Identification of the acid phosphatase (acpA) gene homologues in pathogenic and nonpathogenic Burkholderia spp. facilitates TnphoA mutagenesis

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 thre e species exhibit phosphatase activity when grown on media containing chrom ogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP). Tn5-OT 182 mutagenesis has been utilized to isolate mutants of B. pseudomallei and B. thailandensis unable to hydrolyse XP. Sequence analysis of these mutant s revealed an ORF of 1734 nucleotides demonstrating a high degree of homolo gy to the acpA gene product of Francisella tularensis. PCR primers were des igned based on the B. pseudomallei acpA gene sequence and were used to ampl ify 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 exch ange was used to construct Delta acpA mutants in each of these Burkholderia spp. These mutants were shown to be devoid of phosphatase activity and hav e subsequently allowed for the implementation of phoA fusion transposon mut agenesis systems. Two such systems have been successfully utilized in Burkh olderia spp. for the identification of several genes encoding exported prot eins.