No abstract

Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC50 5.8±0.6ngml(-1)) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC50 44.9±7ngml(-1)). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.

Polyhydroxyalkanoate (PHA) is stored as an important carbon and energy source in bacterial cells. For biomedical applications, gram-positive bacteria can be better sources of PHAs, since they lack outer membrane lipopolysaccharide. Although gram-positive Streptomyces coelicolor A3(2) has been indicated as a high potential PHA producer, pha C gene that encodes the key enzyme PHA synthase in the metabolic pathway is not determined in its genome. BLAST search results of the GenBank database argued that SCO7613 could specify a putative polyhydroxyalkanoate synthase (PhaC) responsible for PHA biosynthesis. Deduced amino acid sequence of SCO7613 showed the presence of conserved lipase box like sequence, 555 GASAG 559 , in which serine residue was present as the active nucleophile. Present study describes deletion of putative S. coelicolor pha C gene via PCR dependent method. We showed that SCO7613 is not an essential gene in S. coelicolor and its deletion affected PHA accumulation negatively although it is not ceased. Transcomplementation abolished the mutant phenotype, demonstrating that the decrease in PHA resulted from the deletion of SCO7613.

Inorganic phosphate is stored as a polyphosphate (polyP) polymer inside every living cell. This polymer is synthesized by the polyP kinase (PPK) enzyme using the terminal phosphate of ATP as substrate and it performs important functions in the cell. In this study, effects of high levels of PPK on Bacillus thuringiensis subsp. israelensis were analyzed. Recombinant Bti ppk, a PPK overproducer, was found to uptake more phosphate into the cell and produce a consistently higher amount of endotoxin than the wild type under culture conditions including a range of temperatures (25 °C, 30 °C, and 37 °C), pH values (pH 5, 6, 8, and 9), and carbon sources (maltose, mannitol, sucrose, and starch). Moreover, this strain was found to overexpress sigB, which might cause a significant increase in the acid tolerance of this microorganism. Spores of Bti ppk were found to be smaller compared to wild-type spores; however, bioassay experiments with third-instar wild Culex pipiens larvae proved that high toxicity is not the result of small spore size. This hypertoxic recombinant Bti strain is very useful for industrial applications, not only because it produces more endotoxin than the wild type under different culture conditions, but also because it is more acid-tolerant under the conditions tested.

IntroductionPolyphosphate (polyP), which is a linear polymer of phosphate (P i ) residues linked by high-energy phosphoanhydride bonds, is present in all organisms from bacteria to humans (Rao et al., 2009). PolyP has been shown to have important roles in a variety of cellular processes including regulation of enzyme activities, storage of P i and energy, adaptive responses to physical and chemical stresses, bacterial survival during the stationary phase, chelation of cations, maintenance of optimal translation efficiency, gene transcription control, and formation and function of cell membrane, channels, and pumps (Kornberg et al., 1999). PolyP has also been shown to be necessary for motility, biofilm formation, and other virulence properties of different bacteria such as Salmonella spp.