Molecular Characterization of Plasmid pBM300 from Bacillus megaterium QM B1551

Strain QM B1551 of Bacillus megaterium contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G+C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea. oriT and ssoT-like regions were detected near two mob genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.     

Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551

An 18 633 bp region containing the replicon from the approximately 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non-essential plasmid DNA has implications for systematics and evolutionary biology.     

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.     

Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes

Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N- or C-terminal domain has been deleted (SleB-ΔN or SleB-ΔC) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-ΔN or SleB-ΔC, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N- or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.Spores of the genera Bacillus and Clostridium emerge from dormancy via the process of germination. The germination process comprises a series of sequential biophysical and biochemical reactions that result irreversibly in the spore losing its properties of metabolic dormancy and extreme resistance to various chemical and physical treatments (24, 34). Germination is initiated by the presumed binding of small molecular germinants, commonly amino acids or sugars, to cognate receptors located within the spore inner membrane (25, 28). In a process that is poorly understood at the molecular level, this interaction leads to a change in the permeability of the inner membrane, resulting in the release of various solutes from the spore core, including metal ions, calcium dipicolinate (Ca-DPA), and some amino acids (32, 33, 35). A degree of rehydration of the core is evident at or around the same time, although this is insufficient to permit a significant degree of vegetative metabolism (9, 31). These events, which appear common to all Bacillus species where examined, comprise stage I of germination (31, 32, 34).The major event in stage II of the germination process from a biochemical perspective involves depolymerization of the spore cortex. The spore cortex is a thick layer of peptidoglycan, characterized by the spore-specific muramic acid lactam (MAL) moiety (37, 38), which, together with the thin inner layer of germ cell wall peptidoglycan (36), forms contiguous layers that entirely envelope the spore protoplast. While the germ cell wall forms the initial cell wall during vegetative outgrowth, the spore cortex serves primarily to maintain the relatively dehydrated status of the spore protoplast during dormancy (13). Dissolution of the cortex permits complete hydration of the spore core and resumption of vegetative metabolism, leading ultimately to shedding of the spore coat and the emergence of a new vegetative cell (34).A number of studies have indicated that spores of various Bacillus species employ two cortex-lytic enzymes (CLEs), SleB and CwlJ, to initiate hydrolysis of the cortex during stage II of the germination process (16, 19, 32). These enzymes are semiredundant; hence, strains bearing null mutations in either structural gene can still degrade the cortex sufficiently to complete germination, whereas double mutant strains do not appear capable of degrading the cortex at all, resulting typically in a decrease of several orders of magnitude in colony-forming ability (15, 19, 32). Other enzymes, including Bacillus cereus/Bacillus anthracis SleL, are also involved in stage II of germination, apparently hydrolyzing peptidoglycan products of SleB and/or CwlJ to smaller peptidoglycan fragments that can more easily permeate through the spore coats to the surrounding germination medium (21).Studies with SleB and SleL purified from dormant and germinating spores indicate that whereas the latter enzyme degrades only cortical fragments of peptidoglycan (7), SleB has a requirement for intact peptidoglycan that has adopted the precise architecture present within the spore (12, 22). These substrate requirements appear to be important in maintenance of the respective autolysins, which are present in the spore in a mature form, in an inactive state during dormancy. Additionally, whereas the molecular mechanism of activation of SleB remains unclear—a change in cortical stress/architecture induced by stage I events has been hypothesized (12)—the efflux of Ca-DPA from the spore core to the cortex/coat boundary where CwlJ is localized (5) appears to be the mechanism by which this CLE is activated. CwlJ can also be activated by high concentrations of exogenous Ca-DPA, presenting an alternative germination pathway that bypasses the germinant receptors (27).The hydrolytic bond specificity of various CLEs has been examined by both direct and indirect biochemical means. Direct assays are typically conducted by incubation of purified or recombinant enzymes with peptidoglycan fragments or suspensions of spores in which the cortex is rendered accessible by first chemically compromising the permeability of the spore coats (7, 12, 22). Subsequent assays for the generation of reducing groups and/or free amino groups can yield information on the probable hydrolytic bond specificity of the respective enzyme(s) being assayed.More recently, the high-performance liquid chromatography/mass spectrometry (HPLC/MS)-based muropeptide analysis technique has been applied to characterize CLE activity during germination of various spore-forming species (2, 4, 10). This methodology has the resolution to reveal fine structural changes that occur to the peptidoglycan in vivo during germination, and when used in combination with CLE null mutant strains, it can be used to indirectly correlate the generation of certain classes of muropeptides, and therefore the hydrolytic bond specificity, with defined CLEs. Muropeptide analysis has revealed, for example, that an intact copy of the sleB gene in B. subtilis and B. anthracis is required for the presence of anhydromuropeptides in the germination exudates of these respective species, indicating that SleB is a lytic transglycosylase or generates substrate for subsequent lytic transglycosylase activity (6, 16). Conversely, B. cereus SleB was characterized as a probable amidase after enzyme purified from germinating spores was found to liberate a large amount of free amino groups when incubated with coat-stripped spores as a substrate (22). The hydrolytic bond specificity of SleB therefore remains ambiguous and perhaps varies between different species.Contrary to these observations, the overall structural architecture of SleB appears to be well conserved between different Bacillus species. Alignment of the primary amino acid sequence from different species indicates that the mature protein comprises an N-terminal domain that is connected to the C-terminal domain by a linker region that is variable in length and amino acid composition (Fig. ​(Fig.1).1). The N-terminal domain is thought to comprise the peptidoglycan binding domain by virtue of two direct sequence repeats that are reminiscent of cell wall-binding motifs observed in other proteins (26). The C-terminal domain shows homology with that of the other major Bacillus CLE, CwlJ, which lacks a corresponding peptidoglycan binding domain and is therefore thought to comprise the catalytic domain (19), although there is as yet no experimental evidence to substantiate this idea.Open in a separate windowFIG. 1.ClustalW alignment of SleB from various Bacillus species. Residues predicted to comprise putative structural domains are denoted. Stars indicate charged residues that were subjected to amino acid substitution in this work. BM, B. megaterium QM B1551; BC, B. cereus W; BCl, B. clausii KSM-K16; BS, B. subtilis 168.In the current study, we have investigated the molecular function of SleB during germination of Bacillus megaterium QM B1551 spores, employing engineered SleB N- and C-terminal deletion strains, site-directed mutagenesis (SDM), and muropeptide analyses. In addition to revealing several cortex-modifying activities during germination of this species, the presented data indicate that while the presence of either the N- or C-terminal domain of SleB is sufficient for the generation of anhydromuropeptides during germination, this may be an indirect effect, and at least a degree of lytic transglycosylase activity may result from the activity of another as yet unidentified enzyme.     

Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.     

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.     

Genetics of leucine biosynthesis in Bacillus megaterium QM B1551.

Genes involved in the biosynthesis of leucine have been mapped in Bacillus megaterium QM B1551, using transducing phage MP13. Mutations were designated leuA, leuB, or leuC on the basis of enzyme assays. Two mutant strains were deficient in the enzyme activities of leuA (alpha-isopropylmalate synthase) and leuC (beta-isopropylmalate dehydrogenase) and so may contain polar mutations. Fine-structure transduction mapping established the gene order leuC-leuB-leuA-ilv-hem-phe. The orientation of the leu genes to the ilv gene is the same as in Bacillus subtilis, but the relationship in respect to two other linked markers, hem and phe, differs.     

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.     

Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores.

Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores.     

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.     

Ionic germination of spores of bacillus megaterium QM B1551

Summary Germination requirements of suspensions of spores of Bac. megaterium QM B1551, a glucose type, have been examined employing a decrease in optical density as the criterion of germination. Low concentrations of deionized glucose showed no germinative powers. Glucose was effective in augmenting germination by a variety of inorganic salts. Salts alone were sufficient germinators, but not all salts were active. Cl^-, Br^-, I^- and NO₃ ^- were efficient germinators. The nature of the cation portion of the germinative salts played a determinant role. A primary role for ions in germination is proposed and a secondary, augmentative action is attributed to glucose.     

Effects of alpha- and beta-D-glucose on germination of spores of Bacillus megaterium QM B1551.

The effects of D-glucose anomers on the germination of dormant spores of Bacillus megaterium QM B1551 were studied, alpha-D-Glucose (1 mM) slightly initiated the germination of the dormant spores during 10 min incubation at 37 degrees C, while about 60% of the dormant spores became germinated with beta-D-glucose (1 mM) in the same conditions. From the above observations and the finding that only a trace amount of alpha- or beta-D-glucose may bind with the dormant spores, it is speculated that the beta-D-glucose-stereospecific receptor site for the germination exists on the surface of the dormant spores of the bacillus.     

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.     

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.     

Sequence of the genes for the beta and epsilon subunits of the ATP synthase of Bacillus megaterium QM B1551

Four of the genes for the subunits of the proton-translocating ATPase of Bacillus megaterium have been cloned into pBR322. Previous studies have shown that two of these genes, for the alpha and beta subunits, can complement Escherichia coli mutants defective in the genes for those subunits (Hawthorne, C.A., and Brusilow, W.S.A. 1985. J. Biol. Chem. 261, 5245-5248). We report here a restriction map of the cloned region and the complete nucleotide sequence of the genes for the beta and epsilon subunits as well as the deduced amino acid sequences and molecular weights of those subunits.     

Deletion mutants of megacinogenic plasmid pBM309 from Bacillus megaterium

A 46.8-kb plasmid, pBM309, of Bacillus megaterium determines the production of a bacteriocin, megacin A, and confers immunity against this antibiotic on the host cells. The megacin A (megA) and megacin A-immunity (megAim) genes were mapped on the physical map of pBM309 by using its deletion derivatives. Both genes were isolated as a 10.6-kb PstI fragment and cloned in Bacillus subtilis vector plasmid pBD9 for expression in B. megaterium.     

Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551: primary roles of gluconokinase and the pentose cycle

The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.