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 degrees 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<0.05). The identified genes involved in L-form growth mapped in the pathways for energy production, iron homeostasis, transporters, DNA repair, membrane biogenesis, and biosynthesis. Our findings shed new insight into the molecular basis of S. aureus unstable L-form formation and may have implications for development of novel drugs targeting S. aureus L-forms for improved treatment.