PanG, a new ketopantoate reductase involved in pantothenate synthesis

Abstract

Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

Fig 1

The biosynthetic pathway in Francisella species and the putative pantothenate operon. (A) The pantothenate biosynthetic pathway consists of two converging arms. PanB, the ketopantoate hydroxymethyltransferase, converts 2-oxoisovalerate with tetrahydrofolate to form 2-dehydropantoate. The substrate 2-dehydropantoate is then converted to (R)-pantoate by a number of enzymes, including IlvC and PanG, which are both capable of ketopantoate reductase (KPR) activity. On the other branch of the pathway, PanD, an aspartate-1-decarboxylase, converts l-aspartic acid to β-alanine. The pathway converges with PanC, the pantothenate synthase that ligates (R)-pantoate with β-alanine to form pantothenate. Molecules were made in PubChem Compound on NCBI, and the pathway was constructed using KEGG metabolic pathway 00770 as a reference (http://www.genome.jp/kegg-bin/show_pathway?FTN_00770). (B) The genomic organization of the putative pantothenate operon is conserved among sequenced Francisella strains containing the panGBCD genes and the pantothenate kinase gene coaX. (C) Synteny diagram of the putative operon in F. novicida, F. tularensis LVS, F. tularensis Schu S4, E. faecalis V583, and C. difficile 630 (35).

Fig 2

F. novicida functional complementation of panB::Tn, panC::Tn, panD::Tn, ilvC::Tn, and panG::Tn. Functional complementation of the pantothenate biosynthetic genes in F. novicida transposon mutant strains grown in 96-well microtiter plates with absorbance (OD₆₀₀) monitored every 15 min in CDM lacking pantothenate (gray) either supplemented with β-alanine (black dotted line), pantolactone (gray dotted line), or calcium pantothenate (black). Shown are growth curves for panB::Tn (A), panC::Tn (B), panD::Tn (C), ilvC::Tn (D), and panG::Tn (E) mutants. (F) Growth of F. novicida U112 and ilvC::Tn in CDM with and without branch chain amino acids: F. novicida U112 in CDM (black), ilvC::Tn in CDM (gray), U112 in CDM without branch chain amino acids (black dotted line), and ilvC::Tn in CDM without branch chain amino acids (gray dotted line). Each growth curve was repeated a minimum of three times, and the graph represents the means of three replicate experiments.

Fig 3

Phylogenetic tree of known ketopantoate reductase proteins and PanG. The phylogenetic tree of known KPR proteins and PanG was generated using Geneious Pro 5.5.6 Tree Builder with the cost matrix set to identity and Jukes-Cantor as the genetic distance model with no outgroups. All IlvC and PanE proteins are annotated on PubMed to be involved in pantothenate synthesis, while all PanG proteins are annotated as hypothetical proteins. Enterococcus faecalis V583 PanG is annotated as hypothetical protein EF1861, Clostridium difficile 630 PanG is annotated as hypothetical protein CD630-15140, Coxiella burnetii RSA 493 PanG is annotated as hypothetical protein CBU_1660, Clostridium botulinum BKT015925 PanG is annotated as hypothetical protein CbC4_0183, and Desulfotomaculum nigrificans DSM 574 PanG is annotated as hypothetical protein DUF2520.

Fig 4

Genetic complementation of the F. novicida ilvC::Tnflp panG::Tn double mutant with F. novicida FTN_1351 panG, F. novicida ilvC, E. faecalis V583 (EF1861) panG, and E. coli panE and genetic complementation of the E. coli ilvC::Flp panE::Kan double mutant with F. novicida panG. Functional complementation experiments were carried out by growing Francisella in CDM lacking pantothenate in 96-well microtiter plates and measuring the absorbance (OD₆₀₀) every 15 min for 30 h. Competent F. novicida isolates were transformed with DNA. (A) pSKI01 carrying F. novicida panG (FTN_1351) driven by its native promoter; (B) pSKI04 carrying ilvC from F. novicida driven by F. tularensis blaB promoter; (C) pEDL71 carrying panG from E. faecalis driven by F. tularensis blaB promoter; (D) pEDL70 carrying panE from E. coli driven by F. tularensis blaB promoter. ilvC::Tn mutant grown in CDM lacking pantothenate containing empty control vector pMP822/pMP831 (black), ilvC::Tnflp panG::Tn double mutant containing an empty control vector (gray), ilvC::Tnflp panG::Tn double mutant containing the respective complementing plasmid (black dotted line), and ilvC::Tnflp panG::Tn double mutant grown in CDM supplemented with pantolactone (gray dotted line). (E) E. coli ilvC::Flp panE::Kan double mutant complemented with pSKI01 carrying F. novicida panG driven by its native promoter. ilvC::Kan mutant grown in M9 medium lacking pantothenate containing an empty control vector (black), ilvC::Flp panE::Kan double mutant containing an empty control vector (gray), ilvC::Flp panE::Kan double mutant containing the respective complementing plasmid (black dotted line), and ilvC::Flp panE::Kan double mutant grown in M9 supplemented with pantolactone (gray dotted line). Each growth curve experiment was repeated three times, and the graph represents the mean of three replicate experiments.

Fig 5

F. novicida CoA levels. CoA concentrations were measured from 50-ml cultures of F. novicida wild-type, ilvC::Tn, panG::Tn, and ilvC::Tnflp panG::Tn strains after 5 h of pantothenate depletion. All strains were grown to the same OD₆₀₀ and were normalized to total protein. The CoA levels represented are the means ± standard deviations (SD) for three independent experiments. Statistical significance was determined by comparing the mutant values to those of wild-type F. novicida. ***, P < 0.0001; **, P < 0.001.

Fig 6

F. tularensis Schu S4, F. tularensis LVS, and F. novicida growth in pantothenate dropout media. Growth curves of F. tularensis subsp. tularensis Schu S4 (black), F. tularensis subsp. holarctica LVS (black dotted line), and F. novicida U112 (gray) were monitored in CDM (A), CDM lacking pantothenate (B), CDM lacking pantothenate supplemented with pantolactone (C), or CDM lacking pantothenate supplemented with β-alanine (D). Each strain was grown in triplicate in 96-well microtiter plates with absorbance (OD₆₀₀) monitored every 15 min over 40 h. Each graph represents the mean of three replicate experiments.

Fig 7

Repair of F. tularensis LVS β-alanine auxotrophy. Growth curves of LVS (gray) and LVS with the native panD_LVS replaced with panD_U112 from F. novicida U112 (black). Each strain was monitored in CDM (A) or CDM lacking pantothenate (B). Both strains were grown in 96-well microtiter plates, and the OD₆₀₀ was determined every 15 min for 24 h. Each graph represents the mean OD₆₀₀ of three replicate experiments.

Fig 8

F. tularensis subsp. tularensis Schu S4 ΔpanG growth and virulence phenotype. (A) Chemical complementation of Schu S4 ΔpanG grown in CDM (black), CDM lacking pantothenate (black dotted line), CDM lacking pantothenate supplemented with β-alanine (gray), and CDM lacking pantothenate supplemented with pantolactone (gray dotted line). (B) Genetic complementation of F. tularensis subsp. tularensis Schu S4 ΔpanG with F. novicida panG expressed by the native promoter in the shuttle vector pMP831. Growth curve of F. tularensis subsp. tularensis Schu S4 (black), F. tularensis subsp. tularensis Schu S4 ΔpanG with pMP831 (empty control vector) (gray), and F. tularensis subsp. tularensis Schu S4 ΔpanG complemented with pSKI01 (black dotted line). All growth curves were done in triplicate, monitoring absorbance (OD₆₀₀) in a 96-well microtiter dish. Graphs represent the mean absorbance at OD₆₀₀. (C to E) Recovery of Schu S4 or Schu S4 ΔpanG mutant in mice following intranasal inoculation. C57BL/6 mice were infected intranasally with either wild-type Schu S4 (black circles) or Schu S4 ΔpanG (gray triangles) at a lethal dose of 50 CFU. Mice were euthanized on days 1 and 3 postinfection, and lung burdens (C), liver burdens (D), and spleen burdens (E) were determined and graphed. Each symbol represents data from a single mouse. There were no significant differences in recovery of mutant versus wild-type organisms from any organ at any time point as determined by the nonparametric Mann-Whitney test.