Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp. israelensis

Abstract

HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria. In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue. To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp. israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine. HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally. The enzyme I and HPr kinase activities of the mutant were not affected. Analysis of the B. thuringiensis subsp. israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose. The mutant strain produced both cry4A and 35 gene transcripts 4 h ahead of the parent strain, but there was no effect on 28 synthesis. In wild-type B. thuringiensis subsp. israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period. The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain. There was a 60 to 70% reduction in the sporulation efficiency of the mutant B. thuringiensis subsp. israelensis strain compared to the wild-type strain.Biological control of dipteran pests in general and mosquitoes in particular has been a subject of primary importance for many years. Bacillus thuringiensis subsp. israelensis has been found to be the most effective microbial strain to date, possessing all the desirable properties of an ideal biocontrol agent. B. thuringiensis subsp. israelensis produces larvicidal crystal proteins in the stationary phase, concomitant with sporulation. The larvicidal activity of the parasporal crystals is attributed to the ␦-endotoxin, composed of at least four major polypeptide species of about 27, 72, 128, and 135 kDa, which act synergistically in the manifestation of toxicity (27,32). The high levels of toxin accumulation are controlled by a variety of mechanisms at the transcriptional, posttranscriptional, and posttranslational levels (2). The genes encoding different protein toxins are normally associated with large plasmids in B. thuringiensis subsp. israelensis (20,26) and are named cry4A, cry4B, cry11A, and cytA. cry4A and cry4B code for the 135-kDa and 125-kDa protoxins, respectively, which are activated by the gut proteases in the alkaline conditions of the insect gut.In sporulating cells of B. thuringiensis, the accumulation of large amounts of toxin proteins is achieved by expression from strong promoters associated with the gene. As in Bacillus subtilis, the developmental process is temporally and spatially regulated at the transcriptional level in B. thuringiensis by successive activati...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…The repressive effect of glucose on Cry4A production disappeared in a ptsH1 mutant (Ser45Ala replacement in B. thuringiensis HPr) (399). In L. casei, the ptsH1 mutation causes a relief from CCR of 6-P-␤-galactosidase and N-acetyl glucosaminidase and from diauxic growth in media containing glucose and either lactose, maltose, or ribose (919).…”

Identification of a Catabolite-Responsive Element Necessary for Regulation of the cry4A Gene of Bacillus thuringiensis subsp. israelensis

“…israelensis (5) and its derepression in an HPr-serine phosphorylation (Hpr-Ser-P)-defective mutant (16) suggested catabolite repression of the ␦-endotoxin gene. To get an insight into the mechanism of cry4A repression, here we analyzed the cry4A gene and its upstream regulatory sequence for potential regulatory elements.…”

“…israelensis (5) demonstrated that glucose and its metabolite fructose 6-phosphate repressed cry4A gene expression through catabolite repression (CR) (16), a global, negative regulatory mechanism of gene transcription in gram-positive bacteria (11). CR establishes priorities in the utilization of carbon and energy sources according to their selective advantage (11) for competitive success of the bacteria in the natural environment.…”

“…Since subunits play a key role in temporal control of Cry4A synthesis as well as sporulation in Bacillus (16,25,34), maintenance of adequate 35 and 28 levels is crucial for the cell. A high transcription rate of cry4A is likely to titrate out the cellular subunit pool and may lead to disruption of the developmental process.…”

Overexpression of PrfA Leads to Growth Inhibition ofListeria monocytogenesin Glucose-Containing Culture Media by Interfering with Glucose Uptake

“…CCR controls, in addition to many genes involved in carbon and nitrogen metabolism (for recent reviews see references 53 and 54), the expression of virulence genes in several pathogenic bacteria (22,32,51) and developmental genes involved in the initiation of sporulation in Bacillus subtilis (16). CCR control in gram-positive bacteria having low GϩC contents de-pends on the regulator protein CcpA (catabolite control protein A).…”