Molecular characterization, metal uptake and copper induced transcriptional activation of efflux determinants in copper resistant isolates of Klebsiella pneumoniae.

An efflux system, comprising cus determinants, plays an important role in pumping out this metal in gram negative bacteria exposed to high concentration of copper. Cus determinants comprise two operons, one regulatory (cusRS) and the other structural (cusCFBA). Although the efflux system has been described in quite a few members of Enterobacteriaceae, little is known about this system in Klebsiella spp. We are describing cus determinants in Klebsiella pneumoniae for the first time and also providing evidence for their metal-induced expression, both under aerobic and anaerobic conditions. Copper resistant K. pneumoniae, capable of copper uptake and later efflux of excessive copper, was isolated from industrial waste water. Expression of both cusRS and cusCFBA was quantified at transcriptional level through real time PCR. The results demonstrated that cus determinants were functional under both aerobic and anaerobic conditions. The mRNA level of both operons increased several fold in the presence of non-lethal as well as sub-lethal copper concentrations. The increase in cusCFBA transcripts was 74.8 fold 15 min after exposure to 3mM Cu(++) under aerobic conditions compared to the 16 fold increase in cusRS under the same conditions. Under anaerobic conditions the cusCFBA transcripts increased 32.65 fold and the cusRS five fold within 15 min after exposure to 3mM Cu(++). It is concluded that cus genetic determinants in K. pneumoniae comprise structural component (cusCFBA) and a regulatory component (cusRS), which show several fold expression under copper induction both under aerobic and anaerobic conditions. Under aerobic conditions, the structural genes express 4.7 fold more than the regulatory genes, whereas under anaerobic conditions, this expression is 6.5 fold. Finally, time course study revealed a novel pattern of immediate up-regulated expression followed by decreased and another increased in the transcript level of both operons of cus determinants in the presence of copper.