Effect of inhibitors of cell envelope synthesis on β‐sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683

Abstract

The role of the lipid bilayer and the peptidoglycan of the mycobacterial cell wall in the permeation of beta-sitosterol into the cell and its transformation to androst-1-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) was studied. Specific inhibitors were used at concentrations affecting the biosynthesis of the assumed target structures, but causing only partial cell growth inhibition or exerting no effect on growth. m-Fluorophenylalanine and DL-norleucine which are known to disorganize the biosynthesis of amphipatic components of the outer layer of the lipid bilayer, used at concentrations 250 micrograms/ml and 400 micrograms/ml, respectively, increased the formation rate of AD+ADD from 0.3 (control) to 0.7 and 0.8 mg products/g dry weight/h. The disorganization of the underlying mycolyl-arabinogalactan structure by the action of the ethambutol at the concentration 40 micrograms/ml, at which the cell growth was apparently not affected, caused the decrease of the product formation from 135 mg/l to 70 mg/l. In the presence of isoniazid (350 micrograms/ml) only trace amounts of AD accumulated during 48 hours of transformation indicating much lower activity than that of the intact cells. The most effective among the tested inhibitors of peptidoglycan synthesis were glycine (15 mg/ml) and vancomycin (150 micrograms/ml) which enhanced the transformation activity of the treated cells nearly three times. Increased transformation rate was also obtained by the action of colistin at concentrations ranging from 10 micrograms/ml to 15 micrograms/ml.

Methyl-β-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria

“…This was in agreement with preserved cell viability and CW basal arabinogalactan skeleton. As shown, the integrity of the mycolyl-arabinogalactan structure is important for the sterol side-chain degradation ability of mycobacteria and related micro-organisms (Sedlaczek et al, 1994).…”

“…This leaflet was shown to hinder the access of sterols to the enzyme system inside the cell (Sedlaczek et al, 1994). Its MCD-mediated disorganization possibly resulted in increased CW permeability for both substrate and products, thus providing an increase in the yield of AD and ADD.…”

“…) and vancomycin (150 mg ml 21 ) resulted in substantial enhancement of sitosterol conversion (Sedlaczek et al, 1994). These agents interacted with the CW peptidoglycan and shifted the ratio of muramic and diaminopimelinic acids (Lisowska et al, 1996;Sedlaczek et al, 1999).…”

“…) resulted in the decrease of AD(D) yield from sitosterol (Sedlaczek et al, 1994). Isoniazid (350 mg ml…”

Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds

“…From this culture, 10 ml was transferred to 90 ml NB medium in 1 litre flasks. At the time of inoculation, β-sitosterol (0n2 g l − "), prepared by homogenization as described previously (Sedlaczek et al, 1994), and one of the polycations were added to the medium, which was then incubated under the conditions described above. The concentration of polycations is shown in Results for the individual configurations.…”