Fig 1. Cell wall synthesis is not essential for L-form growth.

(a) L-forms are highly sensitive to osmotic differences. Walled or L-form cells were exposed to deionized water or Listeria L-form medium (LLM) as control. After 5 min exposure, a ten-fold dilution series was spotted on agar plates to enumerate the surviving bacteria. Whereas no difference between water and LLM treated samples could be observed for walled Listeria, more than 99% of the L-forms died when exposed to deionized water. (b) L-forms can grow in presence of various antibiotics that specifically inhibit transpeptidation (penicillin G; 50 μg/ml), glycosyl transfer (ramoplanin, 6.25 μg/ml) or both (vancomycin, 100 μg/ml). Arrows indicate growing colonies within soft agar tubes. Scale bar, 10 μm. (c) D-alanine dependency of the walled form of a mutant which is deficient in the alanine racemase and the D-amino acid aminotransferase gene. The legend shows the D-alanine concentration in the BHI medium, which only allows growth of walled bacteria. (d) L-forms derived from the D-alanine auxotroph Listeria strain grown in Listeria L-form medium. Scale bar, 10 μm.