Spore-spheroplast transformation of Bacillus megaterium

A new method for transformation of Bacillus megaterium was developed by modification of Chang and Cohen's method. In our method, spore spheroplasts were used as recipient cells instead of the protoplasts of vegetative cells. Longer incubation (60 min) of spore spheroplasts and plasmid DNA before treatment with polyethylene glycol remarkably increased the efficiency of transformation. The frequency of transformation was about 10(4) per microgram of plasmid DNA. A shot-gun-type cloning of chromosome DNA of B. megaterium ATCC 12872 was available in B. megaterium ATCC 19213 strain by this transformation method.     

Biolistic transformation of a procaryote, Bacillus megaterium.

We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.     

Biolistic transformation of a procaryote, Bacillus megaterium

We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.     

Transduction in Bacillus megaterium.

A bacteriophage, MP13, isolated from the soil on $\text{B. megaterium}$ QM B1551 has been found to transduce several auxotrophic markers. Transduction required inactivation of the phage to approximately 0.01% survival with UV light and it was enhanced by the absence of salts that are probably necessary for phage readsorption.     

Phosphatidyltransferase activity in Bacillus megaterium.

Phosphatidyl transfer between phosphatidylethanolamine, phosphatidylglycerol or phosphatidylserine as donors and primary hydroxyl acceptors including ethanolamine, glycerol, serine and Triton X-100 has been shown to be catalysed by membrane particles derived from Bacillus megaterium strains ATCC 13632 and ATCC 14581. The rate of cardiolipin synthesis from phosphatidylglycerol in the presence of ethanolamine was an order of magnitude greater than that of phosphatidylethanolamine formation. Cardiolipin synthesis from phosphatidylethanolamine in the presence of glycerol was also observed, and was 1.5-fold greater than the formation of phosphatidylglycerol. Similar heat lability, effects of pH and of Triton X-100 for phosphatidyl transfer and cardiolipin synthesis indicate that both reactions were catalysed by cardiolipin synthase.     

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.     

Ferrisiderophore reductase activity in Bacillus megaterium.

The release of iron from ferrisiderophores (microbial ferric-chelating iron transport cofactors) by cell-free extracts of Bacillus megaterium was demonstrated. Reductive transfer of iron from ferrisiderophores to the ferrous-chelating agent ferrozine was measured spectrophotometrically. This ferrisiderophore reductase activity (reduced nicotinamide adenine dinucleotide phosphate:ferrisiderophore oxidoreductase) was associated primarily with the cell soluble rather than particulate (membrane) fraction. Ferrisiderophore reductase was inhibited by oxygen and required the addition of a reductant (reduced nicotinamide adenine dinucleotide phosphate was most effective) for maximal activity. The activity was destroyed by both heat and protease treatments and was inhibited by iodoacetamide treatment. Ferrisiderophore reductase activity for several microbial ferrisiderophores was measured; highest activity was displayed for ferrischizokinen, the ferrisiderophore produced by this organism. The Km and Vmax values of the reductase for ferrischizokinen were 2.5 x 10(-4) M and 35.7 nmol/min per mg of the ferrisiderophore reductase reaction. Preliminary fractionation of the cell soluble material by gel filtration chromatography resulted in the demonstration of ferrisiderophore reductase activity in three peaks of different molecular weight. Ferrisiderophore reductase probably mediates entrance of iron into cellular metabolism.     

Red pigment in Bacillus megaterium spores.

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Biosynthesis of phospholipids in Bacillus megaterium.

Information on the biosynthesis of phospholipids in bacteria has been derived principally from the study of Escherichia coli and other gram-negative organisms. We have now carried out a detailed study of the pathways of phospholipid biosynthesis in the gram-positive organism Bacillus megarterium KM in relation to investigations on the biogenesis of lipid asymmetry in membranes. Radioactive precursors such as 32Pi and [3H]palmitate initially label phosphatidylethanolamine much more than phosphatidylglycerol. This raised the possibility that phosphatidylglycerol may be the precursor of phosphatidylethanolamine in a pathway different from that in E. coli. Phosphatidylglycerol is known to be highly reactive metabolically, since it functions as a donor of phosphatidyl residues in the synthesis of cardiolipin and as a donor of glycerophosphate residues in the synthesis of teichoic acids and of membrane-derived oligosaccharides. The large pool of phosphatidylglycerol would dilute the radioactive isotope, slowing the initial rate of incorporation of label into phosphatidylethanolamine. However, assays of cell-free extracts revealed no evidence for such a novel pathway. Instead, phosphatidylserine synthase (cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase) and phosphatidylserine decarboxylase were detected, although at low levels. These results suggest that the pathway in B. megaterium is the same as that in E. coli in which phosphatidylserine, derived from cytidine 5'-diphosphate-diglyceride, is the precursor of phosphatidylethanolamine. The lag in the appearance of label in phosphatidylethanolamine appears to be the effect of a considerable pool of phosphatidylserine (ca. 5 to 10% of the total phospholipid) in certain strains of B. megaterium. The lag in labeling can be correlated with the size of the pool of phosphatidylserine. Pulse-chase experiments in vivo support the conclusion that in B. megaterium phosphatidylserine is not derived from phosphatidylglycerol. Rates of turnover of the membrane phospholipids of B. megaterium have also been studied.     

spoVG sequence of Bacillus megaterium and Bacillus subtilis.

We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis. The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6% amino acid identity. Both genes have putative rho-independent terminators. No significant amino acid or nucleotide homology of either gene was found when compared with sequences contained in either the Genbank or EMBL data bases.     

Dihydrodipicolinate reductases from Bacillus cereus and Bacillus megaterium.

Dyhydrodipicolinate reductases were purified 100-fold from crude extracts of B. cereus and B. megaterium and their properties were compared with those of the reductase from B. subtilis. The molecular weights of the reductases of B. cereus and B. megaterium were fount to be 155,000 and 150,000, respectively. These reductases were shown to be free of flavin, unlike the B. subtilis enzyme, which contains flavin. Both NADPH and NADH acted as coenzymes for these two reductases. NADPH being three or four times more effective than NADH. The Km values for NADPH and dihydrodipicolinate were 8 micrometer and 62 micrometer, respectively, with B. cereus reductase, and 13 micrometer and 59 micrometer with B. megaterium reductase. The pH optima of the enzymes from B. cereus and B. megaterium were pH 7.4 and 7.2, respectively. The reductases were inhibited by dipicolinate noncompetitively with respect to dihydrodipicolinate and the Ki values were 85 micrometer and 140 micrometer, respectively. Lysine and diaminopimelate were not inhibitory. The properties of the reductases from B. cereus and B. megaterium were similar, but they differed considerably from those of the B. subtilis enzyme. However, all three Bacillus reductases were markedly inhibited by dipicolinate, unlike the enzyme from E. coli.     

Transposition of Tn917 in Bacillus megaterium.

Transposon Tn917, carried on plasmid pTV1, was introduced into Bacillus megaterium and transposed efficiently and apparently randomly. Insertional mutations included at least eight different auxotrophic loci, two carbon source loci, and sporulation loci. One trp::Tn917 mutation was further verified as an insertion by both reversion and transduction.     

Glucose-triggered germination of Bacillus megaterium spores.

Triggering of germination in Bacillus megaterium QM B1551 spores with D-glucose was studied. First, the interaction of glucose with spores for less than 1 min resulted in triggering almost 90% of the spores after the glucose was removed by dilution. Therefore only a brief time is needed for glucose to trigger germination, and then the continuous presence of glucose is not necessary. Detectable uptake of glucose began 2 to 3 min after absorbance loss started, and a non-metabolizable glucose analog, methyl-alpha-D-glucopyranoside, triggered germination in the absence of detectable uptake. Several inhibitors that reduced or eliminated glucose uptake did not block triggering of germination. Therefore, glucose uptake may be a relatively late event and not a prerequisite for triggering of germination.     

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     

Thymineless death in Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), S. Kemp, and L. Hogg. Thymineless death in Bacillus megaterium. J. Bacteriol. 87:1079-1086. 1964.-Strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, rapidly loses the ability to multiply when incubated in the absence of thymine, on an otherwise sufficient medium. At 37 C, there is a lag of approximately 60 min, prior to the onset of exponential death (decrease of 1 decade per 50 min). The extent of the decrease in viable count varies from 4 to 5 decades after 5 hr of starvation. The cells die more slowly at 30 C (decrease of 1 decade per 120 min) after a lag of approximately 90 min. Thymine starvation permits substantial net ribonucleic acid (RNA) and protein synthesis, but only slight deoxyribonucleic acid synthesis. In contrast with the changes occurring at 30 C, thymineless death at 37 C is eventually accompanied by a rapid hydrolysis of RNA and by cell lysis. Chloramphenicol inhibits thymineless death at 37 C. Strain T(-)R(1), a derivative of strain KM:T(-), undergoes a very low rate of thymineless death at 37 C (decrease of 1 decade per 240 min). Neither hydrolysis of RNA nor cell lysis occurs during 8 hr of thymine starvation. Strain KM:T(-)H(-) (doubly auxotrophic for thymidine and histidine) requires histidine for maximal thymineless death at 37 C. Preincubation of this strain on the basal medium supplemented with thymidine alone enables the population to become increasingly immune to subsequent thymineless death.     

Microbial transformation of glycyrrhetinic acid derivatives by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741

Glycyrrhetinic acid (GA), the major bioactive pentacyclic triterpene aglycone of licorice root, was known to play a vital role in anti-ulcer, anti-depressant, anti-inflammatory, and anti-allergic. In this study, we semi-synthesized five GA derivatives by a series of chemical reactions. They were selected as substrates for the biotransformation and yielded thirteen metabolites by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741. Their structures were identified on the basis of extensive spectroscopic methods and nine of them were found for the first time. Two main types of reactions, regio- and stereo-selective hydroxylation and glycosylation, especially in the unactivated C-H bonds including C-11, C-19 and C-27, were observed in the biotransformation process, which greatly expand the chemical diversities of GA derivatives. All compounds were tested for their inhibitory effects on nitric oxide (NO) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Among them, olean-12-ene-3β,7β,15α,19α,30-pentol (16) and olean-12-ene-3β,7β,15α,27,30-pentol (17) showed significant inhibitory effect with IC₅₀ values of 0.64 and 0.07 μM, respectively.     

Ribosomal precursor particles of Bacillus megaterium.

Pulse-labeled cells of Bacillus megaterium were converted to protoplasts, and lysates of the protoplasts were analyzed by sucrose gradient sedimentation. Precursor ribonucleoprotein (RNP) particles then appeared predominantly as 50S and 30S precursor ribosomal subunits. Polyacrylamide gel electrophoresis of the ribosomal ribonucleic acid from the 50S and 30S RNP particles confirmed their precursor nature since they were shown to contain precursor 23S and 16S ribosomal ribonucleic acid, respectively. Treatment of protoplast lysates with 0.5% deoxycholate prior to sedimentation analysis resulted in a markedly different radioactivity profile. The 50S RNP particles were no longer present, but 43S particles were observed in addition to increased amounts of pulse-labeled material sedimenting at 30S and slower. Extracts from cells broken in a French press showed a profile from sucrose gradient sedimentation similar to that of the deoxycholate-treated protoplast lysate. These data suggest that the nature of the precursor ribosomal particles appears to be a function of the method of cell disruption or detergent treatment of the cell extract preparation. The observed 50S and 30S RNP particles may be the major precursor ribosomal subunits in vivo; the slower-sedimenting species could result from some form of breakdown or change in the configuration of the 50S and 30S precursors.     

Energy conservation in Bacillus megaterium

1. The respiratory chain energy conservation systems of Bacillus megaterium strains D440 and M have been investigated following growth in batch and continuous culture. Respiratory membranes from these strains contained cytochromes b, aa ₃ , o and b, c, a, o, respectively; both readily oxidised NADH but neither showed any pyridine nucleotide transhydrogenase activity.
2. Whole cells of both strains exhibited endogenous →H^-/O ratios of approximately 4; when loaded with specific substrates the resultant →H⁺/O ratios indicated that proton translocating loops 1 and 2 were present in strain D440 and that loops 2 and 3 were present in strain M.
3. In situ respiratory activities were measured as a function of dilution rate during growth in continuous culture. True molar growth yields with respect to oxygen (Y _{O 2}) of approximately 50 g cells·mole oxygen^-1 were obtained for most of the nutrient limitations employed. Average values for Y _ATP of 12.7 and 10.8 g cells·mole ATP equivalents^-1 were subsequently calculated for strains D440 and M respectively.
4. Energy requirements for maintenance purposes were low in energy-limited cultures but were substantially increased when growth was limited by nitrogen source (NH ₄ ⁺ ). Under the latter conditions there is probably a partial uncoupling of energy-conserving and energy-utilising processes leading to energy wastage.