Molecular Cloning and Analysis of a Putative Siderophore ABC Transporter from Staphylococcus aureus

ABSTRACT From a mass-excised Staphylococcus aureus λZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homology to bacterial siderophore transporter systems. The operon encodes four genes designatedsstA, -B, -C, and -Dencoding two putative cytoplasmic membrane proteins (sstAand sstB), an ATPase (sstC), and a membrane-bound 38-kDa lipoprotein (sstD). Thesst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dependent. SstD was overexpressed inEscherichia coli, purified by Triton X-114 phase partitioning, and used to generate monospecific antisera in rats. Immunoblotting studies located SstD in the membrane fraction ofS. aureus and showed that expression of the lipoprotein was reduced under iron-rich growth conditions. Triton X-114 partitioning studies on isolated membranes provided additional biochemical evidence that SstD in S. aureus is a lipoprotein. Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detected inBacillus subtilis. Expression of SstD in vivo was confirmed by immunoblotting studies with S. aureus recovered from a rat intraperitoneal chamber implant model. To further define the contribution of SstD in promoting growth of S. aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD. Expression of antisense sstD RNA inS. aureus resulted in a decrease in SstD expression under both iron-rich and iron-restricted growth conditions. However, this reduction in SstD levels did not affect 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.

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