Gene ofBacillus Research Articles

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.

Transfer of an insecticidal protein gene ofBacillus thuringiensis into plant-colonizingAzospirillum

A fusion plasmid, pRKC, was constructed, using pACYC184, RSF1010 and a kanamycin-resistance cartridge from pUC4K, to convey thecryIA(a) gene intoAzospirillum spp. With the pRKC plasmid, the number of putative transconjugants obtained inA. lipoferum was about 300-fold higher than inA. brasilense. Conjugation frequency and plasmid stability inA. lipoferum were less for pBTF8, which carries thecryIA(a) gene in the correct orientation for a constitutive promoter, than for pBTF9, which carries the gene in the opposite orientation. Expression of thecryIA(a) gene was not apparent in SDS-PAGE analysis ofA. lipoferum transconjugants harbouring pBTF8. However,Escherichia coli transformants with the pBTF8 rescued fromA. lipoferum transconjugants produced an approximately 135 kDa Cry protein, indicating that thecry gene is intact in the transconjugants.

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus.

Expression of theZymomonas mobilis alcohol dehydrogenase II (adhB) and pyruvate decarboxylase (pdc) genes inBacillus

The genes encodingZymomonas mobilis pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were expressed inBacillus subtilis YB886(pLOI500) under the control of aBacillus SPO2 phage promoter and caused a 50% reduction of growth rate compared with the unmodified vector. Expression was further confirmed by Western blots, activity stains of native gels, and in vitro measurements of alcohol dehydrogenase activity. Additional strains ofBacillus were also transformed, and all produced similar but low levels of these enzymes. Although higher specific activities will be required for efficient ethanol production, no fundamental barriers exist to the expression of theseZ. mobilis genes inBacillus. Two abundant new proteins (ca. mass 33,000 daltons and 14,000 daltons) were observed in Coomassie Blue-stained gels; they are similar in size to the proteins induced by recombinatn products inEscherichia coli.

A genetically engineered mosquitocidal cyanobacterium

Larvae of the mosquitoAedes aegypti ingested, and developed into adults, on a diet of 1O of 14 different species of cyanobacteria includingAgmenellum quadruplicatum PR-6 (=Synechococcus PCC7002). Mosquito larvae ingested and grew on cells of PR-6 adapted to growth in the absence of NaCl. ThecryIVD gene ofBacillus thuringiensis var.israelensis was cloned into a PR-6 expression vector to form pAQRM56, which was transformed into PR-6. Expression of the CryIVD protein in PR-6 was demonstrated by immunocytochemistry and larvicidal activity. Immunogold labelling indicated production of an electron-dense material among the thylakoid membranes of PR-6. Cells of PR-6 carrying pAQRM56 were toxic to the larvae ofA. aeqypti whereas control cells were not. Growth of PR-6 cells carrying pAQRM56 was slower than the growth of control cells and these cells were also larger.

A novel oligonucleotide cassette for the overproduction ofEscherichia coli aspartate transcarbamylase inBacillus stearothermophilus

A novel double-stranded oligonucleotide cassette was devised in order to express heterologous genes inBacillus stearothermophilus. By linking the cassette to the upstream ofEscherichia coli aspartate transcarbamylase gene, this enzyme was overexpressed inB. stearothermophilus cells.

Genetic transfer, inheritance, and phenotypic expression of plasmid RP4::Mu cts genes inBacillus cereus

Genetic transfer, inheritance, and phenotypic expression of plasmid RP4::Mu cts genes inBacillus cereus

Stability of the recombinant plasmids carrying the α-amylase gene inBacillus strains

B. subtilis strains carrying plasmids pBDA 318, pUBA 10, pUBA 11 and pUBA 20 were cultivated for up to 50 generations without antibiotic selection and then screened for the presence of the plasmid and α-amylase gene. The plasmid pUBA 11 proved stable for industrial production, the other plasmids showed structural and segregational instability.

Effect of temperature on plasmid stability and expression of cloned cellulase gene in a recombinantBacillus megaterium.

The expression of carboxymethyl cellulase gene inBacillus megaterium (pCK108) was investigated with respect to temperature in batch culture. The suboptimal temperature supporting maximal cell growth rate was determined to be 30 °C at which stability of the plasmid pCK108 could be maintained stable. However, cellular plasmid contents, production rate of cellulase of the cell, and efficiency of the gene expression increased significantly with increase of the temperature from 30 °C to 44 °C, even though the plasmid stability decreased up to 60% level at the end of the culture.

Stability of the recombinant plasmid carrying theBacillus amyloliquefaciens α-amylase gene inB. subtilis

Theα-amylase gene ofBacillus amyloliquefaciens has previously been cloned into pUB110 to give the recombinant plasmid, pKTH10 (Palva 1982. Gene 19:81–87). Strains transformed by this plasmid are promising candidates for industrialα-amylase production. The stability of pKTH10 was determined in variousB. subtilis strains possessing specific alleles which affect the level ofα-amylase secretion.B. subtilis strains carrying pKTH10 were cultivated in starch-containing medium for up to 50 generations without antibiotic selection and then screened for the presence of pKTH10. The plasmid proved stable enough (< 1.0% cured after 50 generations) for industrial batchwise enzyme production in two strains, but in asacU9 strain (thesacU9 mutation increases concominantly the production ofα-amylase levansucrase and proteases) 99.9% of cells had lost pKTH10 after 50 generations, although the parental plasmid (pUB110) was stable in this strain (0.09% cured after 50 generations). The instability of pKTH10 in thesacU9 strain seems somehow to be related to high expression of the clonedα-amylase gene: when grown in a medium restrictingα-amylase production, only 0.53% ofsacU9 cells had lost pKTH10 after 50 generations.

Release of genetically engineered micro-organisms into the environment

Genetically engineered micro-organisms (GEM) at present are the subject of much public attention. They are being considered for biological control, frost protection of plants, and other applications. There is a need to test such organisms before release to the environment. Examples of GEM includePseudomonas fluorescens into which have been cloned δ-toxin genes ofBacillus thuringiensis, ‘ice-minus bacteria’, and other organisms modified by addition, rearrangement and/or deletion of genetic material. Prior to release, the survival, fate, and effects of GEM in the environment must be established. Because organisms, once released, cannot be recalled or always controlled, it is imperative that a full understanding of the risks be known. Predictive ecology must include the new sub-discipline of molecular microbial ecology, if the need for information prior to release of GEM is to be met. One of the most important aspects of deliberate release which must be considered is the ability to detect and monitor GEM in the environment. It has been discovered that micro-organisms can undergo ‘dormancy’, i.e. enter a viable but non-recoverable stage in the natural environment. New techniques have been developed, employing immunofluorescent/epifluorescent microscopy, coupled with 5S rRNA sequencing, which allow accurate non-genetic detection of GEM. These techniques have been employed in aquatic systems. Charateristics of GEM important in release to the environment include ability to colonize surfaces, transfer genetic material and persist in specific environments. Clearly, the effects of GEM on the environment cannot be precisely predicted, unless the organisms have been so debilitated that they cannot persist in any natural habitat and cannot exchange genetic material with any other organism. It must be recognized that micro-organisms are extremely diverse and versatile. Uniformly applied, standard regulations governing deliberate release of GEM to the environment cannot be applied in the same way as for regulation of chemicals or medical devices. Case-by-case regulation appears to be the best approach for the immediate future. The implications of each organism, in terms of its own biology, will have to be considered.

A cloned 23S rRNA gene fragment of Bacillus subtilis and its use as a hybridization probe of conserved character

A subclone of plasmid p14B8 containing the major part of a 23S rRNA gene ofBacillus subtilis was constructed and designated pJK1. Labeled plasmid pJK1 could be used as a DNA probe with conserved gene sequences. DNA-DNA hybridization experiments between filter-bound DNA from various bacteria and labeled pJK1 showed a good correlation between oligonucleotide sequence analysis of 16S rRNA and DNA homology values. Application of suboptimal or stringent hybridization conditions and an additional short incubation under the same conditions following hybridization yielded the best data for differentiating organisms related toB. subtilis from less or non-related bacteria.

High Production of Thermostable β-Galactosidase of Bacillus stearothermophilus in Bacillus subtilis

By cloning the beta-galactosidase gene of Bacillus stearothermophilus IAM11001 (ATCC 8005) into Bacillus subtilis, enzyme production was enhanced 50 times. beta-Galactosidase could be purified to 80% homogeneity by incubating the cell extract of B. subtilis at 70 degrees C for 15 min, followed by centrifugation to remove the denatured proteins. Because of its heat stability and ease of production, beta-galactosidase is suitable for application in industrial processes.