Genome structure and evolution of the Bacillus cereus group.
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Animals↗
Biomedical subjects
D Lereclus
Publications and source records attributed to D Lereclus.
Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence.
PlcR is a pleiotropic regulator of virulence factors in the insect pathogen Bacillus thuringiensis and in the opportunistic human pathogen Bacillus cereus. It activates the transcription of at least 15 genes encoding extracellular proteins, including phospholipases C, proteases and enterotoxins. Expression of the plcR gene is autoregulated and activated at the onset of stationary phase. Here, we used mini-Tn10 transposition to generate a library of B. thuringiensis mutants, with the goal of characterizing genes involved in the expression of the plcR gene. Three mutant strains were identified carrying distinct mini-Tn10 insertions. The mutations impaired plcR expression and caused a deficient haemolytic phenotype, similar to the phenotype of a B. thuringiensis strain in which the plcR gene had been disrupted. The insertion sites of the three mini-Tn10 transposons mapped in a five-gene operon encoding polypeptides homologous to the components of the oligopeptide permease (Opp) system of Bacillus subtilis, and with a similar structural organization. By analogy, the five B. thuringiensis genes were designated oppA, B, C, D and F. In vitro disruption of the B. thuringiensis oppB gene reproduced the effect of the mini-Tn10 insertions (i.e. the loss of haemolytic activity) and reduced the virulence of the strain against insects. These phenotypes are similar to those of a DeltaplcR mutant. Opp is required for the import of small peptides into the cell. Therefore, plcR expression might be activated at the onset of stationary phase by the uptake of a signalling peptide acting as a quorum-sensing effector. The opp mutations impaired the sporulation efficiency of B. thuringiensis when the cells were cultured in LB medium. Thus, Opp is on the pathway that ultimately regulates Spo0A phosphorylation, as is the case in B. subtilis. However, analysis of plcR expression in DeltaoppB, Deltaspo0A and DeltaoppB Deltaspo0A mutants indicates that Opp is required for plcR expression via a Spo0A-independent mechanism.
Animals↗
The incompatibility between the PlcR- and AtxA-controlled regulons may have selected a nonsense mutation in Bacillus anthracis.
Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are members of the Bacillus cereus group. These bacteria express virulence in diverse ways in mammals and insects. The pathogenic properties of B. cereus and B. thuringiensis in mammals results largely from the secretion of non-specific toxins, including haemolysins, the production of which depends upon a pleiotropic activator PlcR. In B. anthracis, PlcR is inactive because of a nonsense mutation in the plcR gene. This suggests that the phenotypic differences between B. anthracis on the one hand and B. thuringiensis and B. cereus on the other could result at least partly from loss of the PlcR regulon. We expressed a functional PlcR in B. anthracis. This resulted in the transcriptional activation of genes weakly expressed in the absence of PlcR. The transcriptional activation correlated with the induction of enzymatic activities and toxins including haemolysins. The toxicity of a B. anthracis PlcR+ strain was assayed in the mouse subcutaneous and nasal models of infection. It was no greater than that of the parental strain, suggesting that the PlcR regulon has no influence on B. anthracis virulence. The PlcR regulon had dramatic effects on the sporulation of a B. anthracis strain containing the virulence plasmid pXO1. This resulted from incompatible interactions with the major AtxA-controlled virulence regulon. We propose that the PlcR-controlled regulon in B. anthracis has been counterselected on account of its disadvantageous effects.
Animals↗
Survival and conjugation of Bacillus thuringiensis in a soil microcosm.
The survival and conjugation ability of sporogenic and asporogenic Bacillus thuringiensis strains were investigated in broth, in non-amended sterile clay soil monoculture and in mixed soil culture. The 75 kb pHT73 plasmid carrying an erythromycin resistance determinant and a cry1Ac gene was transferred in mating broth and soil microcosm. Survival of strains was assessed in soil monoculture and in mixed soil culture for up to 20 days. Sporogenic strains rapidly formed viable spores which were maintained until the end of the experiment. The asporogenic strains were no longer recovered after 8 days of incubation. This study shows that the environmental impact of asporogenic B. thuringiensis strains is lower than that of sporogenic B. thuringiensis strains. Thus, the use of asporogenic strains may significantly reduce any potential risk (gene transfer, soil and plant contamination) due to the dissemination of B. thuringiensis-based biopesticides in the environment.
Journal Article↗
Regulation of toxin and virulence gene transcription in Bacillus thuringiensis.
Bacillus thuringiensis is a spore-forming bacterium well known for its insecticidal properties and its ability to produce a crystal inclusion during sporulation. The specific activity of B. thuringiensis against insect larvae is due to the crystal proteins (Cry proteins). Two different transcriptional mechanisms (dependent and independent of sporulation) are responsible for cry gene transcription during the stationary phase. In addition to these specific insecticidal toxins, B. thuringiensis produces potential virulence factors including haemolysins, degradative enzymes and enterotoxins. A pleiotropic regulator (PlcR) that activates the transcription of various genes encoding such extracellular proteins has been identified. Its expression at the onset of the stationary phase is dependent on the growth medium and is controlled by the transition state regulator, SpoOA.
Bacillus thuringiensis↗
PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis.
Members of the Bacillus cereus group (B. anthracis, B. cereus, B. mycoides and B. thuringiensis) are well-known pathogens of mammals (B. anthracis and B. cereus) and insects (B. thuringiensis). The specific diseases they cause depend on their capacity to produce specific virulence factors, such as the lethal toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, these Bacillus spp. also produce a variety of proteins, such as phospholipases C, which are known to act as virulence factors in various pathogenic bacteria. Few genes encoding these virulence factors have been characterized in pathogenic Bacillus spp. and little is known about the regulation of their expression. We had previously reported that in B. thuringiensis expression of the phosphatidylinositol-specific phospholipase C gene is regulated by the transcriptional activator PlcR. Here we report the identification of several extracellular virulence factor genes by the virtue of their PlcR-regulated expression. These PlcR-regulated genes encode degradative enzymes, cell-surface proteins and enterotoxins. The PlcR-regulated genes are widely dispersed on the chromosome and therefore do not constitute a pathogenic island. Analysis of the promoter region of the PlcR-regulated genes revealed the presence of a highly conserved palindromic region (TATGNAN4TNCATA), which is presumably the specific recognition target for PlcR activation. We found that the plcR gene is also present in and probably restricted to all the members of the B. cereus group. However, although the polypeptide encoded by the B. cereus PlcR gene is functionally equivalent to the B. thuringiensis regulator, the polypeptide encoded by the B. anthracis gene is truncated and not active as a transcriptional activator. PlcR is the first example described of a pleiotropic regulator involved in the control of extracellular virulence factor expression in pathogenic Bacillus spp. These results have implications for the taxonomic relationships among members of the B. cereus group, the virulence properties of these bacteria and the safety of B. thuringiensis-based biopesticides.
Amino Acid Sequence↗
[Bacillus thuringiensis: a biotechnology model].
This paper is on the different biotechnological approaches that have been used to improve Bacillus thuringiensis (Bt) for the control of agricultural insect pests and have contributed to the successful use of this biological control agent; it describes how a better knowledge of the high diversity of Bt strains and toxins genes together with the development of efficient host-vector systems has made it possible to overcome a number of the problems associated with Bt based insect control measures. First we present an overview of the biology of Bt and of the mode of action of its insecticidal toxins. We then describe some of the progress that has been made in furthering our knowledge of the genetics of Bt and of its insecticidal toxin genes and in the understanding of their regulation. The paper then deals with the use of recombinant DNA technology to develop new Bt strains for more effective pest control or to introduce the genes encoding partial-endotoxins directly into plants to produce insect-resistant trangenic plants. Several examples describing how biotechnology has been used to increase the production of insecticidal proteins in Bt or their persistence in the field by protecting them against UV degradation are presented and discussed. Finally, based on our knowledge of the mechanism of transposition of the Bt transposon Tn4430, we describe the construction of a new generation of recombinant strains of Bt, from which antibiotic resistance genes and other non-Bt DNA sequences were selectively eliminated, using a new generation of site-specific recombination vectors. In the future, continuing improvement of first generation products and research into new sources of resistance is essential to ensure the long-term control of insect pests. Chimeric toxins could also be produced so as to increase toxin activity or direct resistance towards a particular type of insect. The search for new insecticidal toxins, in Bt or other microorganisms, may also provide new weapons for the fight against insect damage.
Agriculture↗
Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance.
The main problems with Bacillus thuringiensis products for pest control are their often narrow activity spectrum, high sensitivity to UV degradation, and low cost effectiveness (high potency required). We constructed a sporulation-deficient SigK(-) B. thuringiensis strain that expressed a chimeric cry1C/Ab gene, the product of which had high activity against various lepidopteran pests, including Spodoptera littoralis (Egyptian cotton leaf worm) and Spodoptera exigua (lesser [beet] armyworm), which are not readily controlled by other Cry delta-endotoxins. The SigK(-) host strain carried the cry1Ac gene, the product of which is highly active against the larvae of the major pests Ostrinia nubilalis (European corn borer) and Heliothis virescens (tobacco budworm). This new strain had greater potency and a broader activity spectrum than the parent strain. The crystals produced by the asporogenic strain remained encapsulated within the cells, which protected them from UV degradation. The cry1C/Ab gene was introduced into the B. thuringiensis host via a site-specific recombination vector so that unwanted DNA was eliminated. Therefore, the final construct contained no sequences of non-B. thuringiensis origin. As the recombinant strain is a mutant blocked at late sporulation, it does not produce viable spores and therefore cannot compete with wild-type B. thuringiensis strains in the environment. It is thus a very safe biopesticide. In field trials, this new recombinant strain protected cabbage and broccoli against a pest complex under natural infestation conditions.
Animals↗
Bacillus popilliae cry18Aa operon is transcribed by sigmaE and sigmaK forms of RNA polymerase from a single initiation site.
Bacillus popilliae is an obligate pathogen for larvae of the insect family Scarabaeidae (Coleoptera). It forms parasporal crystals upon sporulation. The gene cry18Aa coding for the parasporal crystal protein and an upstream open reading frame, orf1, were previously isolated from B.popilliae. Here we report an analysis of cry18Aa transcription in Bacillus thuringiensis. The only transcriptional start site of cry18Aa was found 29 bp upstream of the open reading frame orf1, suggesting that orf1 and cry18Aa are transcribed as an operon. lacZ fusion to the cry18Aa promoter was used to follow the time-course of cry18Aa transcription in wild type B.thuringiensis and in various B.thuringiensis sporulation-deficient mutants (spo0A, sigE or sigK). In wild type B.thuringiensis, the cry18Aa promoter was activated 2 h after the end of exponential growth and the expression lasted to the late sporulation phase. The results of promoter activity in Spo+or Spo-backgrounds together with the results of primer extension experiments suggest that the transcription from this promoter can be driven by both sigmaE and sigmaK types of RNA polymerase at a single start site. The promoter region of cry18Aa operon fits the consensus sequences of both sigmaE and sigmaK dependent promoters of Bacillus.
Animals↗
Bacillus thuringiensis and its pesticidal crystal proteins.
During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.
Amino Acid Sequence↗
Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.
The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.
Bacillus thuringiensis↗
A genetic system that reports transient activation of genes in Bacillus.
Site-specific recombination is a powerful tool for precise excision of DNA fragments. We used this characteristic to construct a genetic system to report the transient activation of a promoter by promoting the stable acquisition of an antibiotic resistance marker by the bacterium. The system is composed of two compatible plasmid derivatives from Gram-positive bacteria. One of the plasmids allows the insertion of promoters upstream from tnpI, which encodes the site-specific recombinase of Tn4430. The second plasmid carries two selectable resistance genes: one is flanked by two site-specific recombination sequences and is lost following recombination; in contrast, the other resistance gene becomes functional after the site-specific recombination event. By inserting conditionally controlled promoters (the xylose-inducible xylA promoter or the plcA promoter whose expression is dependent on the growth medium) upstream of tnpI, we demonstrated that our genetic system responds to signals inducing transcription by conferring a new resistance phenotype to the host bacteria. Thus, this system can be used to identify genes which are transiently or conditionally expressed.
Artificial Gene Fusion↗
A recombinase-mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains.
A TnpI-mediated site-specific recombination system to construct genetically modified Bacillus thuringiensis strains was developed. Recombinant B. thuringiensis strains from which antibiotic resistance genes can be selectively eliminated were obtained in vivo with a new vector based on the specific resolution site of transposon Tn4430. For example, a cryIC gene, whose product is active against Spodoptera littoralis, was introduced into B. thuringiensis Kto harboring a cryIA(c) gene active against Ostrinia nubilalis. The resulting strain had a broader activity spectrum than that of the parental strain. It contained only B. thuringiensis DNA and was free of antibiotic resistance genes. This should facilitate regulatory approval for its development as a commercial biopesticide.
Animals↗
Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector.
Bacillus thuringiensis (Bt) delta-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains. These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed using transformation procedures. However, addition of a new delta-endotoxin gene to strains already harboring other delta-endotoxin genes often resulted in broader-spectrum but less potent products because they produced significantly less of each of the crystal proteins. We report expression of the coding sequence of the sporulation specific cryIC gene from the non-sporulation-dependent cryIIIA promoter. Large amounts of CryIC accumulated in various Bt strains with different genetic backgrounds. Sporulation deficient Spo0A mutants, acrystalliferous derivatives and wild-type Bt strains expressing the engineered cryIII-cryIC gene were obtained. Introduction of the cryIII-cryIC gene whose product is highly active against Spodoptera littoralis into the Kto strain harboring the cryIA(c) gene active against Ostrinia nubilalis resulted in the construction of a new strain with increased potency and broader activity spectrum than the parent strain. Large amounts of each toxin were produced and the expression of the two genes seemed to be summed, presumably because the expression systems of the two genes are different. The plasmid shuttle vector used to introduce the cryIII-cryIC gene into the different Bt hosts utilizes the specific resolution site of transposon Tn4430 to enable construction of recombinant Bt strains that are free of foreign non-Bt DNA. This should facilitate the approval and acceptance for environmental release of the insecticidal recombinant products.
Animals↗
Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis.
The sigE and sigK genes, encoding the sporulation-specific sigma factors sigma 35 and sigma 28 of Bacillus thuringiensis, were each disrupted by inserting a gene conferring resistance to kanamycin into their coding sequences. The B. thuringiensis SigE- and sigK- mutant strains were blocked at different sporulation stages and were unable to sporulate. The SigE-strain was blocked at stage II of sporulation, whereas the SigK- strain was blocked at stage IV. The expression of a cryIAa'-'lacZ transcriptional fusion was analysed in these genetic backgrounds and it was found that both sigma factors are involved in the in vivo transcription of this gene. However, the SigK- strain harbouring the cryIAa gene produced amounts of toxin similar to those produced by the B. thuringiensis Spo+ strain. The toxins accumulated in the mother cell compartment to form a crystal inclusion which remained encapsulated within the cell wall. Thus, transcription from the sigma E-dependent promoter alone (Bt I promoter) is sufficient to support high levels of toxin production in B. thuringiensis.
Bacillus thuringiensis↗
STAB-SD: a Shine-Dalgarno sequence in the 5' untranslated region is a determinant of mRNA stability.
Transcription of the Bacillus thuringiensis cryIIIA toxin gene is initiated at nucleotide position -558 (relative to the cryIIIA start codon). However, the major cryIIIA transcript is an mRNA with its 5' end at nucleotide position -129. Fusions to the lacZ reporter gene revealed that the cryIIIA 5' untranslated region downstream of nucleotide position -129 acts as a 5' mRNA stabilizer. Deletion and mutation analysis suggest that the determinant of stability is a Shine-Dalgarno (SD) sequence close to the 5' end of the stable transcript. This SD sequence, designated STAB-SD, does not direct translation initiation. However, mutations predicted to weaken the putative interaction between the SD sequence and the 3' end of 16S rRNA lead to reduced mRNA stability. The binding of a 30S subunit to STAB-SD may be required for stabilization of cryIIIA mRNA as a transcript with a 5' end at nucleotide position -129. Similar STAB-SD sequences are found in the 5' untranslated regions of other cryIII genes, and in the 5' untranslated regions of genes from Gram-positive bacteria other than B. thuringiensis and may therefore be a widespread determinant of mRNA stability.
Bacillus thuringiensis↗
Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis.
The Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes. Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes. Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background. We used lacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo- genetic backgrounds (spoOA, sigE and sigK). cryIIIA was activated from the end of vegetative growth to stage II of sporulation (t3) in the wild-type strain. Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III. In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase. cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigma E mutant strain. Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors. Furthermore, an event linked with the thE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III.
Bacillus thuringiensis↗
Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase.
A transcriptional analysis of the phosphatidylinositol-specific phospholipase C (plcA) gene of Bacillus thuringiensis indicated that its transcription was activated at the onset of the stationary phase in B. thuringiensis but was not activated in B. subtilis. The B. thuringiensis gene encoding a transcriptional activator required for plcA expression was cloned by using a B. subtilis strain carrying a chromosomal plcA'-'lacZ fusion as a heterologous host for selection. This trans activator (designated PlcR) is a protein of a calculated molecular weight of 33,762 which appears to be distantly related to PreL and NprA, regulator proteins enhancing transcription of neutral protease genes during the stationary phase of a Lactobacillus sp. and B. stearothermophilus, respectively. plcR gene transcription was analyzed in B. thuringiensis and in B. subtilis. PlcR positively regulated its own transcription at the onset of the stationary phase. There is a highly conserved DNA sequence (17 bp) 34 nucleotides upstream from the plcR transcriptional start site and 49 nucleotides upstream from the plcA transcriptional start site. As PlcR positively regulates its own transcription and plcA transcription, this conserved DNA sequence may be the specific recognition target for PlcR activation.
Amino Acid Sequence↗