Glutamate Racemase Mutants of Bacillus anthracis

Abstract

ABSTRACT d -Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ- d -glutamic acid (PDGA) capsule of Bacillus anthracis , the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded by racE1 and racE2 , are each essential for growth of B. anthracis , supplying d -glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion of racE1 or racE2 did not prevent growth of B. anthracis Sterne (pXO1 + pXO2 − ), the noncapsulating vaccine strain, or of B. anthracis Ames (pXO1 + pXO2 + ), a fully virulent, capsulating isolate. While mutants with deletions in racE1 and racE2 were not viable, racE2 deletion delayed vegetative growth of B. anthracis following spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenous d -glutamate. Deletion of racE1 or racE2 from B. anthracis Ames did not affect the production or stereochemical composition of the PDGA capsule. A model is presented whereby B. anthracis , similar to Bacillus subtilis , utilizes two functionally redundant racemase enzymes to synthesize d -glutamic acid for peptidoglycan synthesis. IMPORTANCE Glutamate racemases, enzymes that convert l -glutamate to d -glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent, B. anthracis , requires d -glutamate for the synthesis of peptidoglycan and poly-γ- d -glutamic acid (PDGA) capsule. Here we show that B. anthracis possesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to block B. anthracis growth and achieve therapeutic efficacy.