Analysis of achromobactin biosynthesis by Pseudomonas syringae pv. syringae B728a

Abstract

Pseudomonas syringae pv. syringae B728a is known to produce the siderophore pyoverdine under iron-limited conditions. It has also been proposed that this pathovar has the ability to produce a second siderophore, achromobactin. Here we present genetic and biochemical evidence supporting the hypothesis that P. syringae pv. syringae B728a produces both of these siderophores. We show that strains unable to synthesize either pyoverdine or achromobactin are unable to grow under iron-limiting conditions, which is consistent with these two molecules being the only siderophores synthesized by P. syringae pv. syringae B728a. Enzymes associated with achromobactin biosynthesis were purified and analyzed for substrate recognition. We showed that AcsD, AcsA, and AcsC together are able to condense citrate, ethanolamine, 2,4-diaminobutyrate, and alpha-ketoglutarate into achromobactin. Replacement of ethanolamine with ethylene diamine or 1,3-diaminopropane in these reactions resulted in the formation of achromobactin analogs that were biologically active. This work provides insights into the biosynthetic steps in the formation of achromobactin and is the first in vitro reconstitution of achromobactin biosynthesis.

FIG. 1.

Chemical structure of the siderophore ACR. Portions of the siderophore structure that derive from incorporation of specific precursors are shown. The ethanolamine moiety may be derived directly from ethanolamine or indirectly via decarboxylation of l-serine after condensation with citrate (41).

FIG. 2.

Proposed scheme for the biosynthesis of ACR. The preferential use of l-serine by AcsD is based on recent kinetic analyses of AcsD from D. dadantii (41). However, the initial biosynthesis proposal suggested that l-serine was converted to ethanolamine prior to AcsD activity (5). As shown in this study, AcsD is also able to use ethanolamine as a cosubstrate with citrate (1). The following abbreviations are used: DAB, 2,4-diaminobutyrate; αKG, α-ketoglutarate; PLP, pyridoxal phosphate.

FIG. 3.

Growth of siderophore-deficient B728a strains. (A) Growth in MM. (B) Growth in MM containing 2,2′-dipyridyl. Symbols: closed squares, B728a; open circles, strain ADB1005 (PVD⁻); open squares, strain ADB1006 (ACR⁻); X's, strain ADB1007 (PVD⁻ACR⁻). Trendlines are drawn according to data collected hourly. Data points are shown in 2-h (A) and 4-h (B) intervals for visual clarity.

FIG. 4.

ESI-MS spectra of siderophore purified from PVD-deficient B728a. The observed mass ions are consistent with ACR ([M-H]⁻¹ = 590.14; [M-2H]⁻² = 294.57). amu, atomic mass unit.

FIG. 5.

(A) Schematic representation of the Psyr2580-2595 region of the P. syringae pv. syringae B728a chromosome. Proposed gene nomenclature is shown above the respected ORFs, followed by a four-digit Psyr genome locus tag. (B) Schematic representation of the region of the D. dadantii 3937 chromosome associated with ACR production and transport. Gene nomenclature used is as outlined in Franza et al. (14) and is listed above the respective ORFs. Coding regions involved in ACR biosynthesis and utilization are shown with open arrows.

FIG. 6.

NuPAGE (4 to 15%) Coomassie blue-stained protein gel of N-terminal hexahistidine-tagged AcsA, AcsC, and AcsD. Approximately 4 μg of protein was loaded in each lane. Calculated molecular masses for tagged proteins are as follows: AcsA, 73.9 kDa; AcsC, 72.6 kDa; AcsD, 69.3 kDa.

FIG. 7.

Substrate specificity determination of Acs enzymes. Enzymes were incubated with hydroxylamine, Mg²⁺-ATP, and carboxylic acid substrates and assayed for production of hydroxamates. (A) Results for reaction mixture containing citrate. (B) Results for reaction mixture containing α-ketoglutarate. (C) Results for reaction mixture containing reaction mixture of AcsD incubated with citrate and ethanolamine (left) or with citrate and 2,4-diaminobutyrate (right). mAbs₅₄₀, absorbance at 540 nm (10³).

FIG. 8.

Summary of LC-MS data. “A,” “C,” and “D” represent AcsA, AcsC, and AcsD, respectively. A plus sign (+) indicates that the corresponding enzyme was included in the incubation. (A) MS spectra focused on mass range for detection of [3] (calc. [M-H]⁻¹ = 234.06). (B) MS spectra focused on mass range for detection of [4] (calc. [M-H]⁻¹ = 334.13). (C) MS spectra focused on mass range for detection of [5] or [6] (calc. [M-H]⁻¹ = 462.14). (D) MS spectra focused on mass range for detection of ACR (calc. [M-H]⁻¹ = 590.14). MS data were collected from LC separation at 0.74 min, 0.65 min, 1.85 min, and 4.70 min, respectively, which correspond to the optimal extracted ion signal for the desired mass ion. Each data set was normalized to the intensity of the most-prevalent mass ion present. amu, atomic mass unit.

FIG. 9.

Summary of LC-MS analysis of reactions where l-serine has replaced ethanolamine as a substrate. The AcsA-catalyzed steps and the associated LC-MS mass ion spectra are shown. A mass ion of 7 was also detected (data not shown). The MS spectra for the range of m/z appropriate for the detection of α-ketoglutaryl-diaminobutyryl-citryl-serine and carboxylated ACR are shown. amu, atomic mass unit.