Fine Structure of Bacillus megaterium during Microcycle Sporogenesis

Ultrathin sections were prepared from cultures of Bacillus megaterium QM B1551 undergoing microcycle sporogenesis (initial spore to primary cell to second-stage spore without intervening cell division) on a chemically defined medium. The cytoplasmic core of the dormant spore was surrounded by plasma membrane, cell-wall primordium, cortex, outer cortical layer, and spore coats. Early in the cycle, the coat opened at the germinal groove, the cortex swelled, ribosomes and a chromatinic area associated with large mesosomes (which may later be incorporated into the expanding plasma membrane) appeared in the core, and the cell wall became defined at the site of the cell wall primordium. Poly-β-hydroxybutyrate granules began to appear in the primary cell at about 3 hr. By 7 hr, the forespore of the second-stage spore was delineated by typical double membranes. Between 7 and 12 hr, second-stage cell-wall primordium and cortex developed between the separating forespore membranes. The inner membrane became the plasma membrane of the second-stage spore, and the outer membrane eventually disintegrated within the second-stage spore cortex. A densely staining double layer (spore-coat primordium) developed external to the outer forespore membrane. The inner spore coat and the outer cortical layer of the second-stage spore developed from this primordium. The outer part of the spore coat, probably of sporangial origin, was laid down on the external surface of the inner spore coat. By 12 hr, second-stage spores were almost mature. By 20 hr, the mature endospores, with a thickened outer coat, were often still enclosed by degenerate primary cell wall and by the outer cortical layer and spore coat of the initial spore.     

L-Proline site for triggering Bacillus megaterium spore germination.

Germination of $\text{Bacillus megaterium}$ QM B1551 spores can be triggered by L-proline chloromethyl ketone at ～ 10 fold lower concentrations than L-proline. [³H] L-proline chloromethyl ketone bound to several protein fractions, one of which was decreased in a mutant (JV137) that cannot be triggered by L-proline. Treatment of spores with [³H] acetic anhydride specifically inhibited L-proline triggered germination, and also covalently modified the same protein fraction which appears to be bound to the spore membrane. These results indicate that it is possible to identify a protein fraction in spores that may play a key role in triggering spore germination.     

Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

Previous work demonstrated that Bacillus megaterium QM B1551 spores that are null for the sleB and cwlJ genes, which encode cortex-lytic enzymes (CLEs), either of which is required for efficient cortex hydrolysis in Bacillus spores, could germinate efficiently when complemented with a plasmid-borne copy of ypeB plus the nonlytic portion of sleB encoding the N-terminal domain of SleB (sleB^N). The current study demonstrates that the defective germination phenotype of B. megaterium sleB cwlJ spores can partially be restored when they are complemented with plasmid-borne ypeB alone. However, efficient germination in this genetic background requires the presence of sleL, which in this species was suggested previously to encode a nonlytic epimerase. Recombinant B. megaterium SleL showed little, or no, activity against purified spore sacculi, cortical fragments, or decoated spore substrates. However, analysis of muropeptides generated by the combined activities of recombinant SleB and SleL against spore sacculi revealed that B. megaterium SleL is actually an N-acetylglucosaminidase, albeit with apparent reduced activity compared to that of the homologous Bacillus cereus protein. Additionally, decoated spores were induced to release a significant proportion of dipicolinic acid (DPA) from the spore core when incubated with recombinant SleL plus YpeB, although optimal DPA release required the presence of endogenous CLEs. The physiological basis that underpins this newly identified dependency between SleL and YpeB is not clear, since pulldown assays indicated that the proteins do not interact physically in vitro.     

Outgrowth of Bacillus megaterium Spores in the Presence of Nitrate and Ammonium Ions

Nitrate or ammonium ions, but not nitrite ion, supplied the nitrogen required for outgrowth of Bacillus megaterium QM B1551 spores through the first cell division. This outgrowth was markedly favored by K⁺.     

A unique method for studying the initiation of Bacillus megaterium spore germination.

The sequence of events that characterize initiation of $\text{Bacillus megaterium}$ QM B1551 spore germination can be interrupted by the presence of 1 mM HgCl₂. Whereas these was a complete loss in spore dipicolinic acid, only partial losses in absorbance, refractility, density and resistance to staining occurred. This simple technique allows one to determine the necessity of certain reactions for initiation of germination.     

Surface hydrophobicity of spores of Bacillus spp

The surface hydrophobicity of 12 strains of Bacillus spp. was examined in a hexadecane-aqueous partition system. Mature and germinated spores of Bacillus megaterium QM B1551 transferred to the hexadecane layer, while vegetative and sporulating cells did not. Wild-type spores were more hydrophobic than spores of an exosporium-deficient mutant of B. megaterium QM B1551, although the mutant spores were shown to be hydrophobic to some extent by using increased volumes of hexadecane. This result suggests that the exosporium is more hydrophobic than the spore coat and that the surface hydrophobicity of spores depends mainly on components of the exosporium. The surface hydrophobicity of spores of nine other species of Bacillus was also examined, and spores having an exosporium were more hydrophobic than those lacking an exosporium. Thus measurement of the hydrophobicity of spores by the hexadecane partition method may provide a simple and rapid preliminary means of determining the presence or absence of an exosporium.     

Spore Germination of Bacillus megaterium QM B1551 Mutants

Auxotrophic or antibiotic-resistant mutants of Bacillus megaterium QM B1551 are capable of initiation of germination similar to the parental (QM B1551) prototrophic strain.     

Activation Energy for Glucose-Induced Germination of Bacillus megaterium Spores

The maximum germination rate of Bacillus megaterium QM B1551 spores in glucose increased, and the lag before its attainment decreased, with increasing germination temperature. The activation energy for germination (μ = approximately 20 kcal/mole), based on rate or on lag, was consistent with an enzymatic mechanism.     

Properties of Hg- and Cd-spores of Bacillus megaterium QM B1551

Hg- and Cd-spores of Bacillus megaterium QM B1551 were produced in Schaeffer's medium containing mercuric chloride and cadmium chloride respectively. Metals were added to the medium at 9 hr of incubation (Stage V) to give a final concentration of 50 microM. It was found by electron microscopic and biochemical studies that the coats of both Hg- and Cd-spores were thinner than those of control spores. Of the total Hg and Cd in the spores, 77% of the Hg and 63% of the Cd were detected in the spore coats. Hg- and Cd-spores were less resistant to heat and more sensitive to germinants than control spores. Other properties of Hg- and Cd-spores were similar to those of control spores. These results suggest that the spore coat has some relationship to the heat resistance and germinability of spores.     

Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551

We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly. Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551. Neither of the loci affected regulation of the xylA-lacZ fusions. These results confirm the suitability of the two gene loci for single-copy cloning. Received: 28 October 1996 / Received revision: 29 December 1996 / Accepted: 4 January 1997     

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.     

<Emphasis Type="Italic">Bacillus megaterium</Emphasis>—from simple soil bacterium to industrial protein production host

Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation, germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a “toolbox” of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences make B. megaterium an ideal organism for industrial, environmental, and experimental applications.     

Occurrence of galactosamine-6-phosphate as a constituent of Bacillus megaterium spore coat

Galactosamine-6-phosphate was identified as a component of the coat of the Bacillus megaterium QM B1551 spore. It was one of the main constituents of the outermost layer of the spore coat, but it was absent from the other integuments including the cortex. These findings suggest that galactosamine-6-phosphate comprises the phosphorus-containing skeleton structure of the spore coat.     

An evaluation of respiration chain-associated functions during initiation of germination of Bacillus megaterium spores

In Bacillus megaterium QM B1551, spore germination could be initiated by glucose in the absence of detectable oxygen consumption, ATP synthesis or a pH decrease in the external media, suggesting that none of those reactions were mandatory. In addition, initiation of germination was insensitive to a variety of inhibitors of energy production or protonmotive force uncouplers. Therefore the respiratory chain-associated functions are not prerequisites for initiation of germination but these functions may be necessary to drive energy-dependent transport systems and other biosynthetic reactions during outgrowth.     

ULTRASTRUCTURAL LOCALIZATION OF MINERAL MATTER IN BACTERIAL SPORES BY MICROINCINERATION

The fine localization of mineral matter in spores of Bacillus megaterium and Bacillus cereus was studied by the technique of microincineration adapted for use with the electron microscope. The specimens, which included intact and thin-sectioned spores as well as shed spore coats, were burned either in the conventional way at high temperature or by a new technique using electrically excited oxygen at nearly room temperature. The ash residues were examined by bright field, dark field, and diffraction in the electron microscope and also with the phase contrast microscope. In some cases, the specimen was previewed in both microscopes before incineration. The results do not support a previous report that the mineral elements of the spore are confined to a peripheral layer, but rather indicate that the spore core as well as the coat are mineral-rich. The cortex may be deficient in minerals, but the possibility of artifact prevents a clear decision on this point. Incinerated B. megaterium spores show a highly ordered fine structure displaying 100 A periodicity in the ash of the middle layer of the coat. The nature of this structure is discussed, as is the technique which demonstrated it. The fine definition of the ash patterns, particularly those obtained with the low-temperature, excited-oxygen technique, suggests that microincineration may be generally useful in the study of fine structure.     

Location of Calcium Within Bacillus Spores by Electron Probe X-Ray Microanalysis

Spectroscopic microanalysis of the element-characteristic X rays produced by a scanning electron microprobe was employed to detect calcium and carbon in both intact and thin-sectioned spores of Bacillus cereus T and B. megaterium QM B1551. Linear scan profiles and multilinear scan images of the X-ray emissions for calcium (Ca(Kalpha)) were compared with those for carbon (C(Kalpha)) as an index of mass. Location was accomplished by stereological comparisons with secondary electron images and conventional transmission electron micrographs. Although the elements could be detected at the attogram level theoretically, spatial resolution was limited to approximately 500 to 1,000 nm in an intact spore, e.g., by the primary electron beam diameter, the electron-excited spore microvolume, and the type of specimen support. The resolution was improved to approximately 100 to 200 nm by use of thin-sectioned spores, with precautions to prevent calcium leakage from the specimen during preparations. In both intact and sectioned spores, calcium was distributed throughout the spore, similarly to carbon, and concentrated mainly in a central region corresponding to the spore protoplast.     

Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids ofBacillus megateriumQM B1551

A replicon from one of an array of seven indigenous compatible plasmids ofBacillus megateriumQM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bpHindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3′ end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act intrans.A small region with similarity to theB. subtilischromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressedrepgene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of aB. megateriumreplicon.     

Bioleaching of a Complex Co-Ni-Cu Sulfide Flotation Concentrate by Bacillus megaterium QM B1551 at Neutral pH

Bioleaching of sulfide minerals at neutral pH has been rarely reported. In this study, a bacterium, Bacillus megaterium QM B1551, was isolated from Jinchuan sulfide tailings and used to leach a complex sulfide flotation concentrate for the extraction of Co²⁺, Ni²⁺ and Cu²⁺ at near neutral pH. A total of 38.2% Co, 44.7% Ni and 3.6% Cu were extracted from the sulfide concentrate in 5 days with an initial pH of 6. An enhanced Co²⁺, Ni²⁺ and Cu²⁺ extraction extent was achieved by first bioleaching the concentrate with Bacillus megaterium QM B1551 at 35°C and then followed by chemical leaching with 4 M sulfuric acid at 90°C. As a result, a total of 60.7% Co²⁺, 76.3% Ni²⁺ and 39.8% Cu²⁺ were extracted. On an industrial scale, the profits from the metal recovery by such a combined leaching procedure are optimum if considering the cost-benefit ratio.     

Mechanism of killing of spores of Bacillus cereus and Bacillus megaterium by wet heat

Aims: To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. Methods and Results: Bacillus cereus and B. megaterium spores wet heat‐killed 82–99% gave two bands on equilibrium density gradient centrifugation. The lighter band was absent from spores that were not heat‐treated and increased in intensity upon increased heating times. These spores lacked dipicolinic acid (DPA) were not viable, germinated minimally and had much denatured protein. The spores in the denser band had viabilities as low as 2% of starting spores but retained normal DPA levels and most germinated, albeit slowly. However, these largely dead spores outgrew poorly if at all and synthesized little or no ATP following germination. Conclusions: Wet heat treatment appears to kill spores of B. cereus and B. megaterium by denaturing one or more key proteins, as has been suggested for wet heat killing of Bacillus subtilis spores. Significance and Impact of the Study: This work provides further information on the mechanisms of killing of spores of Bacillus species by wet heat, the most common method for spore inactivation.     

Immunological Detection of 22K Protein in Sporulating Cells of Bacillus megaterium ATCC 12872

The synthesis and deposition of 22,000-dalton (22K) spore coat protein were examined immunochemically on the sporulating cells of Bacillus megaterium ATCC 12872 using the antibody to purified 22K spore coat protein. This antibody cross-reacted with 44K and 25K proteins in immunoblot analysis of dormant spore coat proteins. Immunoblot analysis on the sporulating cells showed that 22K protein was detected from t₈ in forespore coat protein fractions. Sandwich enzyme immunoassay revealed that 22K protein in the spore coat protein fraction appeared at t₆ and reached a plateau at t₉, and 22K protein in the mother cell cytoplasmic fraction was detected at only t₇ and t₈ at a very low level.