Characterization of RbsD of the D-ribose high-affinity transport system in Escherichia coli K12
Abstract
The D-ribose high-affinity transport operon (rbs) of Escherichia coli encodes the proteins necessary for high-affinity transport and utilization of D-ribose. We have overproduced the gene products of the putative transport complex, rbsDAC. However, no evidence exists demonstrating that rbsD is involved in D-ribose transport or utilization. The gene encodes a 15 kDa protein with an amino terminus which resembles the proteolytically cleaved signal sequences of periplasmic and outer membrane proteins. The amino terminus is followed by the pentapeptide AspThrLeuValVal. A similar pentapeptide sequence is present in outer membrane receptor proteins which are constituents of TonB-dependent transport systems and in all colicins which are taken up by a TonB-dependent mechanism. We have overproduced and purified the rbsD protein and deduced its amino terminal sequence. We show that the protein does not contain a cleavable signal sequence and is therefore not likely to be an outer membrane receptor. We have also created a rbs operon mutant at the putative tonB box to determine if D-ribose transport and/or utilization is TonB-dependent. Our results indicate that D-ribose high-affinity transport is not TonB-dependent, but that TonB might be involved in the utilization of D-ribose. Furthermore, we show that when the amino terminal half of RbsD is deleted, both transport and utilization of D-ribose are reduced significantly. We conclude that RbsD is not essential for D-ribose transport and utilization and may not be a component of the high affinity transport complex.
Degree
Ph.D.
Advisors
Hermodson, Purdue University.
Subject Area
Biochemistry|Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.