Escherichia coli possessing the dihydroxyacetone phosphate shunt utilize 5'-deoxynucleosides for growth

Abstract

All organisms utilize S-adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as S-adenosyl-l-homocysteine, 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). A prevalent pathway found in bacteria for the metabolism of MTA and 5dAdo is the dihydroxyacetone phosphate (DHAP) shunt, which converts these compounds into dihydroxyacetone phosphate and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work in other organisms has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Rather, the DHAP shunt in Escherichia coli ATCC 25922, when introduced into E. coli K-12, enables the use of 5dAdo and MTA as a carbon source for growth. When MTA is the substrate, the sulfur component is not significantly recycled back to methionine but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. The DHAP shunt in ATCC 25922 is active under oxic and anoxic conditions. Growth using 5-deoxy-d-ribose was observed during aerobic respiration and anaerobic respiration with Trimethylamine N-oxide (TMAO), but not during fermentation or respiration with nitrate. This suggests the DHAP shunt may only be relevant for extraintestinal pathogenic E. coli lineages with the DHAP shunt that inhabit oxic or TMAO-rich extraintestinal environments. This reveals a heretofore overlooked role of the DHAP shunt in carbon and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt.

Importance: The acquisition and utilization of organic compounds that serve as growth substrates are essential for Escherichia coli to grow and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-l-methionine as a co-substrate by all organisms result in the formation of the 5'-deoxy-nucleoside byproducts, 5'-methylthioadenosine and 5'-deoxyadenosine. All E. coli possess a conserved nucleosidase that cleaves these 5'-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway is found in some extraintestinal pathogenic E. coli, but its function in E. coli possessing it has remained unknown. This study reveals that the DHAP shunt enables the utilization of 5'-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates in E. coli strains with the pathway during aerobic respiration and anaerobic respiration with TMAO, but not fermentative growth. This provides an insight into the diversity of sugar compounds accessible by E. coli with the DHAP shunt and suggests that the DHAP shunt is primarily relevant in oxic or TMAO-rich extraintestinal environments.

Keywords: carbon metabolism; extraintestinal pathogenic E. coli; nucleotide metabolism; sulfur.

Fig 1

Salvage of S-adenosyl-l-methionine (SAM) utilization byproducts, 5-deoxy-pentose sugars, and 6-deoxy-hexose sugars in E. coli. (A) S-adenosyl-l-homocysteine (SAH), produced by methyltransferases, is recycled by the methionine cycle (a.k.a. active methyl cycle) (13, 14). (B and C) The E. coli variation of the DHAP shunt. (B) For adenine salvage and detoxification of 5′-methylthioadenosine (MTA) and 5′-deoxyadenosine (5dAdo), all E. coli possess the multifunctional SAH/MTA/5dAdo nucleosidase (Pfs; a.k.a. MtnN) (15). (C) ExPEC strains possess the Pfs nucleosidase (B) and remainder of the DHAP shunt genes, which together compose a dual-purpose pathway for the conversion of 5′-deoxy-nucleosides in the form of MTA and 5dAdo or corresponding 5-deoxy-pentoses in the form of 5-deoxy-d-ribose and 5-methylthioribose, respectively, into the central carbon metabolite DHAP and acetaldehyde or (2-methylthio)acetaldehyde (11). (D) E. coli analogously metabolize 6-deoxy-hexose sugars in the form of l-fucose and l-rhamnose to DHAP and l-lactaldehyde. During anaerobic growth, l-lactaldehyde is primarily reduced to (S)-1,2-propanediol as a terminal product, whereas during aerobic growth, it is primarily oxidized to l-lactate for carbon assimilation as pyruvate. Enzymes: Pfs (MtnN), SAH/MTA/5dAdo nucleosidase, E.C. 3.2.2.9; LuxS, S-ribosyl-l-homocysteine lyase, E.C. 4.4.1.21; MetH, methionine synthase, E.C. 2.1.1.13; MetE, methionine synthase, E.C. 2.1.1.4; MtnK, 5-methylthioribose/5-deoxyribose kinase, E.C. 2.7.1.100; MtnA, 5-methylthioribose-1-phosphate/5-deoxyribose-1-phosphate isomerase, E.C. 5.3.1.23; Ald2, 5-methylthioribulose-1-phosphate/5-deoxyribulose-1-phosphate aldolase, E.C. 4.1.2.62; ADH, alcohol dehydrogenase, E.C. 1.1.1.1 ; FucI, l-fucose isomerase, E.C. 5.3.1.25; FucK, l-fuculose kinase, E.C. 2.7.1.51; FucA, l-fuculose-1-phosphate aldolase, E.C. 4.1.2.17; RhaA, l-rhamnose isomerase, E.C. 5.3.1.14; RhaB, l-rhamnulose kinase, E.C. 2.7.1.5; RhaD, l-rhamnulose-1-phosphate aldolase, E.C. 4.1.2.19; FucO, (S)−1,2-propanediol oxidoreductase, E.C. 1.1.1.77; and AldA, l-lactaldehyde dehydrogenase, E.C. 1.2.1.22.

Fig 2

Sulfur from MTA cannot be salvaged by E. coli for growth. (A) ATCC 25922 maximum growth achieved after 24 hours measured by optical density at 600 nm when cultured with 1 mM of the indicated sulfur compound as the sole sulfur source. No further growth was observed after 24 hours. Average and standard deviation error bars are for n = 3 independent replicates. (B) Fold difference in the abundance of DHAP shunt-associated metabolites when ATCC 25922 was grown aerobically in the presence of 1 mM sulfate and 1 mM MTA versus grown aerobically in the presence of 1 mM sulfate only. Metabolites were resolved by LC-MS/MS from three independent biological replicates for each growth condition. Values are the average for the three replicates, and the significance of fold change was analyzed by ANOVA with P < 0.05. (C) Reverse-phase HPLC quantification of E. coli ATCC 25922 metabolites when fed aerobically with [¹⁴C-methyl]−5′-methylthioadenosine. Unk, unknown (identified as 2-methylsulfinylethanol by LC-MS/MS, m/z = 109.0319), R_T = 6.7 min; Met, methionine, R_T = 8.0 min; MTR, methylthioribose, R_T = 16.7 min; MT-EtOH, 2-methylthioethanol, R_T = 21.7 min; IS, internal standard, R_T = 24.0 min; and MTA, 5′-methylthioadenosine, R_T = 31.8 min.

Fig 3

Prevention of growth inhibition by SAM utilization byproducts requires Pfs but not the DHAP shunt. Final culture density after 18 hours of the ATCC 25922 wild-type, Δpfs, and ΔK2 (ΔmtnK ΔmtnA Δald2) strains and the K-12 BW25113 wild-type and Δpfs deletion strains grown aerobically with glucose and in the presence of the indicated concentration of either (A) 5′-methylthioadenosine, (B) 5′-deoxyadenosine, or (C) 5-deoxy-d-ribose. Averages and standard deviation error bars are for n = 3 independent replicates. Curves are the nonlinear least squares weighted fit to the Hill equation. (D) ID₅₀ for MTA and 5dAdo in ExPEC ATCC 25922 and commensal K-12 BW25113 strains. ID₅₀ values are from the fit curves in panels A and B to the Hill equation, and the error bars are the parameter 95% confidence interval of the weighted fit. Statistically significant difference, *P < 0.05.

Fig 4

Utilization of 5′-deoxy-nucleosides and 5-deoxy-pentoses for carbon and energy metabolism by E. coli with the DHAP shunt. (A) Growth of either wild-type ATCC 25922, ATCC 25922 ΔK2 (ΔmtnK ΔmtnA Δald2), ΔK2 + pEV (pTETTET empty vector), or ΔK2 + pK2 (pTETTET vector with DHAP shunt mtnK, mtnA, and ald2) on either 25 mM glucose (Glc) or 5 mM 5-deoxy-d-ribose as the carbon source and 50 ng/mL tetracycline. (B–G) Growth of ATCC 25922, ATCC 25922 Δpfs, ATCC 25922 ΔK2, K-12 strain BW25113, or BW25113 Δpfs on 1 mM of (B) glucose, (C) 5dAdo, (F) MTA, (G) or no carbon source. Coordinately, deletion strains were complemented with either an empty control plasmid (pEV), a plasmid containing the mtnK, mtnA, and ald2 genes (pK2), or the pfs gene (pPfs) and grown on 1 mM of (D) glucose or (E) 5dAdo. A total of 75 ng/mL of tetracycline was added for gene expression from the plasmid tetracycline-inducible promoter (D and E).

Fig 5

The ExPEC DHAP shunt is an efficient carbon acquisition pathway for aerobic and microaerophilic conditions. (A) ATCC 25922 growth efficiency in the presence of 5 mM growth substrate measured as dry cell weight of cells generated per gram of substrate under aerobic respiration, anaerobic respiration with 100 mM TMAO, and under anaerobic fermentation growth conditions. EtOH, ethanol; OAc, acetate; adh, acetaldehyde. (B) Ion exclusion HPLC quantification of ³H-labeled metabolites produced upon feeding E. coli ATCC 25922 with [³H-methyl]−5′-deoxyadenosine under anaerobic conditions. After feeding, metabolism was quenched by rapid freezing in liquid nitrogen at the indicated time in minutes. (C and D) ATCC 25922 wild-type and DHAP shunt deletion (ΔK2) strains were grown under varying oxygen concentrations with either (C) 25 mM glucose (Glc) or (D) 5 mM 5-deoxy-d-ribose (5dR) as the sole carbon source. In addition, experiments were performed with no carbon source (NC). In each experiment, 40 mM nitrate was also included, which was shown not to support anaerobic respiration (Fig. S5A). Final optical density measurements at 600 nm (OD₆₀₀) were taken after 24 hours of growth. Averages and standard deviation error bars in panels A, C, and D are for n = 3 independent replicates.

Fig 6

Complementation of K-12 with DHAP shunt genes allows for growth using 5-deoxy-d-ribose as a carbon source. Growth of K-12 strain BW25113 complemented with pEV (pTETTET empty vector) and pK2 (pTETTET with DHAP shunt) using either 5 mM glucose (Glc) or 5 mM 5-deoxy-d-ribose as the carbon source, or no carbon source, and 50 ng/mL tetracycline. Average and standard deviation error bars are for n = 3 independent replicates.