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Burkholderia pseudomallei  and  Burkholderia mallei  are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat  B. pseudomallei  or  B. mallei  infections may not be used as selective agents with the corresponding  Burkholderia  species. Here, we investigated alanine racemase deficient mutants of  B. pseudomallei  and  B. mallei  for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of  B. pseudomallei  K96243 has two annotated alanine racemase genes ( bpsl2179  and  bpss0711 ), and  B. mallei  ATCC 23344 has one ( bma1575 ). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of  Escherichia coli  strain ALA1. Herein, we show that  B. pseudomallei  with in-frame deletions in both  bpsl2179  and  bpss0711 , or  B. mallei  with an in-frame deletion in  bma1575 , requires exogenous  d -alanine for growth. Introduction of  bpsl2179  on a multicopy plasmid into alanine racemase deficient variants of either  Burkholderia  species eliminated the requirement for  d -alanine. During log phase growth without  d -alanine, the viable counts of alanine racemase deficient mutants of  B. pseudomallei and B. mallei  decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with  bpsl2179  as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame Δ flgK  mutant in the alanine racemase deficient variant of  B. pseudomallei  K96243. In murine peritoneal macrophages, wild type  B. mallei  ATCC 23344 was killed much more rapidly than wild type  B. pseudomallei  K96243. In addition, the alanine racemase deficient mutant of  B. pseudomallei  K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.

Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker