Fluorescent Phospholipid Analogs as Microscopic Probes for Detection of the Mycolic Acid-Containing Layer in<i>Corynebacterium glutamicum</i>: Detecting Alterations in the Mycolic Acid-Containing Layer Following Ethambutol Treatment

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  • 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

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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.

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