Deduced roles of AsbA and AsbB in biosynthesis of petrobactinBa.
Database comparisons revealed that AsbA and AsbB are most similar in primary amino acid sequence to IucA and IucC from Escherichia coli
), gene products that are involved in the biosynthesis of aerobactin (Fig. ). Based on genetic studies of E. coli
mutant strains, iucA
was shown to mediate condensation of N
-hydroxy-lysine with citrate, and iucC
was shown to be responsible for linkage of a second N
-hydroxy-lysine molecule with the intermediate generated via the iucA
gene product (8
). Based on the NRPS-independent assembly of petrobactin, the demonstrated role of asb
in its biosynthesis, and the close amino acid sequence relationships of AsbA/AsbB and IucA/IucC, we developed a biosynthetic scheme for assembly of petrobactinBa
(Fig. ). Specifically, we propose that the siderophore is formed by the condensation of 3,4-dihydroxybenzoyl spermidine (3,4-DHB-SPD) with an activated form of citrate (coenzyme A or AMP ester) to form 3,4-dihydroxybenzoyl spermidinyl citrate (3,4-DHB-SPD-CT), followed by the condensation of a second 3,4-DHB-SPD subunit with 3,4-DHB-SPD-CT (again activated as coenzyme A or AMP ester) to form the mature siderophore. This scheme is analogous to the assembly of aerobactin and a number of other siderophores, including rhizobactin 1021, achromobactin, vibrioferrin, alcaligin, and desferrioxamine E, that have similarly annotated genes, forming a family of 40 NRPS-independent siderophore biosynthetic systems (6
FIG. 2. Proposed biosynthetic scheme of petrobactinBa. Spermidine and 3,4-dihydroxybenzoic acid are condensed by a combination of AsbC, -D, -E, and -F, although other factors could be involved. The product of this reaction, 3,4-dihydroxybenzoyl spermidine, is (more ...)
In order to test this hypothesis, in-frame deletion mutants were generated for each of the six genes of the asb operon (see Materials and Methods), as well as a mutant with a deletion that encompassed the entire operon (Fig. ). The B. anthracis Sterne wild type and each of the mutants were grown under iron-depleted conditions, and the cells were removed by filtration. In order to identify accumulated intermediates from the petrobactinBa pathway, the filtrate and cell lysate obtained from each culture were prepared for HPLC, LCMS, and mass spectrometry analysis (see Material and Methods). As anticipated, analysis of the B. anthracis wild-type filtrate provided a single symmetrical HPLC peak corresponding to petrobactinBa (Fig. ) with the expected molecular mass [m/z 719.3, (M+H)+]. Interestingly, inspection of the HPLC chromatograms for the ΔasbA, ΔasbB, and ΔasbAB mutants revealed that all three contained a new coincident product that was not observed in the wild type. Mass spectrometry showed that the metabolites had identical molecular masses[m/z 282.1, (M+H)+] compared to an authentic standard of 3,4-DHB-SPD. In addition to the peak corresponding to 3,4-DHB-SPD, a single asymmetric peak yielding typical UV spectra for 3,4-DHB was observed from the filtrate of the ΔasbB mutant (Fig. ). HPLC purification of this metabolite and MS analysis indicated [molecular mass m/z 456.1, (M+H)+] that it corresponded to the more advanced biosynthetic intermediate 3,4-DHB-SPD-CT. None of the filtrates from the other mutants (ΔasbC, ΔasbD, ΔasbE, ΔasbF, and ΔasbABCDEF mutants) exhibited signals corresponding to either of these precursor metabolites.
Schematic of the various mutants of the asbABCDEF operon. More details for each mutant can be found in Materials and Methods.
FIG. 4. HPLC profiles of filtrates from B. anthracis wild-type Sterne 34F2 and asb mutants. PetrobactinBa (1) was isolated from both the wild type and the ΔasbA mutant. The two isomers of 3,4-dihydroxybenzoyl spermidine (2 and 2′; the structures (more ...)
Interestingly, close inspection of the metabolite analysis for each of the mutant strains revealed that a minor product with the same molecular mass as petrobactinBa [m/z 719.3, (M+H)+] (Fig. ) was generated from the ΔasbA mutant. Production of petrobactinBa in a strain lacking AsbA was surprising. However, it is conceivable that AsbB (or another unknown enzyme) shares enough functional similarity to AsbA to provide partial catalysis. To test this hypothesis, a mutant lacking both AsbA and AsbB (ΔasbAB) was generated, and a siderophore metabolite analysis was performed. No apparent peak corresponding to petrobactinBa was observed for filtrates from the ΔasbAB double mutant strain (Fig. ). Moreover, analysis of cell lysates from the wild type or the ΔasbA mutant strain by LCMS revealed that no petrobactinBa or corresponding biosynthetic intermediates accumulated (data not shown). Further studies confirmed that neither 3,4-DHB-SPD nor 3,4-DHB-SPD-CT could be detected for the cell lysates of any asb mutant. These data demonstrate that petrobactinBa and its biosynthetic intermediates 3,4-DHB-SPD and 3,4-DHB-SPD-CT are effectively excreted from B. anthracis cells.
In order to obtain higher-resolution structural information on the petrobactin biosynthetic intermediates, ESI FTICR MS of HPLC-purified molecules was conducted. Analysis of 3,4-DHB-SPD and 3,4-DHB-SPD-CT from the B. anthracis ΔasbB mutant showed abundant ion peaks at m/z 282.1811 (for 3,4-DHB-SPD) and 456.1976 (for 3,4-DHB-SPD-CT), respectively, which were then assigned as singly protonated ions, indicating the molecular formula C14H23N3O3 (for 3,4-DHB-SPD) or C20H29N3O9 (for 3,4-DHB-SPD-CT) (data not shown). A similar abundant ion peak at m/z 282.1811, corresponding to 3,4-DHB-SPD, was obtained from extracts of ΔasbA and ΔasbAB (data not shown).
To obtain more-detailed fragmentation patterns and structural information on 3,4-DHB-SPD and 3,4-DHB-SPD-CT, they were subjected to SORI-CAD MS/MS. For singly protonated HPLC-purified 3,4-DHB-SPD (Fig. ), major fragment ions were detected at m/z 109.03, 137.02, 194.08, 208.10, 225.12, and 265.16. The fragment ions at m/z 109.03, 137.02, 194.08, and 265.16 could be assigned to the cleavage of one terminal C—C(O) bond, one amide bond, and two amine bonds in 3,4-DHB-SPD (Fig. ). From this pattern, 3,4-DHB is added to the 3-carbon end of spermidine, whereas the fragment ions at m/z 208.10 and 225.12 are unlikely to result from that structure. However, the latter two ions can be produced from an isomeric structure in which 3,4-DHB is added to the 4-carbon end of spermidine (Fig. ). These results suggest that 3,4-DHB-SPD (Fig. ) and its isomeric counterpart (Fig. ) are synthesized in the ΔasbA, ΔasbB, and ΔasbAB mutants as the biosynthetic intermediates.
FIG. 5. FTICR MS/MS of 3,4-DHB-SPD and 3,4-DHB-SPD-CT isolated from the B. anthracis ΔasbB mutant and petrobactinBa from the wild type. A, SORI-CAD mass spectrum from 3,4-DHB-SPD; B, SORI-CAD mass spectrum of 3,4-DHB-SPD-CT; C and D, assignments of selected (more ...)
In SORI-CAD MS/MS evaluation of singly protonated HPLC-purified 3,4-DHB-SPD-CT, major fragment ions at m/z
109.03, 137.02, 194.08, 208.10, 211.11, 265.16, and 282.18 (Fig. ) were detected. The fragments at m/z
109.03, 137.02, 194.08, 211.11, 265.16, and 282.18 can be assigned to cleavage of one terminal C—C(O) bond, two amide bonds, and three amine bonds in 3,4-DHB-SPD (Fig. ), in which 3,4-DHB is attached to the 3-carbon end of spermidine. However, the fragment at m/z
208.10 is unlikely to result from that structure, but it can be formed directly from the isomeric structure of 3,4-DHB-SPD-CT (Fig. ), in which 3,4-DHB is added to the 4-carbon end of spermidine. This result indicates that the B. anthracis
mutant strain synthesizes not only 3,4-DHB-SPD-CT (Fig. ) but also its chemical isomer (Fig. ), as unexpectedly observed in the biosynthesis of two isomeric forms of 3,4-DHB-SP in ΔasbA
, and ΔasbAB
cultures. Significantly, both isomeric forms of 3,4-DHB-SP were observed by direct biochemical conversion to the products (28a
Addition of selected exogenous siderophores restores growth to asb mutants in iron-depleted medium.
While the characterized asb
) had been shown previously to have a growth defect in liquid IDM and failed to produce petrobactin (5
), it remained unclear whether exogenous petrobactin added back to IDM would restore growth. To address this question, exogenous petrobactinBa
(2 μM) was provided, resulting in nearly complete restoration of the mutant strain to wild-type growth levels (Fig. ). These studies were extended by testing a series of heterologous siderophores against the Δasb
strain in IDM to establish their ability to restore growth following addition of petrobactinBa
, the hydroxamate siderophore aerobactin, and the catecholate siderophore salmochelin S4 (Fig. ). Of the four exogenously added siderophores, only salmochelin S4 failed to assist the growth of the Δasb
mutant, with cultures showing the same poor growth kinetics as cultures with no exogenously added siderophore. As expected, petrobactinBa
mutant growth in IDM to virtually equivalent levels. Interestingly, the hydroxamate siderophore aerobactin was also able to restore growth of the Δasb
mutant. This suggests that B. anthracis
, as shown previously with other bacteria (22
), maintains the ability to utilize select chemically distinct heterologous siderophores to meet its iron requirements.
FIG. 6. A. Growth of the Δasb::kmr mutant in IDM with or without the addition of purified petrobactinBa. PetrobactinBa (2 μM) alleviated the growth defect of the Δasb::kmr mutant strain that was unable to produce this siderophore. B. Growth (more ...)
The growth curves in liquid broth described above were obtained by inoculating media with actively growing vegetative cultures of wild-type and Δasb::kmr strains. However, since the dormant spore form of B. anthracis is the actual infectious particle, we assessed the ability of purified petrobactinBa to facilitate outgrowth of newly germinated spores in extreme iron-poor conditions. Accordingly, we employed assays in which sterile paper discs infused with the test molecule (siderophore) were placed onto a solid medium limited for iron onto which either vegetative cells or spores had been spread.
Table compares the results of the ability of spores and vegetative bacilli of wild-type and Δasb::kmr mutant strains to grow on solid medium containing the iron-chelating agent 2,2′-dipyridyl. For these assays, control experiments were conducted that demonstrated wild-type-level germination under all conditions used with no impact on growth due to the test medium (data not shown) containing an iron-chelating agent. Exponentially growing vegetative bacilli of the B. anthracis wild type were able to grow as a light lawn on plates containing 0.5 mM 2,2′-dipyridyl, whereas vegetative bacilli of the Δasb::kmr mutant strain were not able to propagate. Growth of Δasb::kmr vegetative cells was restored with exogenously added petrobactinBa or Fe(II)SO4 (not shown), suggesting that wild-type cells that fail to produce petrobactinBa are unable to overcome the severe iron limitation of this medium.
Growth enhancement of Bacillus anthracis by addition of petrobactinBa to 0.5 mM 2, 2′-dipyridyl medium using disc diffusion assaysa
In contrast, spores of the B. anthracis wild type were not able to outgrow on these plates without the addition of exogenous petrobactinBa or an alternative iron source. Exogenous petrobactinBa enabled outgrowth of spores of both strains, but the wild-type strain exhibited more vigorous growth in a larger zone than the Δasb::kmr mutant. Thus, it appeared that wild-type spores, once they are able to overcome a severely iron-limited growth environment, were able to produce endogenous petrobactinBa for robust growth. In contrast, the Δasb::kmr mutant (both spores and vegetative bacilli) overcame the iron limitation to equivalent levels when exogenous petrobactinBa was provided, but then, unable to produce petrobactinBa, the cells failed to grow further.