Mutation of L-2,3-diaminopropionic acid synthase genes blocks staphyloferrin B synthesis in Staphylococcus aureus

Abstract

Background: Staphylococcus aureus synthesizes two siderophores, staphyloferrin A and staphyloferrin B, that promote iron-restricted growth. Previous work on the biosynthesis of staphyloferrin B has focused on the role of the synthetase enzymes, encoded from within the sbnA-I operon, which build the siderophore from the precursor molecules citrate, alpha-ketoglutarate and L-2,3-diaminopropionic acid. However, no information yet exists on several other enzymes, expressed from the biosynthetic cluster, that are thought to be involved in the synthesis of the precursors (or synthetase substrates) themselves.

Results: Using mutants carrying insertions in sbnA and sbnB, we show that these two genes are essential for the synthesis of staphyloferrin B, and that supplementation of the growth medium with L-2,3-diaminopropionic acid can bypass the block in staphyloferrin B synthesis displayed by the mutants. Several mechanisms are proposed for how the enzymes SbnA, with similarity to cysteine synthase enzymes, and SbnB, with similarity to amino acid dehydrogenases and ornithine cyclodeaminases, function together in the synthesis of this unusual nonproteinogenic amino acid L-2,3-diaminopropionic acid.

Conclusions: Mutation of either sbnA or sbnB result in abrogation of synthesis of staphyloferrin B, a siderophore that contributes to iron-restricted growth of S. aureus. The loss of staphyloferrin B synthesis is due to an inability to synthesize the unusual amino acid L-2,3-diaminopropionic acid which is an important, iron-liganding component of the siderophore structure. It is proposed that SbnA and SbnB function together as an L-Dap synthase in the S. aureus cell.

Figure 1

SbnA and SbnB are essential for synthesis of staphyloferrin B in S. aureus. A) Chemical structure of staphyloferrin B with fundamental components labeled. Asterisks indicate ligands responsible for the octahedral coordination of iron. B) Within the sir-sbn genetic locus, the focus of this study is the characterization of mutations in sbnA (highlighted grey) (encoding a putative cysteine synthase) and sbnB (highlighted in black) (encoding a putative ornithine cyclodeaminase). Together, the products of these two genes are hypothesized to be an L-Dap synthase. C) S. aureus mutants were grown in chelex 100-treated TMS medium containing 10 μM holo-transferrin. In the Δsfa genetic background, growth in this medium is dependent on the production of the siderophore staphyloferrin B. Supplementation of the medium with FeCl₃ allows for equivalent growth for all strains (inset). D) The growth impairment exhibited by S. aureus sbnA or sbnB mutants, in the Δsfa genetic background, can be restored upon complementation in trans with a wild-type copy of the corresponding gene. Plasmid pALC2073 is the vehicle control.

Figure 2

Supplementation of culture medium with L-Dap allows S. aureus sbnA and sbnB mutants to overcome the block in synthesis of staphyloferrin B. A) Bacterial growth curves in chelex 100-treated TMS containing 10 μM holo-transferrin as the sole iron source, with the indicated supplements. B) Siderophore quantification from culture supernatants of iron-starved S. aureus mutants via CAS assay (see Materials and Methods). The inset graph represents culture supernatants from identical strains but grown in medium supplemented with FeCl₃. Siderophore units are normalized to culture density. C) Same as in B) except culture media was supplemented with L-Dap. D) Siderophore-disk diffusion assays. Culture supernatants to be tested were derived from S. aureus Δsfa sbnA::Tc or Δsfa sbnB::Tc strains cultured in medium supplemented with, or without, L-Dap, as indicated, and were spotted onto sterile paper disks before being placed onto TMS agar plates seeded with S. aureus wild-type and siderophore transport mutants, as indicated. Plate disk bioassay is described in Materials and Methods. E) Bacterial growth curves for cultures of S. aureus Δsfa sbnA::Tc and S. aureus Δsfa sbnB::Tc mutants in chelex 100-treated TMS medium containing 10 μM holo-transferrin as the sole iron source, where the medium was supplemented with various hypothesized L-Dap synthase substrates and by-products from Fig. 3, scheme A.

Figure 3

Proposed schemes for SbnA- and SbnB-dependent synthesis of L-Dap. Scheme A is adapted from Thomas et al. [18] for which the functions of SbnA and SbnB are analogous to the proposed functions VioB and VioK, respectively. The proposed functions of SbnA in schemes B-D remain as a β-replacement enzyme while SbnB is proposed to be an NAD⁺-dependent dehydrogenase of the indicated amino acid.