Molecular cloning and analysis of a putative siderophore ABC transporter from Staphylococcus aureus - PubMed (original) (raw)

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

J A Morrissey et al. Infect Immun. 2000 Nov.

Abstract

From a mass-excised Staphylococcus aureus lambdaZapII 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.

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Figures

FIG. 1

FIG. 1

(A) Southern blot analysis showing DNA homology among the S. aureus BB sstABCD operon, other_S. aureus_ strains, and S. epidermidis. Plasmid and genomic DNAs were digested with Mun_I, and the Southern blots were hybridized with a digoxigenin-labeled PCR product amplified from BB genomic DNA using primers 38A for and 38B rev. Lanes: M, λ_Hin_dIII/phiX174 Hae_III DNA markers; 1, S. epidermidis 901; 2, S. aureus BB; 3, S. aureus W; 4, S. aureus RN4220; 5, plasmid pJM10; 6, diluted PCR product used as the probe. (B) Organization of the S. aureus sstABCD ABC transporter operon. (C) The upstream sequences of the sstABCD operon showing one of the putative Fur box sequences in bold and the position of the potential inverted repeat region. The ribosome-binding site and the translational start site are underlined. (D) Alignment of the_sstABCD putative Fur box sequences with the E. coli Fur box consensus sequence (9) and the Fur box sequences from the S. aureus sirA (14) and_fhuA (36) genes. The differences are highlighted.

FIG. 2

FIG. 2

Immunoblots with rat anti-SstD serum showing membrane association, iron regulation, and Triton X-114 solubility of SstD. (A) Membrane fractions of S. aureus BB grown for 6 h under iron-restricted (lane 1) or iron-rich (lane 2) conditions. The 38-kDa SstD lipoprotein is indicated. (B) Triton X-114 extracts of S. aureus BB grown for 18 h in RPMI 1640 medium (lane 2) and_S. aureus_ BB (lane 3) or S. epidermidis 901 (lane 4) grown for 6 h in CRPMI. Lane 1 shows the membrane fraction of S. aureus BB grown for 6 h in CRPMI. The 38-kDa SstD protein is indicated.

FIG. 3

FIG. 3

Immunoblot of Triton-X114 extracts prepared from_S. aureus_, coagulase-negative staphylococci, and B. subtilis grown under iron-restricted conditions in vitro and_S. aureus_ BB grown in vivo. The immunoblots were reacted with monospecific rat antiserum to the 38-kDa SstD lipoprotein. Lanes: 1, S. aureus BB; 2, S. haemolyticus; 3, S. carnosus; 4, B. subtilis; 5, S. hominis; 6, S. warnerii; 7,S. cohni, 8, S. lugdunensis; 9, S. saprophyticus; 10, S. epidermidis; 11, S. aureus BB grown in vivo.

FIG. 4

FIG. 4

(A) Northern blot analysis of antisense sstD_transcripts. Total RNAs were isolated from logarithmically growing_S. aureus strains grown under iron-rich or iron-restricted conditions in vitro. The Northern blot was hybridized with digoxigenin-labeled sstD RNA. The approximately 1-kb antisense transcript is indicated. (B) Western blot analysis of membrane fractions from S. aureus RN6390-B showing the decrease in SstD protein production in the presence of _sstD_antisense RNA. Lanes: 1, RN6390-B (with Fe); 2, RN6390-B (without Fe); 3, RN6390-B pS10 (with Fe); 4, RN6390-B pS10 (without Fe); 5, RN6390-B pS1038 (with Fe); 6, RN6390-B pS1038 (without Fe). (C) Immunoblot of membrane preparations from S. aureus recovered without subculture from the chambers and probed with anti-SstD serum. Lanes: 1, RN6390-B S1038; 2, RN6390-B S10.

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