Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella.

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry(-) with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.

The dlt operon of Bacillus cereus is required for resistance to cationic antimicrobial peptides and for virulence in insects.

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Delta dlt(Bc) mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Delta dlt(Bc) was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Delta dlt(Bc)). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides.

The transcriptional regulator PlcR and its cognate cell-cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR-PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli.

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.

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.

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.

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.

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.

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.

[Bacillus thuringiensis: a biotechnology model]. / Bacillus thuringiensis: un modèle biotechnologique.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.