| As Borrelia burgdorferi, the causative agent of Lyme disease, transitions from its tick vector to a mammalian host, the spirochete up-regulates the expression of a two gene operon that includes the bb0728 and bb0729 genes. The bb0728 gene (cdr), the second gene in the operon, encodes a co-enzyme A disulphide reductase (CoADR) that reduces CoA-disulphides to CoA in an NADH-dependent manner. The function of the gene-product encoded by the first gene in the operon (bb0729) remains uncharacterized, although the gene has been annotated as encoding a transporter from the dicarboxylate/ amino acid: cation (Na+ or H+) symporter (daacs) family based on sequence similarities. Because this operon is up-regulated during the infection of the mammalian host and the cdr gene is essential for virulence, we have been interested in further characterizing the function and role of the bb0729 gene product. To begin this characterization, we have used a mutant in which the bb0729 has been disrupted. Unfortunately, the way in which the clone was made suggests that the both bb0729 and the downstream bb0728 gene may be disrupted in this clone; as part of this study, RT-PCR to determine the level of bb0728 transcription was unsuccessfully attempted. To determine whether the disruption of the bb0729 in this clone changes the growth deficit observed in the clone in which only bb0728 has been inactivated, we compared the growth of these two clones and the wild-type during cultivation under standard (15% oxygen, 6% CO2) and anaerobic (<1% oxygen, 9-13% CO2) conditions; these are conditions under which it is known that the bb0728-deletion mutant has a significant growth defect. We found that the bb0729 mutant used in this study also had a significant growth defect, although not significantly different than that of the bb0728-deletion mutant. We used a shuttle vector containing the bb0729-cdr operon and upstream promoter element to complement the bb0729 deletion mutant. Thus, the newly generated complement was complemented with both bb0729 and bb0728 and found that the complemented mutant had a similar growth pattern to that of the wild/ parent type B. burgdorferi under both conditions. Based on these results, we have confirmed that the operon, as a whole, is essential to maintain the physiological function and metabolism of the bacteria, but more work must be done to further characterize the exact role of bb0729 in this function. |
