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

Abstract

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...

Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

“…The PCR fragments were digested with appropriate restriction enzymes (underlined) and purified as a 0n9 kb HindIII-EcoRI fragment and as a 1 kb EcoRI-BamH1 fragment, respectively. The promoterless lacZ gene was isolated as a 3n2 kb EcoRI DNA fragment from pHT304BhZ (Lereclus et al, 1996). The three DNA fragments were ligated together between the HindIII and BamHI sites of pRN5101.…”

“…PlcR is a pleiotropic regulator of virulence gene expression (Agaisse et al, 1999 ;Lereclus et al, 1996). A 3n8 kb DNA region surrounding the Tn10 insertion site was obtained as two fragments and sequenced, as described in Methods.…”

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 .

“…The EMBL accession numbers for the sequences reported in this paper are given in Table 1. factors in entomopathogenic Bacillus thuringiensis, including the HBL and NHE enterotoxins, phosphatidylcholine-preferring phospholipase C (PC-PLC ; plc) and phosphatidylinositol-specific phospholipase C (PI-PLC ; plcA), are under transcriptional control of a pleiotropic regulator PlcR (Lereclus et al, 1996 ;Agaisse et al, 1999). The PlcR protein has also been shown to regulate its own transcription and may perform its action by binding to a conserved palindromic DNA sequence upstream of the transcription initiation site of the genes subject to its control.…”

Rgg protein structure–function and inhibition by cyclic peptide compounds

“…1A). Gram-positive cytoplasmic pheromone receptors include Bacillus response regulator aspartate phosphatases (Rap), neutral protease regulator (NprR), and phosphatidylinositol-specific phospholipase C gene regulator (PlcR), Enterococcus pheromone-responsive transcription factor (PrgX), and Streptococcus regulator gene of glucosyltransferase (Rgg) (as well as the homologous MutR and GadR) (4)(5)(6)(7)(8)(9)(10)(11). The Rap proteins are phosphatases and transcriptional antiactivators, whereas NprR, PlcR, and PrgX are DNA-binding transcription factors.…”