| D-Alanyl-lipoteichoic acid (LTA) plays a vital role in the growth and physiology of gram-positive bacteria. This macroamphiphile modulates the activities of autolysins, the binding and trafficking of cations and nutrients, and determines the electromechanical properties of the cell wall. The dlt operon (dltA-dltD) has been identified and sequenced in 12 organisms including Lactobacillus rhamnosus and Lactococcus lactis. It encodes four proteins responsible for D-alanylation. They are: the 56 kDa D-alanine:D-alanyl carrier protein ligase (Dcl; DltA), the 8.8 kDa D-alanyl carrier protein (Dcp), the putative transporter (DltB), and DltD. Although the function(s) of D1tB is unknown, it is hypothesized to be an integral membrane protein with 11 transmembrane domains that functions in the translocation of D-alanyl-Dcp to the extramembranal site of D-alanylation. DltB has sequence similarity to a variety of transport systems as well as proteins in the superfamily of membrane bound O-acyl transferases. To understand the function of the putative transporter encoded by dltB, plasmid expression systems for this protein were designed. Catalytic activities, essential residues, and topology of this transporter may be determined with the isolated protein. Initially, Escherichia coli expression vectors were utilized. Although DltB was identified when translated in vitro, no protein could be detected when dltB was expressed in vivo. In contrast, DltB was detected when co-expressed with DltA in L. lactis . The requirement of dltA for dltB expression in this organism may result from the translational coupling of the two genes. The increased levels of DltB cannot be measured with the permeabilized cell assay for D-alanine incorporation, and thus, new assay procedures for this novel protein must be devised. |
