Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Mn2 + improves surfactin production by Bacillus subtilis

Surfactin productivity by Bacillus subtilis was increased from 0.33 g l^–1 to 2.6 g l^–1 by adding 0.01 mM Mn²⁺ to a defined glucose medium. The final yield exceeded that of most reported values for genetically improved strains.     

Discrimination of riboflavin producing Bacillus subtilis strains based on their fed-batch process performances on a millilitre scale

Forty-eight single-use stirred tank bioreactors on a 10-mL scale operated in a magnetically inductive driven bioreaction block and automated with a liquid handler were applied for discrimination of different riboflavin producing Bacillus subtilis strains based on their performances in the parallel fed-batch processes. It was shown that a discrimination of the B. subtilis riboflavin producer strains can efficiently be achieved within one parallel fermentation run based on the integral riboflavin yield after 48 h. The possibility to perform replicates within the parallel fermentation run allows for a robust statistical analysis and is a prerequisite for the discrimination of producer strains under fed-batch process conditions. Within the estimation error, all of the riboflavin producing B. subtilis strains under study showed the same fed-batch process performances on the litre scale compared to the millilitre scale.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.     

Enhanced production of recombinant nattokinase in Bacillus subtilis by the elimination of limiting factors

By systematic investigation, glutamate and a mixture of metal ions were identified as factors limiting the production of nattokinase in Bacillus subtilis. Consequently, in medium supplemented with these materials, the recombinant strain secreted 4 times more nattokinase (260 mg l⁻¹) than when grown in the unsupplemented medium.     

Repeated use of Bacillus subtilis cell walls for copper binding

Purified cell walls from Bacillus subtilis were repeatedly suspended in 5 mm CuCl₂ and, after removing unbound Cu, were suspended in 1% (v/v) HNO₃ to release bound Cu. The walls were then regenerated by washing in H₂O. After five cycles, copper binding actually increased slightly, probably due to enhanced exposure of binding sites in the walls. Thus bacterial walls may be used repeatedly for metal removal during bioremediation of heavy metal pollution.R.J.C. McLean is with the Department of Biology, Southwest Texas State University, 601 University Drive, San Marcos, TX 78666-4616, USA A.M. Campbell is with the Department of Chemical Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. P.T. Khu is with the Department of Mining Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. A.T. Persaud, L.E. Bickerton and D. Beauchemin are with the Department of Chemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

The relative rotation of the ends of Bacillus subtilis during growth

Observation of long single filaments of Bacillus subtilis 168 in depression slide cultures demonstrated that one end rotated relative to the other during growth. This was observed with suspended filaments, filaments attached to glass surfaces and single stranded filaments folded back on themselves growing as a double stranded helix. This extends Mendelson's 1976 conclusion to cases with no alternative interpretation to the hypothesis that as each cell grows, the structure of the peptidoglycan changes to rotate one end relative to the other.     

Buoyant density fluctuations during the cell cycle of Bacillus subtilis

A simple rapid method for preparing synchronous cultures of Bacillus subtilis has been used to investigate changes in density during the cell cycle. Asynchronous cells separated on a stepped Percoll density gradient had a mean cell density of 1.117 g ml^-1±0.004. Samples from a synchronous culture exhibited variation (ca. 1.5%) in mean cell density which was greatest at the onset of cell division. An asynchronous control culture showed little variation in density. These results are discussed in relation to previous work on Escherichia coli.     

Enhanced amylase production by Bacillus subtilis using a dual exponential feeding strategy

A recombinant Bacillus subtilis strain (ATCC 31784) haboring the plasmid pC194 with a thermostable -amylase gene was cultured in a 22-l B. Braun Biostat C fermenter. Traditional batch operations suffer from low cell mass and protein productions because a high initial glucose concentration causes substrate inhibition and also product inhibition due to acetate accumulation. An exponential fed-batch strategy to prevent these inhibitions was developed in this work. The host strain is auxotrophic for phenylalanine, tyrosine and tryptophan. Due to low solubilities of tyrosine and tryptophan in the feed stream, tyrosine and tryptophan were dissolved separately in ammonia water to form a second feed stream. By dual feeding both streams at different exponential feed rates, a high cell density of 17.6 g/l and a final -amylase activity of 41.4 U/ml and the overall biomass yield of 0.39 g cell/g glucose were achieved.     

Conjugal Plasmid Transfer in Bacillus subtilis under Conditions of Soil Microcosms

Conjugal transfer of the small plasmid pUB110 betweenBacillus subtilis strains was studied under conditions of microcosms with sterile and nonsterile soil. Plasmid transfer proved to be possible after soil inoculation with vegetative partner cells or with their spores. Plasmid transfer occurred at temperatures of 30 and 22–23°C.     

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.     

Electron microscopy of isolated cell walls of Bacillus subtilis var. niger

Isolated cell walls of Bacillus subtilis have a striated appearance in the electron microscope. The structure persists when teichoic acids are removed. It is inferred that the structure bears on the arrangement of the peptidoglycan chains.     

Antifungal compounds from Bacillus subtilis B-FS06 inhibiting the growth of Aspergillus flavus

The cell-free culture filtrate (CCF) was prepared from a culture of an Aspergillus flavus antagonist, Bacillus subtilis B-FS06. The CCF inhibited the growth and spore germination of A. flavus at a series of concentrations (10, 25, 50%) (v/v). It still retained the activity after treatment at pH values ranging from 2 to 12 for 24 h or at 100 °C for 30 min. The antifungal activity, however, was reduced by 30% after treatment at 121 °C for 20 min. After purification by anion exchange chromatography, gel filtration chromatography and HPLC, the active compounds revealed six ion peaks: [M–H] m/z = 1006.78, 1020.71, 1034.74, 1049.54, 1056.78, and 1071.64 by using electrospray ionization mass spectrometry (ESI-MS) analysis. In the presence of the active compounds at 200 μg/g, the growth of A. flavus on peanuts was completely inhibited. Ting Zhang and Zhi-Qi Shi contributed equally to this work.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.