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.     

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.     

Role of chromosomal and plasmid-borne receptor homologues in the response of Bacillus megaterium QM B1551 spores to germinants

Spores of Bacillus megaterium QM B1551 germinate in response to a number of trigger compounds, including glucose, proline, leucine, and inorganic salts. An approximate 6-kb region of the 165-kb plasmid was found to harbor a tricistronic receptor operon, gerU, and a monocistronic receptor component, gerVB. The gerU operon was observed to complement the germination response in plasmidless strain PV361 to glucose and leucine, with KBr acting as a cogerminant. Proline recognition is conferred by the monocistronic gerVB gene, the presence of which also improves the germination response to other single-trigger compounds. A chimeric receptor, GerU*, demonstrates interchangeability between receptor components and provides evidence that it is the B protein of the receptor that determines germinant specificity. Introduction of the gerU/gerVB gene cluster to B. megaterium KM extends the range of germinants recognized by this strain to include glucose, proline, and KBr in addition to alanine and leucine. A chromosomally encoded receptor, GerA, the B component of which is predicted to be truncated, was found to be functionally redundant. Similarly, the plasmid-borne antiporter gene, grmA, identified previously as being essential for germination in QM B1551, did not complement the germination defect in the plasmidless variant PV361. Wild-type spores carrying an insertion-deletion mutation in this cistron germinated normally; thus, the role of GrmA in spore germination needs to be reevaluated in this species.     

Identification and control of synthesis of the dsdC activator protein.

Megacins A-216 and A-19213 in Bacillus megaterium are plasmid encoded, as shown by analysis of cured, non-megacinogenic (Meg-) derivatives of strains 216 and ATCC 19213 and by polyethylene glycol-mediated protoplast transformation of Meg- bacteria with plasmid DNA. The results of both techniques implicated a 31-megadalton plasmid, pBM309, in megacin A-216 production and a 29-megadalton plasmid, pBM113, in megacin A-19213 production.     

Bacteriocin from Bacillus megaterium ATCC 19213: comparative studies with megacin A-216

A bacteriocin produced by Bacillus megaterium ATCC 19213 was identified, purified, and compared with megacin A from B. megaterium 216. The ATCC 19213 bacteriocin was inducible with mitomycin C and showed phospholipase A activity. Both megacin A-216 and megacin A-19213 contained two dissimilar polypeptide subunits. Megacin A-216 contains a 30,000-dalton alpha subunit and a 15,000-dalton beta subunit. Megacin A-19213 is composed of an alpha subunit 18,000 daltons in mass and a beta subunit about 7,500 daltons in mass. No sequence similarities between alpha and beta subunits of either megacin were detected. The two megacins were further distinguished by quantitative differences in activity spectra and by immunodiffusion analyses.     

Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis.

Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(alpha)-phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine.     

Transport of D- and L-tryptophan in Bacillus megaterium by an inducible permease.

Tryptophan-grown cells of Bacillus megaterium ATCC 19213 contain a permease system that transports both D- and L-tryptophan and is inhibited by sodium azide. Arginine-grown cells contain little tryptophan permease activity, suggesting that the system is inducible. Arginine represses the tryptophan permease as well as the transport system for leucine and phenylalanine. Kynurenine was a more effective inducer of the tryptophan transport system than either D- or L-tryptophan.     

Mineralization and heat resistance of bacterial spores.

The heat resistances of the fully demineralized H-form spores of Bacillus megaterium ATCC 19213, B. subtilis var. niger, and B. stearothermophilus ATCC 7953 were compared with those of vegetative cells and native spores to assess the components of resistance due to the mineral-free spore state, presumably mainly from dehydration of the spore core, and to mineralization. Mineralization greatly increased heat resistance at lower killing temperatures but appeared to have much less effect at higher ones.     

Siderophore-Mediated Aluminum Uptake by Bacillus megaterium ATCC 19213

The bacterium Bacillus megaterium ATCC 19213 is known to produce two hydroxamate siderophores, schizokinen and N-deoxyschizokinen, under iron-limited conditions. In addition to their high affinity for ferric ions, these siderophores chelate aluminum. Aluminum was absorbed by B. megaterium ATCC 19213 through the siderophore transport receptor, providing an extra pathway for aluminum accumulation into iron-deficient bacteria. At low concentrations of the metal, siderophore-mediated uptake was the dominant process for aluminum accumulation. At high concentrations of aluminum, passive transport dominated and siderophore production slowed the passive transport of aluminum into the cell. Siderophore production was affected by the aluminum content in the media. High concentrations of aluminum increased production of siderophores in iron-limited cultures, and this production continued into stationary phase. Aluminum did not stimulate siderophore production in iron-replete cultures. The production of siderophores markedly affected aluminum uptake. This has direct implications on the toxicity of heavy metals under iron-deficient conditions.     

Inhibition of cortex hydrolysis during spore germination by CdCl2

When the spores of Bacillus megaterium QM B1551 (ATCC 12872) were incubated with 5 mM CdCl2 at 30 C, they underwent the early germination events, such as loss of heat resistance and release of calcium dipicolinate, in the same way as when they were germinated by glucose + KNO3. However, germination by CdCl2 caused no increase in the reducing groups in the cortex and no excretion of glucosamine-containing materials due to the hydrolysis of the cortex peptidoglycan. Addition of CdCl2 at any time during germination by glucose + KNO3 inhibited the release of glucosamine-containing materials from the spores, whereas removal of cadmium from the CdCl2-germinated spores by treatment with cysteine restored the hydrolysis of peptidoglycan. These results suggested that CdCl2 caused the early events of spore germination but prevented the spores from undergoing the events following germination by inhibiting the enzymatic lysis of the cortex peptidoglycan. The conclusion from the study is that cortex degradation is not always required for the initiation of germination.     

Germinability of coat-lacking spores of Bacillus megaterium

Upon treatment with acid, the germinability of both intact and coat-lacking spores of Bacillus megaterium ATCC 19213 exhibited similar features. Namely, when the spores previously germinated by alanine in the presence of phosphate buffer were converted to H-spores by treatment with nitric acid, germination proceeded at a very low speed in a same germination medium. When H-spores converted to Ca-spores by treatment with calcium acetate and subsequently germinated, germination proceeded at a speed higher than that of native spores and occurred even in the absence of buffer. These results suggest that the site of exchangeable cations concerned with germinability must not exist in the coat.     

Hydroxamate Recognition During Iron Transport from Hydroxamate-Iron Chelates

Kinetics of radioactive iron transport from three structurally different secondary hydroxamate-iron chelates (schizokinen-iron, produced by Bacillus megaterium ATCC 19213; Desferal-iron, produced by an actinomycete; and aerobactin-iron, produced by Aerobacter aerogenes 62-1) revealed that B. megaterium SK11 (a mutant which cannot synthesize schizokinen) has a specific transport system for utilization of ferric hydroxamates with a recognition capacity based on the chemical structure of the hydroxamate. Both Desferal and schizokinen enhanced iron uptake in this organism; however, Desferal-iron delivered only one-sixth the level of iron incorporated from the schizokinen-iron chelate. Desferal-iron did not generate the rapid rates of iron transport noted with schizokinen-iron at elevated iron concentrations. Assays containing large excesses of either iron-free Desferal or iron-free schizokinen suggested that the iron-free hydroxamate may compete with the ferric hydroxamate for acceptance by the transport system although the system has greater affinity for the iron chelate. Aerobactin-iron did not stimulate iron uptake in B. megaterium SK11 and aerobactin inhibited growth of this organism, indicating that B. megaterium SK11 cannot efficiently process the aerobactin-iron chelate.     

Cloning and Sequencing of the Spore Germination Gene of Bacillus megaterium ATCC 12872: Similarities to the NaH-Antiporter Gene of Enterococcus hirae

The germination mutant TM-31 of Bacillus megaterium ATCC 12872, was isolated by transposon Tn917 insertional mutagenesis. Glucose, L -proline, L -leucine and KNO₃ germinated TM-31 poorly. The DNA in the region of the Tn917 insertion was cloned, and its nucleotide sequence determined. One major open reading frame was present on the cloned DNA. The hydrophobic protein encoded is presumably membrane-associated. A homology search revealed that the gene encoded in the region of the Tn917 insertion is homologous to napA of Enterococcus hirae. napA codes for the NaH-antiporter. It is hypothesized that transport of cations must play an important role in spore germination in B. megaterium ATCC 12872.     

17K-spore coat protein antigen in sporulating cells of Bacillus megaterium ATCC 19213

Using immunological techniques, we studied the behavior of spore coat protein during sporulation of Bacillus megaterium ATCC 19213. Antibody specific to the main coat protein of 17,000 daltons was prepared and used to demonstrate that the spore coat protein was synthesized and deposited at a later stage during sporulation.     

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.     

Comparison of various properties of low-molecular-weight proteins from dormant spores of several Bacillus species.

Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared. All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan. The proteins could be subdivided into two groups: group I (B. megaterium A and C proteins, B. cereus A protein, and B. subtilis alpha and beta proteins) and group II (B. cereus and B. megaterium B proteins and B. subtilis gamma protein). Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline. Similarly, proteins in each group were more closely related immunologically. However, antisera against a B. megaterium group I protein cross-reacted more strongly with the B. megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B. megaterium group II protein cross-reacted most strongly with B. megaterium group I proteins. Analysis of the primary sequences at the amino termini and in the regions of the B. cereus and B. subtilis proteins cleaved by the B. megaterium spore protease revealed that the B. cereus A protein was most similar to the B. megaterium A and C proteins, and the B. cereus B protein and the B. subtilis gamma protein were most similar to the B. megaterium B protein. However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.     

环状芽孢杆菌α—羟基—γ—氨丁酰酰化酶基因的克隆和表达

本文报道了以质粒pUB110为载体,以枯草芽孢杆菌(Bacillus subtilis 168)作为受体菌,对丁酰苷菌素产生菌(Bacillus circulans NRRL-B3312)总DNA进行了鸟枪克隆,在所获得的转化子中,No.733转化子经薄层层析,生物显迹和质谱分析表明,它具有将卡那霉素A生物转化成为丁胺卡那霉素的能力,说明该转化子所含重组质粒pUBC733的插入片段中含有a-羟基-r-氨丁酰(HABA)酰化酶基因,HABA酰化酶基因已经在枯草芽孢杆菌中获得了克隆和表达。该重组质粒分子量为7.3kb,插入片段为2.8kb,经Southern分子杂交确证此片段确来源于环状芽孢杆菌,已构建了该质粒限制性内切酶图谱。     

Appearance of uridine 5'-diphospho-N-acetylglucosamine-4-epimerase during sporulation of Bacillus megaterium

In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high-performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion-pair (or anion-exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.     

Characterization and deposition of the proteins in the outermost layer of Bacillus megaterium spore

It was proved that three spore coat proteins of 48, 36, and 22 kDa (P48, P36, and P22) were the components of the outermost layer (OL) of Bacillus megaterium ATCC 12872 spore by analysis of the isolated OL. And it was indicated that these proteins were deposited not by disulfide bond, but by ionic and/or hydrophobic bonds on the spore. Among them, P36 and P22 were expected to be located on the very surface of the spore by immunological analysis. In the OL deficient mutant of B. megaterium ATCC 12872, MAE05, whose spore was lacking in these OL proteins and galactosamine-6-phosphate polymer, both P36 and P22 were present in the mother cell cytoplasm and deposited on the forespores, but they disappeared with the lysis of mother cells. An OL protein-releasing factor having proteolytic activity was detected in the culture supernatant at the late sporulating stage of both the wild-type and the mutant strains. But the factor could not act on the proteins of the mature spores and the forespores at t10 (tn indicates n hr after the end of exponential growth) of the wild-type strain. Moreover, P36 and P22 were found in the spores of a revertant of MAE05 which could form galactosamine-6-phosphate polymer, suggesting that this sugar polymer played the role in protecting the OL proteins against the protease-like substance after the deposition.