Resistance to copper toxicity in a spontaneously generated mutant of Salmonella Typhimurium

The objective of this investigation was to define an experimental system using analytical electron microscopy as a complementary tool to traditional biochemical methods. Specifically, the system studied was the phenomenon of resistance to copper toxicity in a spontaneously generated mutant of Salmonella typhimurium strain LT2.

It was observed that this mutant, henceforth called CF, showed normal growth rates in minimal medium containing 0.01 M copper citrate, whereas the wildtype cells showed signs of metal toxicity at 10^-5 M copper citrate levels. It was also noted that these mutants had the innate ability to actively accumulate the copper, which led to the possibility that these cells could be examined using energy dispersive X-ray spectroscopy, to determine if and where this accumulation of copper may be localized.

The accumulation of copper was highest at a period approximately 10 hours into resting phase, which will be refered to as PRP(copper peak of resting phase). Upon cell fractionization, a significant amount of copper was seen in both the cell extract and the cell membrane fraction. At PRP, copper levels from cell extracts of CF grown in copper showed up to a 60-fold increase over cell extracts from CF mutants grown in minimal medium that had not been supplemented with copper. It was found that CF cells harvested at PRP contain at least two copper binding factors, one of high molecular weight and one of low molecular weight. The low molecular weight copper binder is the result of an inducible response to growth in copper and has an apparent molecular weight smaller than that of cytochrome c.

Also when comparing accumulation of copper in exponential or log phase growth of the wildtype vs. CF : the wildtype strain accumulates relatively high copper levels until mid-log phase and then growth ceases ; CF shows low levels of copper accumulation throughout log phase. This may indicate a possible copper exclusion phenomenon in the CF mutant which aids its adjustment to high copper levels in the growth medium.