Knockout of the alanine racemase gene in Aeromonas hydrophila HBNUAh01 results in cell wall damage and enhanced membrane permeability

This study focused on the alanine racemase gene ( alr -2), which is involved in the synthesis of d-alanine that forms the backbone of the cell wall. A stable alr -2 knockout mutant of Aeromonas hydrophila HBNUAh01 was constructed. When the mutant was supplemented with d-alanine, growth was unaffected; deprivation of d-alanine caused the growth arrest of the starved mutant cells, but not cell lysis. No alanine racemase activity was detected in the culture of the mutant. Additionally, a membrane permeability assay showed increasing damage to the cell wall during d-alanine starvation. No such damage was observed in the wild type during culture. Scanning and transmission electron microscopy analyses revealed deficiencies of the cell envelope and perforation of the cell wall. Leakage of UV-absorbing substances from the mutants was also observed. Thus, the partial viability of the mutants and their independence of d-alanine for growth indicated that inactivation of alr -2 does not impose an auxotrophic requirement for d-alanine.