Molecular characterization and sequence of phosphatidylinositol‐specific phospholipase C of <i>Bacillus thuringiensis</i>

Lechner, Max; Kupke, Thomas; S, Stefanovic; Götz, Friedrich

doi:10.1111/j.1365-2958.1989.tb00209.x

Cited by 44 publications

(28 citation statements)

References 29 publications

(14 reference statements)

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“…These results suggest that pathogenesis in mice and insects requires common PlcR controls pathogenicity extracellular factors that depend on the plcR regulon. Thus, PlcR-regulated genes, such as those encoding the enterotoxins Hbl (Beecher et al, 1995b) and Nhe (Granum et al, 1999 ;, the phospholipase PI-PLC (Kuppe et al, 1989 ;Lechner et al, 1989) and the cereolysin AB component (Gilmore et al, 1989), may be responsible for pathogenicity. These four genes were found in the B. thuringiensis 407 and B. cereus ATCC 14579 strains used in this study (Agaisse et al, 1999 ;.…”

Section: Discussionmentioning

confidence: 99%

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects

Ramisse²,

et al. 2000

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Bacillus thuringiensis has been widely used for 40 years as a safe biopesticide for controlling agricultural pests and mosquitoes because it produces insecticidal crystal proteins. However, spores have also been shown to contribute to overall entomopathogenicity. Here, the opportunistic properties of acrystalliferous B. thuringiensis Cry N and Bacillus cereus strains were investigated in an insect species, Galleria mellonella, and in a mammal, BALB/c mice. In both animal models, the pathogenicity of the two bacterial species was similar. Mutant strains were constructed in which the plcR gene, encoding a pleiotropic regulator of extracellular factors, was disrupted. In larvae, coingestion of 10 6 spores of the parental strain with a sublethal concentration of Cry1C toxin caused 70 % mortality whereas only 7 % mortality was recorded if spores of the ∆plcR mutant strain were used. In mice, nasal instillation of 10 8 spores of the parental strain caused 100 % mortality whereas instillation with the same number of ∆plcR strain spores caused much lower or no mortality. Similar effects were obtained if vegetative cells were used instead of spores.The cause of death is unknown and is unlikely to be due to actual growth of the bacteria in mice. The lesions caused by B. thuringiensis supernatant in infected mice suggested that haemolytic toxins were involved. The cytolytic properties of strains of B. thuringiensis and B. cereus, using sheep, horse and human erythrocytes and G. mellonella haemocytes, were therefore investigated. The level of cytolytic activity is highly reduced in ∆plcR strains.Together, the results indicate that the pathogenicity of B. thuringiensis strain 407 and B. cereus strain ATCC 14579 is controlled by PlcR.

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Section: Discussionmentioning

confidence: 99%

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects

Ramisse²,

et al. 2000

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“…The DNA regions upstream of plcR and plcA and the 5h ends of these genes were cloned and sequenced directly from chromosomal DNA by inverse PCR using appropriate primers. Flanking sequences from known DNA fragments covering the 3h ends and downstream regions of plcR (this paper) and the PI-PLC-encoding plcA gene (Henner et al, 1988 ;Kuppe et al, 1989 ;Lechner et al, 1989 ;Lo$ vgren et al, 1998) were scanned for suitable restriction cleavage sites, and chromosomal DNA (5 µg) from B. cereus ATCC 14579 was completely digested with the chosen restriction enzyme(s). The resulting DNA fragments were extracted with phenol and religated in a volume of 200 µl by incubation with 1 U T4 DNA ligase (Roche Diagnostics) for 12 h at 14 mC to preferentially obtain circular molecules.…”

Section: Methodsmentioning

confidence: 99%

Sequence analysis of three Bacillus cereus loci carrying PlcR-regulated genes encoding degradative enzymes and enterotoxin The EMBL accession numbers for the sequences reported in this paper are given in Table 1 T1 .

et al. 1999

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PlcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase. Among the genes regulated by PlcR are : plcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC) ; plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC) ; nhe, encoding the non-haemolytic enterotoxin ; hbl, encoding haemolytic enterotoxin BL (HBL) ; and genes specifying a putative S-layer like surface protein and a putative extracellular RNase. By analysing 371 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted. Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified. The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes. Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR. Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.

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“…Genomic DNA of B. thuringiensis 407 was used as a template for amplification by PCR of the 220-bp fragment upstream from the plcA gene. The primers (designated PLC1 and PLC2) were deduced from the nucleotide sequence previously published (22). Primer PLC1 (5Ј-CCCCAAGCTTAGATCTATAAATATGAGAATAAAGAT G-3Ј) creates a HindIII site and a BglII site upstream from the promoter region and is complementary to sequences beginning 229 nucleotides 5Ј to the plcA start codon.…”

Section: Bacterialmentioning

confidence: 99%

“…The plcA genes encoding PI-PLCs from Bacillus cereus, B. thuringiensis, Staphylococcus aureus, and Listeria monocytogenes have been cloned and sequenced (6,15,21,22,32), and the deduced amino acid sequences show extensive similarity (about 50%). The PI-PLC of L. monocytogenes contributes to the growth of bacteria in infected cells and is therefore considered to be a virulence factor (45).…”

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confidence: 99%

Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase

et al. 1996

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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.Phospholipases C that specifically cleave phosphatidylinositol (PI-PLCs) have been isolated from several gram-positive bacteria (for a review, see reference 48). The plcA genes encoding PI-PLCs from Bacillus cereus, B. thuringiensis, Staphylococcus aureus, and Listeria monocytogenes have been cloned and sequenced (6,15,21,22,32), and the deduced amino acid sequences show extensive similarity (about 50%). The PI-PLC of L. monocytogenes contributes to the growth of bacteria in infected cells and is therefore considered to be a virulence factor (45). Expression of plcA in L. monocytogenes is positively regulated during the growth phase by the pleiotropic transcriptional activator PrfA (33). The S. aureus PI-PLC is also a potential virulence factor. Its production is positively regulated by Agr, a pleiotropic regulator which activates expression of several staphylococcal exoproteins at the end of the exponential growth (6, 41).B. thuringiensis is known for its entomopathogenic properties which are partly due to the production of a variety of larvicidal crystal proteins designated Cry (for reviews, see references 17 and 26). When ingested by susceptible insect larvae, these crystal proteins are dissolved in the insect gut and activated. They bind to specific receptors on the surface of the midgut epithelial cells, forming transmembrane pores and causing cell lysis. This toxic effect either kills the susceptible insects or weakens them, creating favorable conditions for the germination of spores in the gut environment. Thereafter, the bacteria can invade the hemocoel from the gut and cause septicemia. The contribution of the B. thuringiensis spores to overall virulence has been demonstrated: insecticidal activity against some lepidopteran species requ...

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Molecular characterization and sequence of phosphatidylinositol‐specific phospholipase C of Bacillus thuringiensis

Cited by 44 publications

References 29 publications

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects

Sequence analysis of three Bacillus cereus loci carrying PlcR-regulated genes encoding degradative enzymes and enterotoxin The EMBL accession numbers for the sequences reported in this paper are given in Table 1 T1 .

Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase

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