Molecular cloning and analysis of a putative siderophore ABC transporter from Staphylococcus aureus

From a mass-excised Staphylococcus aureus lambda ZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homol ogy to bacterial siderophore transporter systems. The operon encodes four g enes designated sstA, -B, -C, and -D encoding two putative cytoplasmic memb rane proteins (sstA and sstB), an ATPase (sstC), and a membrane-bound 38-kD a lipoprotein sstD). The sst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dep endent. SstD was overexpressed in Escherichia call, purified by Triton X-11 4 phase partitioning, and used to generate monospecific antisera in rats. I mmunoblotting studies located SstD in the membrane fraction of S. aureus an d showed that expression of the lipoprotein was reduced under iron-rich gro wth conditions. Triton X-114 partitioning studies on isolated membranes pro vided additional biochemical evidence that SstD) in S. aureus is a lipoprot ein. Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detecte d in Bacillus subtilis. Expression of SstD in vivo was confirmed by immunob lotting studies with S. aureus recovered from a rat intraperitoneal chamber implant model. To further define the contribution of SstD in promoting gro wth of S. aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD. Expression of antisense sstD RNA in S. aureus resulted in a decrease in SstD expression under both iron-rich and iron-res tricted growth conditions. However, this reduction in SstD levels did not a ffect the growth of S. aureus in vitro in an iron-limited growth medium or when grown in an intraperitoneal rat chamber implant model in vivo.