Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates

Various nutrients belonging to three categories, carbon, organic nitrogen and complex organic sources, were investigated for the first time in terms of their effect on the co-production of extracellular thermostable alpha-amylase and beta-galactosidase by Bacillus subtilis, a bacterium isolated from fresh sheep's milk. Among the organic nitrogen sources tested, tryptone and corn steep liquor favored their production. Substitution of soluble starch by various starchy substrates, such as corn flour, had a positive effect on both enzyme yields. Furthermore, a two-fold higher production of both enzymes was achieved when corn steep liquor or tryptone was used in combination with the different flours. Among the divalent cations examined, calcium ions appeared to be vital for alpha-amylase production. The crude alpha-amylase and beta-galactosidase produced by this B. subtilis strain exhibited maximal activities at 135 degrees C and 65 degrees C, respectively, and were also found to be significantly stable at elevated temperatures.     

Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis

The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the beta-galactosidase gene, lacZ, of Escherichia coli so that the expression of beta-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of beta-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID::lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spo0 lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make beta-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA----[spoIIG, spoIIE]----[spoIID, spoIIB, spoIIF].     

Promoter-probe plasmid for Bacillus subtilis.

We have constructed a promoter-probe expression vector for Bacillus subtilis. This plasmid, pCED6, can be used to fuse various DNA sequences to the structural gene of Escherichia coli beta-galactosidase, permitting analysis of the promoter activity of such sequences. pCED6 replicates and confers drug resistances in both E. coli and B. subtilis.     

Structural plasmid instability in recombination- and repair-deficient strains of Bacillus subtilis

Plasmid pGP1, containing a fusion between the penicillinase gene of Bacillus licheniformis and the beta-galactosidase gene of Escherichia coli, was constructed. This plasmid enabled a study of structural plasmid instability in Bacillus subtilis wild-type cells and a variety of B. subtilis strains, defective in recombination- and DNA-repair functions. Large differences with respect to the level of stability of this plasmid were observed in the various genetic backgrounds.     

Regulation of expression of genes coding for small, acid-soluble proteins of Bacillus subtilis spores: studies using lacZ gene fusions.

We constructed in-frame translational fusions of the Escherichia coli lacZ gene with four genes (sspA, sspB, sspD, and sspE) which code for small, acid-soluble spore proteins of Bacillus subtilis, and integrated these fusions into the chromosomes of various B. subtilis strains. With single copies of the fusions in wild-type B. subtilis, beta-galactosidase was synthesized only during sporulation, with the amounts accumulated being sspB much greater than sspE greater than or equal to sspA greater than or equal to sspD. Greater than 97% of the beta-galactosidase was found in the developing forespore, and the great majority was incorporated into mature spores. Less than 2% of the maximum amount of beta-galactosidase was made when these fusions were introduced into B. subtilis strains blocked in stages 0 and II of sporulation, as well as in some stage III mutants. Other stage III mutants, as well as stage IV and V mutants, had no effect on beta-galactosidase synthesis. Increasing the copy number of the sspA-, sspD-, or sspE-lacZ fusions (up to 17-fold for sspE-lacZ) in wild-type B. subtilis resulted in a parallel increase in the amount of beta-galactosidase accumulated (again only in sporulation and with greater than 95% in the developing forespore), with no significant effect on wild-type small, acid-soluble spore protein production. Similarly, the absence of one or more wild-type ssp genes or the presence of multiple copies of wild-type ssp genes had no effect on the expression of the lacZ fusions tested. These data indicate that these ssp-lacZ fusions escape the autoregulation seen for the intact sspA and sspB genes. Strikingly, the kinetics of beta-galactosidase synthesis were identical for all four ssp-lacZ fusions and paralleled those of glucose dehydrogenase synthesis. Similarly, all asporogenous mutants tested had identical effects on both glucose dehydrogenase and ssp-lacZ fusion expression.     

Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.     

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.     

The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Isolation and characterization of mutations in the gene encoding an endogenous Bacillus subtilis beta-galactosidase and its regulator.

We have isolated mutations that appear to inactivate the gene (lacA) encoding an endogenous beta-galactosidase activity in Bacillus subtilis and in a closely linked negative regulatory element (lacR). Both genes map to the hisA-thrA region. The lacA mutations may help to avoid some of the problems arising from the use of the Escherichia coli lacZ gene as a reporter gene in B. subtilis.     

Isolation and characterization of the lacA gene encoding beta-galactosidase in Bacillus subtilis and a regulator gene, lacR.

We have isolated transposon insertions in the lacA gene encoding an endogenous beta-galactosidase of Bacillus subtilis. Upstream of the putative operon containing lacA is a negative regulator, lacR, which encodes a product related to a family of regulators that includes the lactose repressor, lacI, of Escherichia coli. New strains with insertions in the lacA gene should be of use in studies using lacZ fusions in B. subtilis.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Export of extracellular levansucrase by Bacillus subtilis: inhibition by cerulenin and quinacrine.

Bacillus subtilis B secretes an inducible, extracellular enzyme, levansucrase. Inhibition studies were undertaken to investigate the possible mechanism of release of this enzyme. The antibiotic cerulenin, at a concentration of 10 micrograms/ml, totally inhibited de novo lipid synthesis in B. subtilis B for at least 1 h, while only slightly reducing protein and RNA synthesis. At this concentration cerulenin, added concomitantly with the inducer sucrose, prevented the release of levansucrase for at least 150 min. This was not due to the prevention of inducer uptake by the cells. The release of the enzyme was also independent of cell division. In B. subtilis 1007 the induction of beta-galactosidase by 5 mM lactose was not prevented by cerulenin. Preliminary evidence indicated the association of a lipid moiety with the enzyme as it passes through the cytoplasmic membrane. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing protease of Bacillus licheniformis, inhibited levansucrase release from B. subtilis B, but had no effect on lipid synthesis.     

An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis

The recently described beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis [Haima et al., Gene 86 (1990) 63-69]was optimized in several ways. First, the efficiency of translation of the lac Z delta M15 gene was improved. Second, the plasmid-borne lacZ delta M15 gene was segregationally stabilized by integration into the B. subtilis chromosome. Third, a new lacZ alpha complementing cloning vector was constructed, containing more unique target sites. It was shown that large heterologous DNA fragments (up to at least 29 kb) could be cloned with lacZ alpha-complementing vectors carrying the replication functions of the cryptic B. subtilis plasmid pTA1060, and that these inserts were structurally stably maintained for at least 100 generations of growth.     

枯草芽孢杆菌微生态制剂发酵研究进展

微生态制剂是饲用抗生素的绿色有效替代品。枯草芽孢杆菌在逆境中可形成抗逆性强的芽孢,在生产和应用过程中保持高活性,是一种高效的微生态制剂菌种。提高枯草芽孢杆菌活菌数及芽孢率是保证微生态制剂产品质量的关键。本文综述了枯草芽孢杆菌芽孢形成的分子生物学机制及影响芽孢形成的重要因素,进一步比较枯草芽孢杆菌微生态制剂不同发酵方式的特点,重点阐述了提高枯草芽孢杆菌有效生物量的工艺优化,最后介绍了枯草芽孢杆菌微生态制剂的应用,并对将来研究思路进行了讨论。