Conditions and mutations affecting Staphylococcus aureus L-form formation

Staphylococcus aureus is a prominent human pathogen and is known to form L-forms in vitro and in vivo during infection. However, the conditions of L-form formation are not optimal and the mechanisms of L-form formation in this organism are unknown. Here, we optimized the conditions of S. aureus unstable L-form formation, constructed a transposon mutant library and screened for mutants defective in unstable L-form formation. Our results revealed that 20 % sucrose, 3.5 % sodium chloride, 750-1000 U penicillin and 33 °C were optimal conditions for L-form formation. Stationary phase cultures of S. aureus formed L-forms better than exponential phase cultures. The S. aureus L-form colonies showed typical 'fried-egg' morphology and the cells had deficient cell wall, showed morphological diversity, and stained Gram-negative. The mutant library screens identified 15 mutants deficient in L-form formation and sequencing analysis identified mutations in eight genes and three intergenic regions. Real-time PCR analysis indicated that, with the exception of gntK, seven genes including glpF, glpK, NWMN_0623, NWMN_0843, NWMN_0333, NWMN_0872 and NWMN_1269 were preferentially expressed in L-forms as compared with normal cell-walled form (P