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Infection and Immunity, November 2000, p. 6281-6288, Vol. 68, No. 11
Institute of Infections and
Immunity,1 Department of Applied
Biochemistry and Food Science,2 and
School of Pharmaceutical Sciences,3
University of Nottingham, Nottingham NG7 2UH, United Kingdom
Received 26 June 2000/Returned for modification 24 July
2000/Accepted 16 August 2000
From a mass-excised Staphylococcus aureus
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular Cloning and Analysis of a Putative
Siderophore ABC Transporter from Staphylococcus
aureus
ZapII
expression library, we cloned an operon encoding a novel ABC
transporter with significant homology to bacterial siderophore
transporter systems. The operon encodes four genes designated
sstA, -B, -C, and -D
encoding two putative cytoplasmic membrane proteins (sstA and sstB), an ATPase (sstC), and a
membrane-bound 38-kDa 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 dependent. SstD was overexpressed in
Escherichia coli, purified by Triton X-114 phase
partitioning, and used to generate monospecific antisera in rats.
Immunoblotting studies located SstD in the membrane fraction of
S. 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 in
Bacillus 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 in
S. 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.
*
Corresponding author. Present address: Department of
Microbiology & Immunology, University of Leicester, P.O. Box 138, Medical Sciences Building, University Rd., Leicester LE1 9HN, United
Kingdom. Phone: 44-116-2522943. Fax: 44-116-2525030. E-mail:
jam26{at}le.ac.uk.
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