Studies on the region involved in the transport activity of Escherichia coli TolC by chimeric protein analysis

Gram-negative bacteria possess the outer membrane protein TolC which acts as an exit duct across the outer membrane. However, the region involved in the transport activity of TolC has remained unclear. We analyzed this region by creating chimeric TolCs. First, we expressed the genes for TolCs of Vibrio parahaemolyticus (vp-tolC) and Salmonella typhimurium (sal-tolC) in Escherichia coli. The levels of sequence identity in the mature region of VP-TolC/EC-TolC and Sal-TolC/EC-TolC with maximum matching are 43% and 90%, respectively. We found that the transport activity of VP-TolC was weak compared with that of TolC of E. coli (EC-TolC) although the transport activity of Sal-TolC was similar to that of EC-TolC. A comparison of the sequence of the three tolCs showed that the sequence around the periplasmic region covering Asn-188 to Lys-214 of EC-TolC is lowly identical to that of VP-TolC although the region of EC-TolC is almost identical to that of Sal-TolC. We think, therefore, that the region covering Asn-188 to Lys-214 of EC-TolC may have an important role to express its transport activity in E. coli. To examine the possibility, we divided the region of EC-TolC into three and exchanged the gene for each portion with that of vp-tolC. These mutant ec-tolCs were expressed in E. coli and the activity of each chimeric TolC was measured. The results showed that the portion covering Val-198 to Lys-214 of EC-TolC is deeply involved in the transport activity.