Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect

Several Bacillus strains belonging to the B. subtilis/amytoliquefaciens group isolated from plant-pathogen-infested soil possess plant-growth-promoting activity [Krebs, B. et al. (11998) J Plant Dis Prot 105, 181-197]. Three out of the four strains investigated were identified as B. amylotiquefaciens and were able to degrade extracellular phytate (myo-inositol hexakisphosphate). The highest extracellular phytase activity was detected in strain FZB45, and diluted culture filtrates of this strain stimulated growth of maize seedlings under phosphate limitation in the presence of phytate. The amino acid sequence deduced from the phytase phyA gene cloned from FZB45 displayed a high degree of similarity to known Bacillus phytases. Weak similarity between FZB45 phytase and B. subtilis alkaline phosphatase IV pointed to a possible common origin of these two enzymes. The recombinant protein expressed by B. subtilis MU331 displayed 3(l)-phytase activity yielding D/L-Ins(1,2,4,5,6)P5 as the first product of phytate hydrolysis. A phytase-negative mutant strain, FZB45/M2, whose phyA gene is disrupted, was generated by replacing the entire wild-type gene on the chromosome of FZB45 with a km::phyA fragment, and culture filtrates obtained from FZB45/M2 did not stimulate plant growth. In addition, the growth of maize seedlings was promoted in the presence of purified phytase and the absence of culture filtrate. These genetic and biochemical experiments provide strong evidence that phytase activity of B. amytoliquefaciens FZB45 is important for plant growth stimulation under phosphate limitation.