Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement

An efficient method for gene replacement in Bacillus megaterium was developed and used to inactivate the chromosomal neutral protease gene (nprM) from strain DSM319. A temperature-dependent suicide vector was constructed to allow replacement of the normal chromosomal copy with an altered version of the nprM gene. One mutant B. megaterium MS941 was selected for further characterization. Measurement of extracellular protease activity from strain MS941 indicated the existence of an additional minor extracellular protease in B. megaterium. Inhibitor studies revealed that this minor protease, comprising only 1.4% of the wild-type total extracellular protease activities, is a serine-type enzyme.Data presented in this contribution are part of a doctoral thesis of the Naturwissenschaftliche Fakultät Münster, Germany (KDW)     

Cloning, expression, and carbon catabolite repression of the bamM gene encoding beta-amylase of Bacillus megaterium DSM319

The bamM gene from Bacillus megaterium DSM319 encoding an extracellular beta-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 degrees C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE--retaining unchanged the amino acid sequence--provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.     

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.     

Cloning and sequencing of the leu C and npr M genes and a putative spo IV gene from Bacillus megaterium DSM319

The leuC gene, encoding 3-isopropylmalate dehydrogenase, the nprM gene (neutral protease) and a sporulation gene coding for a putative spoIV protein (spoIV) from Bacillus megaterium DSM319 were cloned and the nucleotide sequences were determined. The leuC gene is 1101 bp in length, preceded by a ribosome binding site; no promoter consensus sequence could be found. The nucleotide sequence from nprM when compared to the recently published gene from B. megaterium ATCC 14581 exhibited only a 17-base pair deviation. From a sporulation mutant isolated after transposonmutagenesis with transposon Tn917 the insertion site of the transposon was cloned and adjacent chromosomal fragments were characterized. An open reading frame that encodes for a putative spo protein of 247 amino-acid residues was identified.Sequence data presented in this contribution are part of doctoral theses of the Naturwissenschaftliche Fakultät Münster, Germany nprM (KDW); leuC and spoIV (MB)     

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     

Isolation and characterization of a unique division mutant of Bacillus megaterium.

A filamentous division mutant, PV302, of Bacillus megaterium QM B1551 was isolated while screening for sporulation-defective mutants after nitrosoguanidine mutagenesis. Both phase-contrast and electron microscopy revealed that the mutant produced small spherical cells as well as filaments. It also accumulated large amounts of poly-beta-hydroxybutyrate. Poly-beta-hydroxybutyrate accounted for 16% of the dry weight of the mutant strain even after 28 h growth. In comparison to the parental strain, the division mutant also showed both an inability to sporulate and a reduced growth rate. All these phenotypes transduced together. Revertants gained the ability to sporulate, divide, and grow normally. Transductional mapping of the mutation, designated div-1, established a new linkage group for B. megaterium consisting of div-1 and the pyrimidine biosynthesis genes pyrD BCF. The spherical cells were separated from filaments by sucrose gradients and were tested for nucleic acid content and viability. The purified spherical cell fraction contained one-fifth the amount of DNA per mg protein as compared with the filamentous cell fraction and was shown to contain both non-viable minicells and some cells capable of growing after a lag of about 4 h. This suggests that the mutation not only causes defects in septum placement and sporulation, but may possibly affect DNA partitioning.     

Functional analysis of the response regulator DegU in Bacillus megaterium DSM319 and comparative secretome analysis of degSU mutants

We functionally analysed the two-component regulatory system DegSU (historically SacU) in Bacillus megaterium DSM319 by generating a genetic knock out as well as a sacU32 mutation. The latter—known to cause a hypersecretion phenotype in Bacillus subtilis—had no influence on extracellular protease and amylase activity in B. megaterium. Since the B. megaterium DegU complemented a Bacillus licheniformis ∆degSU mutant, functionality of the protein was proven. Expression of the sacB encoded levansucrase was found to be dependent on DegSU in B. megaterium. Consistently, the fusion of the sacB promoter to gfp revealed a strong increase in GFP-expression in the sacU32 strain. On 2 D-gels of the secretome, a large number of intracellular proteins was seen. The culture medium contained only 42 secreted proteins which can be assigned to polypeptides involved in the metabolism of the cell wall, polypeptides with proteolytic activities and those with unknown functions. Though overall protease activity matches with the wild type, two proteolytic enzymes (Vpr and YwaD) are missing in the secretome of the ∆degSU strain, while other degradative enzymes are not affected. In line with such findings, no increase of proteolytic or other degradative enzymes was seen in the sacU32 mutant. Thus, compared to B. subtilis and B. licheniformis, the number of extracellular proteins influenced by DegSU is surprisingly low in B. megaterium, a feature, probably advantageous as to the use of the sacU32 mutant for production of secreted proteins.     

Isolation and characterization of outermost layer deficient mutant spores of Bacillus megaterium

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.     

Aminotransferase from a deletion mutant in the histidine operon

The imidazolylacetolphosphate:l-glutamate aminotransferase from the deletion mutant hisHB22 has been partially characterized. Although genetic studies have not yet shown the deletion to involve the structural information for this enzyme, physical studies indicate that an abnormal enzyme is produced. Evidence is presented which, together with previous data on the characterization of the enzyme, indicates that the catalytic integrity of the enzyme is intact, and that the low specific activity seen in cell extracts is due to formation of an enzyme which has a reduced coenzyme content. It is suggested that this reduced coenzyme content is due primarily to a reduced affinity of the enzyme (nascent or apo-) for its coenzyme, and that the coenzyme must be incorporated into the enzyme at the moment of synthesis for formation of a functional protein.     

Properties of a thermosensitive asporogenous filamentous mutant of Bacillus megaterium

Mutant TH14 of Bacillus megaterium ATCC 19213 is thermosensitive and defective in cell-division septation and spore formation at the restrictive temperature (39 C). As a consequence, the mutant forms multinucleate aseptate filaments and is asporogenic. The mutation does not result in any qualitative compositional changes in extractable membrane proteins. At the restrictive temperature, the mutant membrane has a reduced content of a small molecular weight protein(s). A membrane protein(s) with a molecular weight of nearly 80,000 appears to be partially derepressed in the mutant grown at the restrictive temperature. In addition, numerous unidentified spherical inclusions of fairly uniform size (diameter approximately 100 nm) are present in the cytoplasm at the restrictive temperature. They are especially concentrated at only one pole of each filament. Filamentous growth of the mutant is less sensitive to penicillin than growth in the rod form. Growth in either form is equally sensitive to d-cycloserine at the concentrations used for selection of the mutant. Temperature shift-up experiments suggest that one to two rounds of deoxyribonucleic acid (DNA) replication occur before the phenotypic expression of the mutation occurs. The septations after these replication events can be either two-division septations or a single-division septation plus a subsequent sporulation septation. This conclusion, coupled with previously reported work, supports the hypothesis that the early stages of sporulation represent a modified cell division.     

A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA)

In the genome of Bacillus megaterium DSM319, a strain who has recently been sequenced to fully exploit its potential for biotechnological purposes, we identified a gene encoding the cytochrome P450 CYP106A1 as well as genes encoding potential redox partners of CYP106A1. We cloned, expressed, and purified CYP106A1 and five potential autologous redox partners, one flavodoxin and four ferredoxins. The flavodoxin and three ferredoxins were able to support the activity of CYP106A1 displaying the first cloned natural redox partners of a cytochrome P450 from B. megaterium. The CYP106A1 system was able to convert the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) belonging to the main bioactive constituents of Boswellia serrata gum resin extracts, which are used to treat inflammatory disorders and arthritic diseases. In order to provide sufficient amounts of the KBA products to characterize them structurally by NMR spectroscopy, recombinant whole-cell biocatalysts were constructed based on B. megaterium MS941. The main product has been identified as 7β-hydroxy-KBA, while the side product (～20 %) was shown to be a mixture of 7β,15α-dihydroxy-KBA and 15α-hydroxy-KBA. Without further optimization 560.7 mg l^?1 day^?1 of the main product, 7β-hydroxy-KBA, could be obtained thus providing a suitable starting point for the efficient production of modified KBA by chemical tailoring to produce novel KBA derivatives with increased bioavailability and this way more efficient drugs.     

The restriction modification system of Bacillus licheniformis MS1 and generation of a readily transformable deletion mutant

Restriction modification systems (R-M systems), consisting of a restriction endonuclease and a cognate methyltransferase, constitute an effective means of a cell to protect itself from foreign DNA. Identification, characterization, and deletion of the restriction modification system BliMSI, a putative isoschizomer of ClaI from Caryophanon latum, were performed in the wild isolate Bacillus licheniformis MS1. BliMSI was produced as recombinant protein in Escherichia coli, purified, and in vitro analysis demonstrated identical restriction endonuclease activity as for ClaI. A recombinant E. coli strain, expressing the heterologous bliMSIM gene, was constructed and used as the host for in vivo methylation of plasmids prior to their introduction into B. licheniformis to improve transformation efficiencies. The establishment of suicide plasmids in the latter was rendered possible. The subsequent deletion of the restriction endonuclease encoding gene, bliMSIR, caused doubled transformation efficiencies in the respective mutant B. licheniformis MS2 (∆bliMSIR). Along with above in vivo methylation, the establishment of further gene deletions (∆upp, ∆yqfD) was performed. The constructed triple mutant (∆bliMSIR, ∆upp, ∆yqfD) enables rapid genome manipulation, a requirement for genetic engineering of industrially important strains.

     

A complete deletion mutant of the Escherichia coli dnaKdnaJ operon

Southern hydridization analyses of genomic DNAs from various dnaJ mutants of Escherichia coli showed that mutant K7052, which has well characterized dnaK706 and dnaJ705 double mutantions, is a deletion mutant. The deletion is about 8.0 kb long and encompasses the whole of the dnaKdnaJ operon.     

Isolation and characterization of forespores from Bacillus megaterium

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine. Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension.