using sterile P-deficient soil led us to conclude that inoculation to soil of the mixture of B. flexus and B. megaterium may improve P nutrition and growth to a level previously attained by the addition of soluble P-fertilizer at 40 w/v P. A non-sterile experiment showed a beneficial response with B. megaterium but not with B. flexus. We propose utilizing these bacteria in P-deficient alkaline soils in future field trials in order to evaluate their potential as biofertilizers.     

The Glucomannokinase of the Gram-Negative Ruminal Bacterium,Prevotella bryantii B14, and its Sequence Conservation with Regulatory Glucokinases of Gram-Positive Bacteria

The glucomannokinase gene of Prevotella bryantii B₁4, a strictly anaerobic ruminal bacterium, was amplified with degenerate PCR primers. The degenerate PCR primers were based on the N-terminal amino acid residues of the purified glucomannokinase and the C-terminus of other bacterial glucokinases. The PCR product had a molecular weight of approximately 550 bp. Because the purified glucomannokinase was composed of dimers with a monomer molecular weight of 34.5 kDa, the PCR product accounted for approximately 60% of the glucomannokinase DNA sequence. The B₁4 sequence had significant similarity with the glucokinases of several aerobic, Gram-positive bacteria (Streptomyces coelicolor,Staphylococcus xylosus , Bacillus subtilis, Bacillus megaterium). Previous work indicated that the Gram-positive glucokinases and the B₁4 glucomannokinase have a role in catabolite repression. These presumptive regulatory kinases formed a cluster that was distinct from other bacterial glucokinases. Amino acid sequence analyses indicated that the regulatory glucokinase genes (including the P. bryantii B₁4 glucomannokinase gene) had a mean sequence similarity of 42 ± 2.4%. The regulatory kinases had some highly conserved regions with other bacterial glucokinases, but the mean similarity was lower (17 ± 2.0%). The role of regulatory kinases in catabolite repression has not been precisely defined, but sequence comparisons suggest a common theme may exist. Because P. bryantii B₁4 is a Gram-negative bacterium, it appears that regulatory glucokinases may be widely distributed in eubacteria and not restricted to Gram-positive species.     

Bacillus megaterium Has Both a Functional BluB Protein Required for DMB Synthesis and a Related Flavoprotein That Forms a Stable Radical Species

Despite the extensive study of the biosynthesis of the complex molecule B₁₂ (cobalamin), the mechanism by which the lower ligand 5,6-dimethylbenzimidazole (DMB) is formed has remained something of a mystery. However, recent work has identified and characterized a DMB-synthase (BluB) responsible for the oxygen-dependent, single enzyme conversion of FMN to DMB. In this work, we have identified BluB homologs from the aerobic purple, nonsulfur, photosynthetic bacterium Rhodobacter capsulatus and the aerobic soil bacterium Bacillus megaterium and have demonstrated DMB synthesis by the use of a novel complementation assay in which a B₁₂ deficient strain, substituted with the precursor cobinamide is recovered either by the addition of DMB or by the recombinant expression of a bluB gene. The DMB-synthetic activity of the purified recombinant BluB enzymes was further confirmed in vitro by providing the enzyme with FMNH₂ and oxygen and observing the formation of DMB by HPLC. The formation of a 4a-peroxyflavin intermediate, the first step in the oxygen dependent mechanism of DMB biosynthesis, is reported here and is the first intermediate in the enzyme catalysed reaction to be demonstrated experimentally to date. The identification and characterization of an FMN-binding protein found on the cobI operon of B. megaterium, CbiY, is also detailed, revealing an FMN-containing enzyme which is able to stabilize a blue flavin semiquinone upon reduction with a 1-electron donor.     

Glucose 6-Phosphate Accumulation in Mycobacteria: IMPLICATIONS FOR A NOVEL F420-DEPENDENT ANTI-OXIDANT DEFENSE SYSTEM*

Glucose 6-phosphate (G6P) is a metabolic intermediate with many possible cellular fates. In mycobacteria, G6P is a substrate for an enzyme, F₄₂₀-dependent glucose-6-phosphate dehydrogenase (Fgd), found in few bacterial genera. Intracellular G6P levels in six Mycobacterium sp. were remarkably higher (∼17–130-fold) than Escherichia coli and Bacillus megaterium. The high G6P level in Mycobacterium smegmatis may result from 10–25-fold higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase when grown on glucose, glycerol, or acetate compared with B. megaterium and E. coli. In M. smegmatis this coincided with up-regulation of the first gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, when acetate was the carbon source, suggesting a cellular program for maintaining high G6P levels. G6P was depleted in cells under oxidative stress induced by redox cycling agents plumbagin and menadione, whereas an fgd mutant of M. smegmatis used G6P less well under such conditions. The fgd mutant was more sensitive to these agents and, in contrast to wild type, was defective in its ability to reduce extracellular plumbagin and menadione. These data suggest that intracellular G6P in mycobacteria serves as a source of reducing power and, with the mycobacteria-specific Fgd-F₄₂₀ system, plays a protective role against oxidative stress.     

Aquimarina atlantica sp. nov., isolated from surface seawater of the Atlantic Ocean

A taxonomic study was carried out on strain 22II-S11-z7^T, which was isolated from the surface seawater of the Atlantic Ocean. The bacterium was found to be Gram-negative, oxidase negative and catalase positive, long-rod shaped, and gliding. Growth was observed at salinities of 1–5 % and at temperatures of 10–41 °C. The isolate was capable of hydrolysing gelatin and Tween 80 and able to reduce nitrate to nitrite, but unable to degrade aesculin. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 22II-S11-z7^T belongs to the genus Aquimarina, with highest sequence similarity to Aquimarina megaterium XH134^T (98.31 %), followed by Aquimarina macrocephali JAMB N27^T (96.59 %); other species of the genus Aquimarina shared 93.63–96.08 % sequence similarity. The ANI value between strain 22II-S11-z7^T and A. megaterium XH134^T was found to be 91.86–91.81 %. The DNA–DNA hybridization estimated value between strain 22II-S11-z7^T and A. megaterium XH134^T was 47.7 ± 2.6 %. The principal fatty acids were identified as Summed Feature 3 (C_16:1 ω7c/ω6c, as defined by the MIDI system; 8.1 %), SummedFeature 9 (iso-C_17:1 ω7c/C_16:110-methyl; 6.8 %), iso-C_15:0 G (11.3 %), iso-C_15:0 (24.9 %), iso-C_16:0 (5.7 %), C_16:0 (5.2 %), iso-C_15:0 3OH (6.4 %) and iso-C_17:0 3OH (21.5 %). The G+C content of the chromosomal DNA was determined to be 32.99 mol %. The respiratory quinone was determined to be MK-6 (100 %). Phosphatidylethanolamine, two unidentified aminolipids, five unidentified phospholipids and two unidentified lipids were found to be present. The combined genotypic and phenotypic data show that strain 22II-S11-z7^T represents a novel species within the genus Aquimarina, for which the name Aquimarina atlantica sp. nov. is proposed, with the type strain 22II-S11-z7^T (=MCCC 1A09239^T = KCTC 42003^T).