Determination of genes Involved in bacterial phosphate solubilisation

Abstract

Agricultural systems depend on continued inputs of phosphate fertiliser to maintain productivity. However, due to ever increasing global demand, the finite reserves of phosphate rock are being rapidly depleted. This is compounded by the fact that half of the soluble phosphate in fertiliser applied to soil is converted to sparingly-soluble minerals such as calcium phosphate which are unavailable for plant uptake. It is therefore important to investigate strategies that will improve the utilisation of phosphate in soil-plant systems. While it has been demonstrated that phosphate-solubilising bacteria can utilise sparingly-soluble phosphate minerals in soil, the mechanisms by which this occur remain unclear. In this project, 105 rhizobacteria were assessed for a number of plant growth-promoting traits, such as production of 1-aminocyclopropane-1-carboxylate deaminase, phytase, and inorganic phosphate solubilisation using plate screening assays. Ten of the most effective phosphate-solubilising isolates were further assessed using an in vitro hydroxyapatite liquid culture assay. Analysis of culture filtrates revealed that the effective phosphate-solubilising isolates, Pseudomonas spp. and Burkholderia sp., were predominantly secreting 2-keto-gluconic acid rather than the expected gluconic acid. In addition, a yet to be characterised organic acid was secreted by several Pseudomonas spp. strains which may also be involved in phosphate solubilisation. In an attempt to identify novel genes involved in phosphate solubilisation, three isolates of different bacterial genera, Enterobacter sp. Wi28, Pseudomonas sp. Ha200 and Burkholderia sp. Ha185, derived from diverse geographic locations were subjected to random transposon mutagenesis. This enabled the identification of two unique uncharacterised genes from Burkholderia sp. Ha185. One mutant, hemX::Tn5(F18), with a mutation in hemX gene involved in haem biosynthesis, exhibited an almost complete abolition in hydroxyapatite solubilisation. The second mutant bxpC::Tn5(F13) exhibited partial and delayed solubilisation. The translated product of the bxpC gene encodes a novel hypothetical protein with unknown function. However, based on a combination of organic acid, qRT-PCR and bioinformatics analysis, it is hypothesised that BxpC is a potential cargo protein involved in protection and transport of the calcium bound 2-ketogluconic acid compound. Through the use of a Burkholderia sp. Ha185 GFP-tagged strain in conjunction with a gnotobiotic in vivo plant assay developed in this study, the colonisation pattern of Burkholderia sp. Ha185 and its derivatives on ryegrass roots over three week duration was defined. The preliminary characterisation of the bxpC and hemX identified in the study and defining the interaction of Burkholderia sp. Ha185 on the plant root provides a greater understanding of phosphate cycling by bacteria and its relationship to the plant. The novel pathways and findings of this study have provided further insights into the underlying mechanisms of inorganic phosphate solubilisation in soil-plant systems.