Fluorescent phospholipid analogs as microscopic probes for detection of the mycolic acid-containing layer in Corynebacterium glutamicum: detecting alterations in the mycolic acid-containing layer following ethambutol treatment.
Corynebacterium glutamicum belongs to the mycolic acid-containing actinomycetes, which also include Mycobacterium, Nocardia, and Rhodococcus. The cells of this group possess a cell wall with a thick outer layer composed primarily of mycolic acid, which functions as a permeability barrier. To investigate the mechanism of mycolic acid-containing layer (mycolate layer) formation, we have developed a fluorescence microscopic technique detecting the mycolate layer in situ. The staining specificity of fluorescence-labeled phospholipid analogs was determined by simultaneous staining with the hydrophobic fluorescent dye Nile Red and peptidoglycan-staining fluorescence-conjugated vancomycin. We found that fluorescence-labeled phospholipid analogs preferentially stain the mycolate layer. Using this technique, we observed the effect of the anti-mycobacterial drug ethambutol on C. glutamicum mycolate-layer formation. Ethambutol interfered specifically with mycolate-layer formation on the division planes and cell poles, while the side-wall mycolate layer was not severely affected. This indicates that mycolate-layer formation occurs mainly on division planes and cell poles in C. glutamicum, where the peptidoglycan layer is actively synthesized.
Kumagai
Yutaro
Y
Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Japan.
Hirasawa
Takashi
T
Hayakawa
Kenshi
K
Nagai
Kazuo
K
Wachi
Masaaki
M
D000890:Anti-Infective Agents / Q000494:pharmacology; D002473:Cell Wall / Q000737:chemistry*; D048230:Corynebacterium glutamicum / Q000166:cytology*; D004977:Ethambutol / Q000494:pharmacology*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence / Q000379:methods; D009171:Mycolic Acids / Q000032:analysis*; D010078:Oxazines; D010743:Phospholipids
D000890:Anti-Infective Agents; D005456:Fluorescent Dyes; D009171:Mycolic Acids; D010078:Oxazines; D010743:Phospholipids; D004977:Ethambutol; C044808:nile red