Overexpression of Bacillus thuringiensis HknA, a histidine protein kinase homology, bypasses early Spo mutations that result in CryIIIA overproduction

Abstract

The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed protein that is toxic to coleopteran insect larvae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the asporogenous B. thuringiensis subsp. morrisoni strain EG1351, which harbors the native cryIIIA-encoding 88-MDa plasmid, showed a 2.5-fold overproduction of the CryIIIA protein compared with that of an isogenic wild-type strain. Further studies showed that neither CryIIIA protein synthesis nor CryIIIA protein processing was affected in strain EG1351 during vegetative growth. In an attempt to characterize the EG1351 mutation by complementation of function, the hknA gene was identified and cloned from a B. thuringiensis cosmid library. Primer extension analysis of hknA mRNA in wild-type B. thuringiensis demonstrated that the hknA gene is transcribed during vegetative growth from a sigma A-like promoter. Multiple copies of either the hknA gene or the Bacillus subtilis kinA (spoIIJ) gene were shown to bypass the sporulation defect in strain EG1351 as well as a spo0F mutation in B. thuringiensis EG1634. Additional studies showed that the hknA gene was not defective in strain EG1351. The results of this study suggest that hknA encodes a novel histidine protein kinase involved in B. thuringiensis sporulation. We also propose that the CryIIIA-overproducing phenotype of strain EG1351 is most likely due to a defect in the phosphorylation of Spo0A and confirm that CryIIIA production is not dependent on sporulation.

Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis

“…Class 4 HK 39 clustered closest to HKs of non-spore-forming bacteria, such as AtoS of Escherichia coli. However, overexpression of a HK 39 orthologue in B. thuringiensis EG1351 has been shown to bypass sporulation defects and a spo0F mutation in different B. thuringiensis strains (Malvar et al, 1994). In addition, it has recently been shown that HKs 39, 40, 48 (KinD orthologue), 49 (KinB orthologue) and 50 are capable of inducing sporulation in B. anthracis (Brunsing et al, 2005).…”

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

“…Another E E -driven operon, the glycogen operon, which is repressed by glucose in sporulating cells of B. subtilis, also supports the need for metabolic control of the stationary-phase phenomenon (19). In the light of the remarkable conservation of sigma subunits between B. thuringiensis and B. subtilis, the temporal regulation of sporulation in B. thuringiensis through the cascade of sigma factors is believed to be closely similar to that of B. subtilis (1,21,23,24). Homologs of several important transcription factors described in B. subtilis have been identified in B. thuringiensis also (1).…”

Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis

“…Our previous studies have shown that CryIIIA protein production is increased 2.5-fold in the asporogenous B. thuringiensis strain EG1351 (9). We have suggested that this is a consequence of the sporulation-independent transcription of the cryIIL4 gene (4,12,13), the terminal stationary phase arising from an early Spo-defect in EG1351, and the failure to induce sporulation-specific proteases (9). Additionally, our observations that either multicopy hknA or kinA can bypass the sporulation defect in EG1351 and the spoOF defect in EG1634 have led us to propose that the EG1351 defect occurs in the SpoOA phosphorylation cascade (9).…”

“…The transposon vector pEG922 (3) containing the recently described class II B. thuringiensis transposon TnS401 (3) was used to construct a transposoninsertion library in B. thuringiensis EG10368 essentially as described by Youngman (16). Subsequent screening of the library on nutrient sporulation medium (NSM) plates identified 25 colonies with a translucent appearance, characteristic of sporulation mutants (9), with sporulation frequencies of less that 104 spores per ml. To identify the B. thuringiensis DNA at each transposition site, chromosomal DNAs prepared from each of the Spo-isolates (7) and oligonucleotide primer TM1 (Integrated DNA Technologies Inc. [ Fig.…”

“…Our previous findings (9) extension analysis (9) was used to monitor spoOF mRNA levels in EG1668 (hknA) and the isogenic wild-type strain EG10368 during the vegetative and early stationary phases of growth (Fig. 2B).…”