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PubMed2000

Coexpression of cyt1Aa of Bacillus thuringiensis subsp. israelensis with Bacillus sphaericus binary toxin gene in acrystalliferous strain of B. thuringiensis.

The cyt1Aa gene of Bacillus thuringiensis subsp. israelensis and binary toxin gene of Bacillus sphaericus C3-41 were introduced into an acrystalliferous strain of B. thuringiensis independently and in combination by using shuttle vector pBU4. SDS-PAGE and Western blot analysis proved that cyt1Aa and binary toxin genes coexpressed during the sporulation of the recombinant. Transformant strain expressing the Cyt1Aa and binary toxin proteins in combination was more toxic to susceptible and resistant Culex pipiens quinquefasciatus than the transformants expressing Cyt1Aa protein or binary toxin proteins independently. It was suggested that large amount of production of Cyt1Aa protein and binary toxin proteins possibly interacted synergistically, thereby increasing its mosquitocidal toxicity significantly.

Animals↗

PubMed2002

Isolation and characterization of Bacillus cereus-like bacteria from faecal samples from greenhouse workers who are using Bacillus thuringiensis-based insecticides.

OBJECTIVES: Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B. thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD). METHODS: Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis. RESULTS: Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product. CONCLUSIONS: The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces. The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.

Bacillus cereus↗

PubMed1999

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter.

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na+ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na+/H+ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs.

ATP-Binding Cassette Transporters↗

PubMed1992

Unsaturated and branched chain-fatty acids in temperature adaptation of Bacillus subtilis and Bacillus megaterium.

The effect of growth temperature on the cellular fatty acid profiles of Bacillus subtilis and Bacillus megaterium was studied over a temperature range from 40 to 10 degrees C. As the growth temperature of B. subtilis was reduced, the lower-melting point anteiso-acids increased, while the higher-melting point iso-acids decreased. Consequently the ratio of branched- to straight-chain acids was unaffected by temperature, although changes in the position of fatty acid branching and the degree of unsaturated branched-chain fatty acids occurred. In B. megaterium a more complicated, biphasic behaviour was observed. Saturated, straight-chain and iso-branched acids decreased only from 40 degrees C down to 20-26 degrees C, and anteiso-acids decreased only from 20-26 degrees C to 10 degrees C, while unsaturated acids increased over the whole temperature range studied. Thus, in B. megaterium total branched-chain acids decreased and straight-chain acids increased as temperature decreased. However, the overall cellular content of lower-melting point fatty acids increased with decreasing temperature in both bacilli, and unsaturated fatty acids appeared to be essential components in the adaptation of the microbes to changes in temperatures. Since changes in the relative amounts of branched- and straight-chain fatty acid biosynthesis are known to reflect differences in fatty acid primers, temperature seems to affect not only the activity of the fatty acid desaturases but also the formation or availability of these primers. The results indicate, however, that notable species-specific regulatory features exist in this genus of bacteria.

Adaptation, Physiological↗

PubMed1990

Bacillus amyloliquefaciens alpha-amylase signal sequence fused in frame with human proinsulin is properly processed by Bacillus subtilis cells.

The plasmid pBINS1, containing the promoter, SD and leader peptide sequences of Bacillus amyloliquefaciens alpha-amylase gene and 267 bp long sequence coding for human proinsulin directs the efficient synthesis of hybrid preproinsulin, as well as quantitative secretion of proinsulin outside of protease-deficient Bacillus subtilis AJ73 cells. The recombinant proinsulin has been isolated from the culture medium and its N-terminal sequence shown to be identical with that of natural human prohormone.

Amino Acid Sequence↗

PubMed1982

Evaluation of a microfluorometer in immunofluorescence assays of individual spores of Bacillus anthracis and Bacillus cereus.

A microfluorometer was constructed by modifying a standard fluorescent microscope with a fibre optic eyepiece and a simple photometric system. It was evaluated in direct immunofluorescence assays of Bacillus anthracis and Bacillus cereus spores immobilised on multispot microscope slides. From measurements of stable fluorescent crystals comparable in size to the spores, it was inferred that the fluorescence intensity of a stained bacterium could be measured with good precision. Fluctuation of a exciting light from a mercury vapour lamp did not contribute significantly to the distribution of fluorescence measurements obtained when samples of 20 spores were assessed. Attempts to correlate spore size with fluorescence intensity suggest that spore fluorescence does not increase in a 1 : 1 ratio with surface area; it is therefore possible that the density of antigenic sites on the surface decreases with increasing spore size. It is concluded that differences in the observed fluorescence of individual spores truly reflect differences in fluorescent antibody binding, but the relative contribution of antigenic variability and of artefacts of the staining procedure remain unknown.

Animals↗

PubMed1990

Comparison of development of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus in mosquito larvae.

Immunofluorescent staining was used with thin sections of paraffin-embedded specimens to detect the development of Bacillus thuringiensis var. israelensis and Bacillus sphaericus in the gut of mosquito larvae. The third- and fourth-instar larvae of Aedes aegypti, Anopheles maculatus, and Culex quinquefasciatus were fed either vegetative cells or spores of the bacteria. Spore germination, multiplication, and sporulation were studied in the larvae of each species. The spores of B. thuringiensis var. israelensis and B. sphaericus strain 2297 could germinate and cells could sporulate in the larval body. The vegetative cells of B. sphaericus strain 810428 were also able to produce spores in the mosquito larval gut, but the germination of spores could not be detected in the larvae. Multiplication of all bacterial species was observed after the larvae died. Growth of the bacteria in distilled water containing crude extracts of larvae made from each species was compared with that in synthetic medium (nutrient broth). They could produce spores and toxins in all the media used and the toxins had larvicidal activity against the target mosquitos Ae. aegypti, An. maculatus, and C. quinquefasciatus.

Animals↗

PubMed1991

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis.

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.

Bacillus subtilis↗

PubMed1992

spoVG sequence of Bacillus megaterium and Bacillus subtilis.

We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis. The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6% amino acid identity. Both genes have putative rho-independent terminators. No significant amino acid or nucleotide homology of either gene was found when compared with sequences contained in either the Genbank or EMBL data bases.

Amino Acid Sequence↗

PubMed1991

Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis.

Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(alpha)-phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine.

Bacillus megaterium↗

PubMed1992

Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing.

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated.

Bacillus anthracis↗

PubMed1983

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase.

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.

Bacillus↗

PubMed1984

In vivo transcription initiation and termination sites of an alpha-amylase gene from Bacillus amyloliquefaciens cloned in Bacillus subtilis.

The alpha-amylase gene, originally isolated by molecular cloning from chromosomal DNA of Bacillus amyloliquefaciens, is efficiently expressed from its own promoter in a Bacillus subtilis host when present in the multicopy plasmid vector pUB110. The flanking regions of this gene were sequenced and the ends of the in vivo-generated messenger RNA were mapped by the S1 procedure. Outside the coding sequence, the mRNA for alpha-amylase contains about 30 nucleotides at the 5' end and 51 nucleotides at the 3' end. The promoter region has -10 sequence TAAAAT starting eleven nucleotides upstream from the transcription start point, pppU, and the -35 hexanucleotide TTGTTA is separated from it by 16 nucleotides. As indicated by its sequence, the terminator is bidirectional and of the rho-independent kind, and the mRNA can form a long hairpin structure at the very 3' end. The 3' terminus of the transcript does not seem to include a U stretch, although the DNA template codes for U3AU6 at the 3' end of the hairpin sequence. The bulk of the amylase mRNA does not contain any 3'-terminal poly(A).

Bacillus↗

PubMed1986

The beta-glucanase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis.

The nucleotide sequence of a 1583-bp DNA fragment containing gene bg1A for endo-beta-1,3-1,4-glucanase (EC 3.2.1.73) of Bacillus amyloliquefaciens strain BE20/78, a high producer of secreted enzymes, has been determined. The gene bg1A comprises an open reading frame (ORF) of 717 bp (= 239 codons) starting with ATG at 469 up to the translation stop codon TAA at 1188. Upstream from the translation initiation codon ATG, the ribosome-binding sequence 5'-AAAAAAGGGGG-3' and two putative bglA promoters have been identified. A box of eleven AT out of twelve base pairs (bp) precedes the -35 region of promoter P1. Beyond the translation stop codon UAA, a sequence of 69 bp can be folded into a hook-like stem-loop structure which probably functions as a transcriptional terminator. The ORF region of the gene bglA reveals about 90% homology with another beta-glucanase gene, bglS of Bacillus subtilis C120 sequenced by Murphy et al. (1984). Three regions of frequent amino acid (aa) changes are indicated. However, the major difference between these is a set of deletions within the non-coding region separating the bglA gene from an unknown preceding ORF and by one deletion shortening the proposed signal peptide by three aa (Pro-Tyr-Leu-). The putative transcription terminator of gene bglA completely lacks homology with a B. subtilis bglS gene. The signification of deletions erasing the 'sacR-homology region' in B. amyloliquefaciens, which have been detected in proximity of the beta-glucanase gene of B. subtilis by Steinmetz and Aymerich (1986), is discussed.

Bacillus↗

PubMed1995

Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis.

The cloning and sequence of the 5' untranslated region (5'-UTR) of the Bacillus licheniformis (Bl) 6816 subtilisin Carlsberg gene (subC) are reported here. The 5' and 3' ends of subC transcripts were characterized, and the promoter identified. Expression was studied using a fused lacZ reporter gene integrated into the chromosome of heterologous host Bacillus subtilis (Bs). beta Gal activities of mutants deleted within the promoter region identified a region which is required for stimulation by the transcriptional activator proteins, DegU and DegQ. This region is close to the transcription start point (tsp), and is adjacent to a sequence homologous to that involved in DegU/Q stimulation of the Bs subtilisin gene, aprE. Expression of subC in Bs was optimized by the use of heterologous promoter and by the deletion of UTR sequences predicted to be involved in secondary structures in the native subC mRNA. Sequence comparison with other subtilisin Carlsberg-type-encoding genes revealed a high degree of conservation of the entire 5'-UTR, including regulatory sequences and promoter, as well as part of the structural gene.

Amino Acid Sequence↗

PubMed1995

Cloning and characterization of a Bacillus thuringiensis homolog of the spoIIID gene from Bacillus subtilis.

The SpoIIID protein of Bacillus subtilis (Bs) is a small DNA-binding protein that is essential for gene expression of the mother cell compartment during sporulation. We have cloned a DNA fragment from Bacillus thuringiensis (Bt) that showed a specific hybridization with the Bs spoIIID gene. Sequence analysis found an open reading frame encoding 90 amino acids (aa), which are 89% identical to the deduced aa sequence of Bs spoIIID. Upstream from the transcription start point (tsp), a nucleotide sequence highly homologous to the consensus sequence motif for the sigma 35-recognized promoters was found. Northern blot analysis has indicated that the expression of the gene is induced only at the midsporulation stage, and that the gene constitutes an operon with a downstream gene, mreB. The Bs strain carrying the spoIIID delta erm or spoIIID83 mutation completely restored sporulation ability upon introduction of the spoIIID homologous gene from Bt. These results strongly suggest that the gene we have cloned is a Bt homolog of spoIIID.

Amino Acid Sequence↗

PubMed1994

Sequences of ccpA and two downstream Bacillus megaterium genes with homology to the motAB operon from Bacillus subtilis.

A regulatory gene with 69% nucleotide sequence identity to the Bacillus subtilis ccpA was cloned from Bacillus megaterium by complementation of a mutant relieved of catabolite repression. Sequencing of the gene and its adjacent regions revealed two additional open reading frames (ORFs) downstream from ccpA. These three genes are presumably in one operon. ORF1 and ORF2 show homology to two genes downstream from ccpA in B. subtilis, as well as to the B. subtilis motA and motB genes, respectively.

Amino Acid Sequence↗

PubMed1996

Nucleotide sequence of the Bacillus coagulans homologue of the spoIIA operon of Bacillus subtilis.

The spoIIA locus of Bacillus coagulans (Bc) was cloned into pTZ18R and the nucleotide sequence was determined. To clone the operon, one PCR primer corresponding to the C-terminal region of SpoIIAB, and a second corresponding to a region near the middle of SpoIIAC, were designed on the basis of the three previously published Bacillus spoIIA sequences. The Bc spoIIA sequence contains three open reading frames coding for putative proteins of 116, 147 and 252 aa.

Amino Acid Sequence↗