Alternative spermidine biosynthetic route is critical for growth of Campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota

Abstract

The availability of fully sequenced bacterial genomes has revealed that many species known to synthesize the polyamine spermidine lack the spermidine biosynthetic enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We found that such species possess orthologues of the sym-norspermidine biosynthetic enzymes carboxynorspermidine dehydrogenase and carboxynorspermidine decarboxylase. By deleting these genes in the food-borne pathogen Campylobacter jejuni, we found that the carboxynorspermidine decarboxylase orthologue is responsible for synthesizing spermidine and not sym-norspermidine in vivo. In polyamine auxotrophic gene deletion strains of C. jejuni, growth is highly compromised but can be restored by exogenous sym-homospermidine and to a lesser extent by sym-norspermidine. The alternative spermidine biosynthetic pathway is present in many bacterial phyla and is the dominant spermidine route in the human gut, stomach, and oral microbiomes, and it appears to have supplanted the S-adenosylmethionine decarboxylase/spermidine synthase pathway in the gut microbiota. Approximately half of the gut Firmicutes species appear to be polyamine auxotrophs, but all encode the potABCD spermidine/putrescine transporter. Orthologues encoding carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase are found clustered with an array of diverse putrescine biosynthetic genes in different bacterial genomes, consistent with a role in spermidine, rather than sym-norspermidine biosynthesis. Due to the pervasiveness of ε-proteobacteria in deep sea hydrothermal vents and to the ubiquity of the alternative spermidine biosynthetic pathway in that phylum, the carboxyspermidine route is also dominant in deep sea hydrothermal vents. The carboxyspermidine pathway for polyamine biosynthesis is found in diverse human pathogens, and this alternative spermidine biosynthetic route presents an attractive target for developing novel antimicrobial compounds.

FIGURE 1.

Bacterial polyamines and decarboxylated S-adenosylmethionine pathway for spermidine biosynthesis. A, aminopropyl groups are shown in red; aminobutyl groups are shown in blue. B, biosynthesis of spermidine from putrescine by transfer of an aminopropyl group from decarboxylated S-adenosylmethionine.

SCHEME 1.

Synthetic strategy for sym-homospermidine.

FIGURE 2.

Spermidine biosynthesis in C. jejuni. A, HPLC of polyamines from C. jejuni wild-type (81116) cells grown in polyamine-deficient medium. Polyamines were extracted from log phase (A_{600 nm} 0.2 to 0.3) and stationary phase (A_{600 nm} 0.9 to 1.0) cells. R, fluorescent labeling dye; Spd, spermidine; IS, internal standard (1,7-diaminoheptane). B, putative pathway for spermidine biosynthesis in C. jejuni. Candidate C. jejuni open reading frames encoding pathway enzymes are indicated. ADC, arginine decarboxylase; AIH, agmatine deiminase/iminohydrolase; NCPAH, N-carbamoylputrescine amidohydrolase; CASDH, carboxyspermidine dehydrogenase; CASDC, carboxyspermidine decarboxylase. The aminopropyl group transfer is shown in blue.

FIGURE 3.

HPLCs of polyamines from C. jejuni gene deletion mutants grown in polyamine-deficient medium. Cells were grown as described under “Experimental Procedures.” R, fluorescent labeling dye; Spd, spermidine; IS, internal standard (1,7-diaminoheptane).

FIGURE 4.

Cell growth in C. jejuni deletion mutants. Cells were grown in polyamine-deficient medium ± polyamines as described under “Experimental Procedures.” Data represent the means of triplicate cultures ± standard deviation. A, cell growth for wild-type (81116), ΔC8J_0715 (ADC), ΔC8J_0166 (CASDH) and ΔC8J_1418 (CASDC) deletion strains, and the genetically complemented strain c_ΔC8J_1418. a, wild-type parental strain; b, ΔC8J_1418 (CASDC); c, ΔC8J_1418 (CASDC) plus spermidine (Spd); d, ΔC8J_0715 (ADC); e, ΔC8J_0715 (ADC) plus agmatine (Agm); f, ΔC8J_0715 (ADC) plus spermidine; g, ΔC8J_0166 (CASDH); h, genetically complemented ΔC8J_1418 (CASDC), i.e. (c_ΔC8J_1418). Where added to the medium, spermidine and agmatine were at 500 μm final concentration. B, HPLCs of C. jejuni gene deletion strain ΔC8J_1418 (CASDC) and the same strain genetically complemented by expressing a chromosomally located copy of the CASDC-encoding ORF (c_ΔC8J_1418). C, a, C. jejuni 81116 wild-type parental strain; b, ΔC8J_0890 (NCPAH); c, ΔC8J_0890 plus 500 μm spermidine (Spd); d, ΔC8J_0892 (AIH); e, ΔC8J_0892 plus 500 μm spermidine. D, a, C. jejuni 81116 wild-type parental strain; b, ΔC8J_1418 (CASDC); c, ΔC8J_1418 plus 500 μm spermidine; d, ΔC8J_1418 plus 500 μm sym-norspermidine; e, ΔC8J_1418 plus 500 μm sym-homospermidine.

FIGURE 5.

Growth restoration of C. jejuni polyamine auxotrophic strains by exogenous polyamines. Cells were grown in polyamine-deficient medium ± polyamines as described under “Experimental Procedures.” A, dose response of cell growth in ΔC8J_1418 (CASDC) gene deletion mutant in polyamine-deficient medium supplemented with spermidine or sym-norspermidine. a, C. jejuni wild-type parental strain (81116); b, ΔC8J_1418 (CASDC); c, ΔC8J_1418 plus 500 μm spermidine; d, ΔC8J_1418 plus 750 μm spermidine; e, ΔC8J_1418 1000 μm spermidine; f, ΔC8J_1418 plus 500 μm sym-norspermidine; g, ΔC8J_1418 plus 750 μm sym-norspermidine; h, ΔC8J_1418 plus 1000 μm sym-norspermidine. B, dose response of cell growth in ΔC8J_0715 (ADC) gene deletion mutant in polyamine-deficient medium supplemented with polyamines. a, C. jejuni wild-type parental strain (81116); b, ΔC8J_0715; c, ΔC8J_0715 plus 500 μm agmatine; d, ΔC8J_0715 plus 500 μm putrescine; e, ΔC8J_0715 plus 500 μm cadaverine; f, ΔC8J_0715 plus 500 μm 1,3-diaminopropane; g, ΔC8J_0715 plus 500 μm spermidine; h, ΔC8J_0715 plus 500 μm sym-norspermidine.

FIGURE 6.

Polyamine-related gene clusters containing CASDH and CASDC orthologues in bacterial genomes. Protein accession numbers are shown below the first and last ORFs. Bacterial phyla are listed in parentheses. AUH, agmatine ureohydrolase; AAT, aspartate aminotransferase-fold; AR, alanine racemase-fold; hypo, hypothetical protein.

FIGURE 7.

Spermidine biosynthetic pathways in the human gut microbiota. Protein accession numbers are given for CASDH, CASDC, AdoMetDC, and SpdSyn ORFs in the 55 most abundant, ubiquitous bacterial species in the human gut (21). Firmicutes species (F) are shown in pink and Bacteroidetes species in green (B). Absent ORFs are not colored.

FIGURE 8.

Polyamine biosynthetic pathway configurations in human gut microbiota species. aAR-ADC, ancestral alanine racemase-fold biosynthetic arginine decarboxylase (23); AR-ADC, alanine racemase-fold biosynthetic arginine decarboxylase (23); biosynthetic aspartate aminotransferase-fold arginine decarboxylase (23); AUH, agmatine ureohydrolase.