Biosynthetic Analysis of the Petrobactin Siderophore Pathway from Bacillusanthracis

Abstract

The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus (asbABCDEF) were constructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis when strains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew to a very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis asb mutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenous petrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl citrate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxybenzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This work sets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomal peptide synthetase-independent biosynthetic system.

Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore

“…Disc Diffusion Assay. These growth assays were performed as described previously with some modifications (44) and are detailed in SI Methods.…”

Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis

“…1C) as a first step in petrobactin biosynthesis (12). AsbB is similar in activity to AsbA but classified as a type C NIS synthetase due to its preference for advanced intermediates in siderophore biosynthesis as substrates including 1 (11,17). Both AsbA and AsbB incorporate the asymmetric polyamine spermidine from only one (N8) of its two primary amino group termini (Fig.…”

“…Conversely, a second siderophore, petrobactin (Fig. 1A), is essential for virulence within current infection models (6,10) and has since become a focus of multiple microbiological, genetic, and biochemical studies (3,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)20). This mixed catechol-carboxylate siderophore was first isolated from the Gram-negative marine microbe Marinobacter hydrocarbonoclasticus (21).…”

Genetic analysis of petrobactin transport inBacillus anthracis

“…Although B. anthracis encodes the biosynthesis machinery for two siderophores, only one of these, petrobactin, is required for efficient bacterial growth in low-iron conditions and for full virulence in a murine model of anthrax infection (Cendrowski et al, 2004;Lee et al, 2007). As this siderophore plays such an important role in B. anthracis pathogenesis, we sought to elucidate the role of two proteins recently implicated in petrobactin binding.…”

“…Construction of 0.05 and growth was monitored hourly by measurement of optical density at 600 nm. A mutant strain deficient for petrobactin biosynthesis (DasbABCDEF) was also included as a control (Cendrowski et al, 2004;Lee et al, 2007). Cultures of wild-type B. anthracis are capable of rapid growth under these conditions, reaching high density within four hours ( Fig.…”