Burkholderia pseudomallei and Burkholderia mallei are pathogens responsible for disease in both humans and animals. Burkholderia thailandensis, while phylogenetically similar, is considered avirulent in comparison. These three species exhibit phosphatase activity when grown on media containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP). Tn5-OT182 mutagenesis has been utilized to isolate mutants of B. pseudomallei and B. thailandensis unable to hydrolyse XP. Sequence analysis of these mutants revealed an ORF of 1734 nucleotides demonstrating a high degree of homology to the acpA gene product of Francisella tularensis. PCR primers were designed based on the B. pseudomallei acpA gene sequence and were used to amplify an acpA homologue from B. mallei. The predicted amino acid sequence of B. pseudomallei AcpA differed from those of the predicted B. thailandensis AcpA and B. mallei AcpA by 15 and 3 amino acids, respectively. Allelic exchange was used to construct DeltaacpA mutants in each of these Burkholderia spp. These mutants were shown to be devoid of phosphatase activity and have subsequently allowed for the implementation of phoA fusion transposon mutagenesis systems. Two such systems have been successfully utilized in Burkholderia spp. for the identification of several genes encoding exported proteins.