The STM4195 gene product (PanS) transports coenzyme A precursors in Salmonella enterica

Abstract

Coenzyme A (CoA) is a ubiquitous coenzyme involved in fundamental metabolic processes. CoA is synthesized from pantothenic acid by a pathway that is largely conserved among bacteria and eukaryotes and consists of five enzymatic steps. While higher organisms, including humans, must scavenge pantothenate from the environment, most bacteria and plants are capable of de novo pantothenate biosynthesis. In Salmonella enterica, precursors to pantothenate can be salvaged, but subsequent intermediates are not transported due to their phosphorylated state, and thus the pathway from pantothenate to CoA is considered essential. Genetic analyses identified the STM4195 gene product of Salmonella enterica serovar Typhimurium as a transporter of pantothenate precursors, ketopantoate and pantoate and, to a lesser extent, pantothenate. Further results indicated that STM4195 transports a product of CoA degradation that serves as a precursor to CoA and enters the biosynthetic pathway between PanC and CoaBC (dfp). The relevant CoA derivative is distinguishable from pantothenate, pantetheine, and pantethine and has spectral properties indicating the adenine moiety of CoA is intact. Taken together, the results presented here provide evidence of a transport mechanism for the uptake of ketopantoate, pantoate, and pantothenate and demonstrate a role for STM4195 in the salvage of a CoA derivative of unknown structure. The STM4195 gene is renamed panS to reflect participation in pantothenate salvage that was uncovered herein.

Importance: This manuscript describes a transporter for two pantothenate precursors in addition to the existence and transport of a salvageable coenzyme A (CoA) derivative. Specifically, these studies defined a function for an STM protein in S. enterica that was distinct from the annotated role and led to its designation as PanS (pantothenate salvage). The presence of a salvageable CoA derivative and a transporter for it suggests the possibility that this compound is present in the environment and may serve a role in CoA synthesis for some organisms. As such, this work raises important question about CoA salvage that can be pursued with future studies in bacteria and other organisms.

FIG 1

Pantothenate and CoA biosynthesis in Salmonella enterica. Known biosynthetic enzymes, salvage enzymes, and metabolic intermediates involved in CoA biosynthesis and salvage are depicted.

FIG 2

The STM4195p1 allele restores growth to a panE ilvC strain. Cultures were grown in glucose minimal medium containing BCAA with no additional supplements (A), vitamin-free CAA (0.2%) (B), or pantothenate (100 μM) (C). Wild-type LT2 (Wt), panE ilvC (DM6486), and panE ilvC STM4195p1 (DM13708) strains are shown in each panel. The data displayed are representative of three independent cultures.

FIG 3

Mutations in the chromosome upstream of STM4195 restored growth to a panE ilvC strain when grown in the presence of CAA. (A) The open reading frame organization in the relevant genomic region is depicted. (B) The sequence upstream of the STM4195 coding region has been expanded. The predicted −10/−35 promoter regions are underlined. Lowercase letters depict the regulatory binding site for the small RNA repressor, GcvB (41). Allele STM4195p1 contains an IS10-R insertion, indicated by the inverted triangle, inserted 24 bp upstream of the STM4195 start codon (#). The coding sequence of the transposase gene encoded by IS10-R is indicated, as is the promoter reading out of the IS10-R coding region (pOUT). The double-underlined region indicates the 9-bp insertion site recognition sequence that is found duplicated at both ends of the IS10-R insertion (31). The position of allele STM4195p3 (C to T) is denoted with an asterisk and is in the predicted −10 region, 76 bp upstream of the STM4195 start codon.

FIG 4

Expression of STM4195 in trans suppresses growth defect in the presence of CAA. panE ilvC (squares) strains containing pDM1397 (DM13947; filled) or the vector only control (DM13948; empty) and panC (triangles) strains containing pDM1397 (DM13950; filled) or vector only control (DM13951; empty) were grown in minimal medium containing CAA (0.2% [wt/vol]) and arabinose (1%). Growth was compared relative to that of the wild type (circles). The data displayed are representative of three independent cultures.

FIG 5

Alignment of STM4195 from Salmonella enterica and a homolog from Neisseria meningitidis (ASBT_NM; NMB0705). Asterisks indicate residues from ASBT_NM that coordinate sodium with their side chains (38). Hash symbols indicate residues from ASBT_NM important for binding taurocholate (38). Identical residues and similar residues are highlighted black and gray, respectively.

FIG 6

Expression of STM4195 facilitates ketopantoate and pantoate uptake. (A) Supplements were added at the indicated concentrations to panE ilvC STM4195 strains containing pDM1397 (DM14206) or the empty vector control (DM14207) grown in glucose minimal medium containing BCAA and 1% arabinose. (B) panB STM4195 strains carrying pDM1397 (DM14273) or the empty vector control (DM14274) were examined as described for panel A. The data from three independent cultures are represented as the average and standard deviation of the OD₆₅₀ at 12 h (all cultures reached stationary phase at ∼12 h).

FIG 7

Heated CoA produces substrates of STM4195. A 100 mM solution of CoA dissolved in water was heated at 98°C, and 5-μl aliquots were taken at the indicated time points and spotted on soft-agar overlays of panE ilvC STM4195 pDM1397 (DM14206) (A) and panE ilvC STM4195 pEmpty (DM14207) (B) strains grown on glucose minimal medium containing BCAA and 1% arabinose. Five microliters of pantothenate (Pan; 100 μM) was added as a positive control. Pictures were taken after 48 h of growth at 37°C.

FIG 8

HPLC separation of CoA breakdown products. (A) A 100-μl portion of 100 mM untreated CoA was separated by using an anion-exchange column while monitoring the absorbance at 259 nm. The inset displays the absorption spectrum of pure CoA (eluted at ∼15 min). (B) A 100-μl portion of 100 mM heat-treated CoA (98°C for 5 h) was separated using an anion-exchange column while monitoring the absorbance at 259 nm. Peak fractions were collected and tested for biological activity. The arrow indicates the only peak that displayed biological activity in STM4195-dependent growth assays. (C) The biologically active fraction collected in panel B was further resolved by reversed-phase chromatography while monitoring the absorbance at 259 nm. The arrow indicates the peak that displayed STM4195-dependent biological activity. The inset displays the absorption spectrum of the relevant peak from panel C, with a maximal absorbance at 259 nm.

FIG 9

STM4195-dependent growth requires CoaBC (dfp). STM4195-dependent growth of panC and dfp strains was tested on glucose minimal medium containing BCAA, 1% arabinose, and 1.25 mM heated CoA or 100 μM pantetheine. (A) Growth of panC strains carrying pDM1397 (DM13950; filled symbols) or pEmpty (DM13951; open symbols). (B) Growth of dfp strains carrying pDM1397 (DM14459; filled symbols) or pEmpty (DM14460; open symbols). The growth data are representative of three independent cultures.

FIG 10

Model of STM4195-mediated salvage of a CoA derived product(s). STM4195 facilitates the uptake of a substrate derived from CoA that has spectral properties indicative of an intact adenine moiety. Spontaneous or enzymatic cleavage is required to release a compound that feeds into the pathway upstream of CoaBC.