The bacteriumBurkholderiasp. Ha185 readily solubilizes inorganic phosphate by releasing the low molecular weight organic anion, 2-ketogluconate. Using random transposon mutagenesis andin silicoanalysis, a mutation that caused almost complete abolition of phosphate solubilization was located withinhemX, which is part of thehemoperon.Burkholderiasp. Ha185 HemX is a multidomain protein, predicted to encode a bifunctional uroporphyrinogen-III synthetase/uroporphyrin-III C-methyltransferase, which has not previously been implicated in phosphate solubilization. Complementation ofhemXrestored the ability of the mutant to solubilize phosphate in both plate and liquid cultures. Based on a combination of organic-anion profiling, quantitative polymerase chain reaction andin silicoanalyses,hemXwas confirmed to be solely responsible for hydroxyapatite solubilization inBurkholderiasp. Ha185. It is proposed that the biosynthesis of a yet to be determined redox cofactor by HemX is the main pathway for generating 2-ketogluconate via a haem-dependent gluconate 2-dehydrogenase inBurkholderiasp. Ha185.