The Gram-positive bacterium, Rhodococcus sp. 33, was selected for further study to identify the characteristics conferring it with high tolerance to concentrations of benzene. Since most organic solvents, like benzene, are lipophilic, they tend to accumulate within lipid membranes where they express toxicity. The mechanisms conferring this Rhodococcus with resistance to benzene were hypothesised to be located within the subcellular region of this bacterium - cell wall, membrane, and cell-bound polymer. Therefore, this investigation was instigated to identify these mechanisms. To accomplish this, the development of methodologies to isolate highly purified cell wall and membrane fractions, from the organism, were required. To corroborate this investigation, a total of 6 benzene-sensitive mutants were prepared from Rhodococcus sp. 33 and their characteristics compared to those of the parent wild-type strain. 1-D PAGE analysis of proteins revealed various benzene-induced wall, membrane, and cytoplasmic proteins in the w-t. A protein band, with an approximate molecular weight of 58 kDa, was identified to be absent in the most sensitive mutant isolated (mutant M2b). Interestingly, much of this research showed that the benzene-catabolising enzymes played an insignificant role in tolerating the benzene. Gas chromatography and mass spectrometry of whole cell-derived fatty acids revealed that benzene induced an increase in the ratios of saturated/unsaturated fatty acids. Moreover, protein determinations revealed that benzene induced an increase in the concentration of total membrane protein. These increases are suggestive as possible mechanisms to decrease the fluidity of the cell membrane. This was further supported by the observed increase in the generalised polarisation (GP) of laurdan fluorescence in the membranes during growth of the organism with benzene, which is correlated with a decrease in membrane fluidity. The organism was also found to synthesise hexadecenoic acid, 16:1w6c (11 - 13% of total fatty acids), an uncommon fatty acid and previously unreported in other Rhodococcus spp. Analysis of the organism's cell-bound extracellular polymer revealed it to be composed of polysaccharide with biosurfactant-like properties. Its function is proposed to act as a surfactant layer outside the cell, concentrating the benzene within its matrix and reducing benzene's contact with the cell membrane.
| Identifer | oai:union.ndltd.org:ADTP/258494 |
| Date | January 1999 |
| Creators | Guti??rrez, Jos?? Antonio, School of Microbiology & Immunology, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Jos?? Antonio Guti??rrez, http://unsworks.unsw.edu.au/copyright |
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