Evolution and Multiplicity of Arginine Decarboxylases in Polyamine Biosynthesis and Essential Role in Bacillus subtilis Biofilm Formation

Materials

All materials were of the highest grade available and were purchased from Sigma unless otherwise stated. l-[1-¹⁴C]Ornithine hydrochloride (57.1 mCi/mmol), l-[U-¹⁴C]arginine monohydrochloride (346 mCi/mmol), and l-[U-¹⁴C]lysine monohydrochloride (320 mCi/mmol) were purchased from PerkinElmer Life Sciences.

Bacterial Strains and Growth Conditions

E. coli XL2 Blue and BL21 (DE3) pLysS were purchased from Stratagene and Novagen, respectively. B. subtilis 168 was a kind gift from Dr. Stephan Bornemann (John Innes Centre), and B. subtilis genomic DNA was prepared using standard procedures. The original B. subtilis 168 ΔspeA (BSIP 7010) and ΔyaaO knock-out strains were a kind gift of Drs. Agnieszka Sekowska and Antoine Danchin (Institut Pasteur; the ΔyaaO gene knock-out strain was originally made by Dr. Asai Kei). Strains of B. subtilis were grown routinely in Luria-Bertani (39) medium (10 g/liter NaCl, 5 g/liter yeast extract, and 10 g/liter tryptone) at 37 °C, unless otherwise stated. Where appropriate, MSgg medium (5 mm potassium phosphate and 100 mm MOPS at pH 7.0 supplemented with 2 mm MgCl₂, 700 μm CaCl₂, 50 μm MnCl₂, 50 μm FeCl₃, 1 μm ZnCl₂, 2 μm thiamine, 0.5% glycerol, 0.5% glutamate) (41) was used for analysis of biofilm formation. The B. subtilis strains used in this study for biofilm analysis were the strain 3610 (prototroph) obtained from the Bacillus Genetic Resource Center and strain NRS3088 (3610 ΔspeA (spc); created in this study). Phage transductions into the B. subtilis strain background NCIB3610 were conducted as described previously (40). When required, antibiotics were used at the following concentrations: erythromycin (1 μg/ml) with lincomycin (25 μg/ml) and spectinomycin (100 μg/ml). When required, polyamines were added to the MSgg medium at a concentration of 0.5 mm where specified.

Biofilm Image Analysis

Analysis of biofilm formation was performed essentially as described (41, 42). Strains of B. subtilis to be tested were grown to mid-late exponential phase in LB liquid medium inoculated from a freshly streaked LB solid medium plate. Ten μl of the undiluted culture was spotted onto an MSgg plate with 1.5% agar and incubated at 37 °C for the time indicated. For pellicle analysis, 1.5 μl of the same culture was added to 1.5 ml of MSgg medium in a 24-well tissue culture dish, placed at 37 °C for the time period indicated. Images were captured using a Leica MZ16 FA stereoscope using LAS software version 2.7.1.

Cloning and Gene Synthesis

The open reading frame (43) of B. subtilis speA (NP_389346) was amplified by PCR from genomic DNA. Putative speA ORFs from Clostridium difficile 630 (YP_001087362), Chloroflexus auranticus J-10-fl (YP_001634722), and Gramella forsetii KT0803 (YP_863630) were synthesized by Genscript (Piscataway, NJ) with codons optimized for expression in E. coli. The speA ORFs from B. subtilis and C. difficile were subcloned into the BamHI and XhoI sites of pET21a, and those from Chloroflexus aurantiacus and G. forsetti were subcloned into the NdeI and BamHI sites of pET15b.

Protein Expression and Purification

For expression of putative decarboxylases, E. coli BL21 was transformed with protein expression plasmids and grown to an A_{600 nm} of 0.3 in LB liquid medium containing 100 μg/ml ampicillin at 37 °C. Protein expression was induced with 0.4 mm isopropyl 1-thio-β-d-galactopyranoside, and cultures were incubated for a further 3 h at 37 °C. T7-tagged proteins were purified using the T7·Tag affinity purification kit (Novagen) according to the manufacturer's instructions. For purification of His-tagged proteins, cells were resuspended in 20 mm sodium phosphate (pH 8.0) containing 500 mm NaCl, 20 mm imidazole, and 0.02% (v/v) Brij35 before being broken by sonication. The cell lysate was cleared by centrifugation and applied to a HiTrap^TM chelating HP column (GE Healthcare) that had been charged with Ni²⁺ and equilibrated with the above buffer. The column was washed with the above buffer, and bound protein was eluted using a 0.02–1 m imidazole gradient. Purified proteins were concentrated using Amicon Ultra-4 centrifugal filter units, buffer-exchanged with 20 mm Tris·HCl (pH 7.5) containing 20% (v/v) glycerol and 2 mm dithiothreitol and stored at −80 °C.

Polyamine Analysis

For HPLC analysis, polyamines were labeled using the AccQ-Fluor^TM reagent kit (Waters Corp., Milford, MA) according to the manufacturer's instructions. Labeling reactions contained 5 μl of stopped enzyme assay and 1.25 μm 1,7-diaminoheptane as an internal standard and were heated to 55 °C for 10 min. Derivatized polyamine samples were analyzed by HPLC using a reverse phase C18 column (Luna 5 μ, Phenomenex) on a Dionex Summit HPLC System. Polyamine separation was performed using 10 μl of derivatized sample. The system was operated at 33 °C and equilibrated with Eluent A (70 mm acetic acid, 25 mm triethylamine, pH 4.82) at 1.2 ml/min. Elution was performed using the following linear gradients of Eluent B (80% acetonitrile): 22% for 5 min, 39% for 12 min with 6% methanol, 33% for 30 s with 14% methanol, 10% for 6.5 min with 70% methanol, and finally 100% for 21 min. Polyamines were monitored by fluorescence (Dionex RF 2000 detector) with a 248-nm excitation filter and a 398-nm emission filter and identified by comparison of retention times with known standards that were derivatized and analyzed in parallel with the enzyme assays.

Enzyme Assays

Amino acid decarboxylase activity was measured using a stopped ¹⁴CO₂ release assay (44). Unless otherwise stated, assays were buffered in 50 mm HEPES (pH 7.5) containing 50 mm NaCl, 2 mm DTT, and 0.1 mm pyridoxal 5′-phosphate (PLP) and contained 3.7 kBq l-[1-¹⁴C]ornithine hydrochloride, l-[U-¹⁴C]arginine monohydrochloride, or l-[U-¹⁴C]lysine monohydrochloride, 0.04–10 mm unlabeled substrate, and 0.1–5 μg of purified protein. Reactions were incubated at 30–80 °C for 10 min before being stopped by the addition of 5% (v/v) trichloroacetic acid, and ¹⁴CO₂ release was quantified by liquid scintillation counting. For determining the pH optima of enzymes, reactions were performed in the following series of buffers: 50 mm sodium acetate (pH 3.5, 4.5, or 5.5), 50 mm MES (pH 6.5), 50 mm HEPES (pH 7.5), and 50 mm CHES (pH 8.5 or 9.5).

Molecular Modeling

Enzyme structures were presented using the molecular graphics program PyMOL (45).

Bioinformatics Analysis

Sequence alignment and neighbor joining tree building were performed as described previously (16).

An Ancestral Form of the Alanine Racemase Fold of Biosynthetic Arginine Decarboxylase

The biosynthetic AR-fold ADC has an interdomain insertion of between 90 and 105 amino acids compared with other enzymes of the same structural class (16). This insertion is located between the β/α-barrel N-terminal domain and the C-terminal β-barrel domain, relative to the known structures of DAPDC, ODC, L/ODC, and CANSDC. Thus, AR-fold ADC proteins are usually at least 100 amino acids bigger than other members of the family. The position of the interdomain insertion in the crystal structure of the V. vulnificus ADC monomer (20) is shown in Fig. 1A. A four-helix bundle structure is assumed by the insertion peptide chain. We noted previously (16) that species of the Chloroflexi phylum do not possess orthologues of ADC, ODC, or CANSDC but do possess orthologues of DAPDC. Closer inspection of Chloroflexi genomes revealed that they contain genes encoding two paralogues of DAPDC-like sequences, relatively diverged from one another. One paralogue, although considerably shorter than ADC orthologues from other species due to lack of the interdomain insertion, exhibits closer similarity with the longer insertion-containing ADC orthologues than with DAPDC. BLASTP sequence comparison analysis revealed that members of the Bacteroidetes phylum, mostly in the Sphingobacteria and Flavobacteria classes but also including Alistipes putredinis and Alistipes shahii of the Bacteroidia class, possess genes encoding short ADC-like orthologues lacking an interdomain insertion. The short putative ADCs from both the Chloroflexi and Bacteroidetes phyla possess active site residues in the “specificity helix” of the active site pocket that resemble the long form ADCs rather than DAPDC (20). Fig. 2 shows a neighbor joining tree of representative short and long ADC, DAPDC, ODC, and L/ODC proteins of the AR-fold. An alignment of the same sequences around the interdomain insertion region of the long ADCs is shown in Fig. 3, and the full alignment is displayed in supplemental Fig. S1.

Representative speA orthologues encoding short ADCs lacking the interdomain insertion, from the Chloroflexi species C. aurantiacus J-10-fl (YP_001634722; 500 amino acids) and the Flavobacterium G. forsetii KT0803 (YP_863630; 467 amino acids), were synthesized with E. coli optimized codons, expressed in E. coli with N-terminal His tags, and purified. When assayed for ADC activity, the C. auranticus and G. forsetii proteins decarboxylated arginine with specific activities of 1.5 and 0.39 μmol min⁻¹ mg⁻¹ protein, respectively, at pH 7.5 and 37 °C. The C. auranticus enzyme was also able to decarboxylate ornithine and lysine, at 1.4 and 1.9%, respectively, of its main ADC activity, and activity with meso-diaminopimelate was less than 2% of that with arginine. Dependence of the C. auranticus and G. forsetii ADC enzyme activities on pH and temperature was determined (supplemental Fig. S2). The enzymes differed substantially in their temperature optima; C. auranticus ADC was most active at ≥80 °C (the practical upper temperature limit for the assay), whereas G. forsetii ADC was most active at 30 °C (supplemental Fig. S2A). Attempts to determine the kinetic constants for C. auranticus ADC at 80 °C were unsuccessful due to enzyme instability at this temperature. There was a large difference in the pH optima of the two enzymes, with maximal activity for C. auranticus ADC at pH 6.5 and G. forsetii ADC at pH 8.5 (supplemental Fig. S2B). Thermal stability of C. auranticus ADC was assessed by assaying for decarboxylation of arginine (at 70 °C) after incubation of the enzyme at 70 or 80 °C for 0, 5, 10, or 30 min. At 70 °C, full enzyme activity was retained, even after 30 min of incubation (supplemental Fig. S2C), indicating that C. auranticus ADC has high thermal stability. However, enzyme activity was almost completely abolished within 5 min by incubating the enzyme at 80 °C (supplemental Fig. S2C). Optimal growth temperature of C. auranticus is between 52 and 60 °C (46), whereas G. forsetii is a marine bacterium found in surface waters (47). Kinetic constants for the C. auranticus and G. forsetii ADCs were determined at pH 6.5 and 70 °C and at pH 8.5 and 30 °C, respectively (Table 1). Relative k_cat/K_m values indicate that the C. auranticus ADC is ∼80-fold more efficient at decarboxylating arginine than the G. forsetii ADC. However, the long form AR-fold ADC from V. vulnificus assayed previously with arginine at 37 °C exhibited a k_cat of 20 s⁻¹, a K_m of 0.20 mm, and a k_cat/K_m value of 100,000 (16), indicating that the V. vulnificus ADC is ∼5.5-fold more efficient than the C. auranticus ADC. Assay of the G. forsetii ADC in the presence of a 5 mm concentration of the ADC product agmatine revealed that the reaction product inhibited activity by ∼30%. To determine the affinity of the product/inhibitor for the two ADC enzymes, kinetic constants were calculated at various concentrations of agmatine, giving K_i values of 0.92 ± 0.2 and 1.9 ± 0.4 mm for C. auranticus and G. forsetii ADCs, respectively. Differences in K_i values for agmatine were consistent with the substrate affinities of the two enzymes.

The short form AR-fold ADC found in the Chloroflexi phylum and Bacteroidetes phyla is probably the ancestral form of the AR-fold ADC enzyme. It is likely that the 90–105-amino acid insertion in the long form ADC is a polarized transition (i.e. it is much less probable that the insertion was removed without trace to produce a short form ADC than that a preexisting short form ADC acquired an insertion). Within the Chloroflexi, agmatine is converted to putrescine by AIH/NCPAH. Within the Bacteroidetes, Sphingobacteria contain only AIH/NCPAH orthologues, whereas Flavobacteria possess only AUH orthologues. There is one species of Sphingobacteria encoding the long form ADC (Rhodothermus marinus DSM 4252; Figs. 2 and 3). An aberrant form of AR-fold ADC is encoded in Francisella species of the γ-Proteobacteria; it has an insertion of only 20 amino acids relative to the short ADCs but is more similar to the long form ADC. It is found in a gene cluster containing an AIH gene disrupted by a frameshift (Fig. 4), with the implication that the ADC may not function for putrescine biosynthesis in these species. Gene clusters containing long and short form AR-fold speA ORFs together with other polyamine metabolic genes are common (Fig. 4).

Ancestral Biosynthetic Arginine Decarboxylases of the Aspartate Aminotransferase Fold

The E. coli AAT-fold ADC is an acid-inducible enzyme with a monomer size of 756 amino acids. This ADC activity is not involved in polyamine biosynthesis but in an acid resistance system. Five AAT-fold basic amino acid decarboxylases are encoded in the E. coli genome: the acid-inducible ADC, acid-inducible and constitutive LDCs, and acid-inducible and constitutive ODCs. Each decarboxylase monomer is roughly of the same size: acid-inducible ADC, 756 aa; constitutive LDC, 713 aa; acid-inducible LDC, 715 aa; acid-inducible ODC, 732 aa; constitutive ODC, 711 aa. A polyamine biosynthetic ADC exhibiting sequence similarity to the E. coli AAT-fold LDC was identified previously in the firmicute bacterium B. subtilis (48). The biosynthetic AAT-fold ADC enzyme identity was established by disruption of the encoding speA gene and subsequent analysis of polyamine levels in the gene knock-out strain (48). No biochemical characterization of the enzyme was performed in that study (48); therefore, we characterized the kinetic behavior of the B. subtilis 168 biosynthetic ADC (NP_389346), cloned from genomic DNA and expressed from pET21a in E. coli and purified as an N-terminal T7-tagged fusion protein. The B. subtilis biosynthetic ADC was found to have an optimal pH of 7.7 and optimal temperature of >75 °C (supplemental Fig. S3), and at 37 °C, the K_m of the substrate arginine was 0.63 mm and the k_cat was 0.21 s⁻¹ (Table 2). Many of the firmicute bacteria contain two AAT-fold basic amino acid decarboxylase paralogues. The second paralogue in B. subtilis, encoded by the yaaO gene, was shown previously to play no detectable role in polyamine biosynthesis (48). There are two AAT-fold basic amino acid decarboxylase paralogues in the genome of C. difficile 630 (YP_001087362 and YP_001090072). The YP_001087362 open reading frame is clustered immediately adjacent to open reading frames orthologous to the polyamine biosynthetic enzymes AdoMetDC, spermidine synthase, and AUH (Fig. 5). We therefore expressed YP_001087362 as an N-terminally T7-tagged protein in E. coli and purified the recombinant enzyme. The C. difficile YP_001087362 protein is a functional ADC (Table 2) but with relatively low efficiency compared with the B. subtilis enzyme (K_m = 3.3 mm, k_cat = 0.018 s⁻¹ at 60 °C). Another AAT-fold ADC was recently identified in the firmicute Selenomonas ruminantium (49). Its K_m for arginine was even higher than the C. difficile ADC, at 5.6 mm, and the optimal temperature was 60 °C.

Biosynthetic AAT-fold ADCs from the Firmicutes are considerably shorter than the acid-inducible AAT-fold ADC from E. coli and the biosynthetic AAT-fold ODC from Lactobacillus 30a (S. ruminantium ADC, 485 aa (BAD80720); B. subtilis ADC, 490 aa (P21885); and C. difficile ADC, 491 aa (YP_001087362)). A crystal structure of the Lactobacillus 30a ODC revealed that the functional unit is a dimer, with the active sites formed across the interface between the two monomers. The enzyme assembles into a dodecamer composed of six dimers (50). Similarly, the acid-inducible ADC of E. coli is a decamer composed of five dimers (51). Monomer forms of both the E. coli acid-inducible ADC and the Lactobacillus 30a biosynthetic ODC are composed of five structural domains: the wing, linker, PLP-binding region, the aspartate aminotransferase-like small domain, and the C terminus (50, 51) (supplemental Fig. S4). The wing domain is required for multimeric assembly (Fig. 1B) and is also required for acid induction of activity in the E. coli ADC (51). Lactobacillus 30a biosynthetic ODC is very similar in amino acid sequence to E. coli biosynthetic and acid-inducible ODCs, and probably the Lactobacillus 30a ODC was acquired by horizontal transfer because only the Lactobacilli within the Firmicutes possess the long form biosynthetic AAT-fold ODC. When the firmicute B. subtilis, C. difficile, and S. ruminantium biosynthetic ADCs are aligned with the Lactobacillus 30a ODC and acid-inducible ADC of E. coli, it is clear that the firmicute biosynthetic ADCs lack completely the wing domain of the E. coli and Lactobacillus 30a enzymes (supplemental Fig. S4). In addition, the biosynthetic AAT-fold ADCs have small deletions in each of the other four domains relative to the E. coli acid-inducible ADC and Lactobacillus 30a ODC enzymes (supplemental Fig. S4).

A sequence comparison analysis using BLASTP of either the E. coli acid-inducible ADC or the Lactobacillus 30a ODC shows clearly that the wing domain of both proteins exhibits sequence homology to the signal receiver domains of response regulators, such as CheY (so-called REC domains). An alignment of the E. coli acid-inducible ADC wing domain with REC domains of diverse response regulator proteins is shown in supplemental Fig. S5. A number of aspartate residues are conserved between the ADC wing domain and the other REC domains and may be involved in metal binding. Comparison of the crystal structure of the acid-inducible ADC wing domain with other structures, using the Vector Alignment Search Tool at the National Center for Biological Information, shows an even greater similarity than sequence-based searches to the structures of many diverse REC domain-containing proteins. It was shown previously that the wing domain protrudes from the rest of the E. coli acid-inducible ADC protein structure (51). It is thus most likely that the acid-inducible ADC wing domain was acquired by fusion of a REC domain protein to the N terminus of a firmicute short form biosynthetic AAT-fold ADC. Because the wing domains are not found in other AAT-like proteins (51), it is very probable that the shorter biosynthetic ADCs found in the Firmicutes are ancestral to the longer acid-inducible ADC and the constitutive and inducible LDCs and ODCs found in E. coli. Potential orthologues of the firmicute biosynthetic AAT-fold ADCs are also encoded in Cyanobacteria, forming a distinct cyanobacterial clade. Genes for the corresponding enzymes from the filamentous cyanobacterium Anabaena variabilis ATCC 29413 (YP_322672; 488 aa) and Nostoc punctiforme PCC 73102 (YP_001864209; 504 aa) were synthesized with E. coli-optimized codons, expressed from pET21a in E. coli, and purified as T7-tagged proteins. Neither enzyme displayed any activity with arginine, ornithine, or lysine (results not shown), and they appear to be uninvolved in polyamine metabolism, similar to the AAT-fold ADC-like protein encoded by the yaaO gene of B. subtilis described above.

The Biosynthetic AAT-fold ADC of B. subtilis Is Essential for Biofilm Development

A gene disruption mutant of the B. subtilis 168 biosynthetic ADC was made previously by Sekowska et al. (48); however, the effect of the speA gene deletion on growth was not reported. We analyzed aerobic growth, in polyamine-free defined minimal medium, for both the biosynthetic ADC gene deletion (ΔspeA) strain made by Sekowska et al. (48) and a deletion strain of the related yaaO gene (ΔyaaO). Deletion of either speA or yaaO had no discernible effect on aerobic growth of planktonic cells in liquid medium (Fig. 6, top), although polyamines were completely absent from the ΔspeA cells (Fig. 6, bottom). This experiment also confirmed that deletion of yaaO has no effect on polyamine levels in ΔyaaO cells grown aerobically in polyamine-free medium. Polyamines are critical for biofilm formation in the γ-Proteobacteria Y. pestis and V. cholerae (37, 38, 52, 53). Therefore, the effect of disruption of speA on B. subtilis biofilm formation was examined. Phage transduction was used to replace the speA (ADC) gene of B. subtilis NCIB3610 with the ΔspeA region of B. subtilis 168 to facilitate biofilm analysis. The B. subtilis NCIB3610 strain forms complex and robust biofilms in comparison with the laboratory isolate 168 (41). Polyamine depletion in the ΔspeA gene knock-out strain resulted in abolition of biofilm formation using both complex colony architecture and pellicle formation as two independent measures of biofilm formation capability (Fig. 7). The negative impact on biofilm formation was fully reversed by the addition of 0.5 mm agmatine or spermidine to the growth medium (Fig. 8). The addition of putrescine to the growth medium did not restore biofilm formation, which may be due to lack of uptake. Although biofilm formation in the B. subtilis ΔspeA NCIB3610 background was abolished (Fig. 7A), aerobic growth of planktonic cells of the same ΔspeA NCIB3610 strain in polyamine-free liquid growth medium was unaffected (Fig. 7B).

The Phylogenetic Distribution of Polyamine Biosynthetic ADCs

In addition to the AR-fold and AAT-fold pyridoxal 5′-phosphate-dependent ADCs, there are two types of pyruvoyl-dependent ADCs found in archaea and some bacteria. Pyruvoyl-dependent decarboxylases autocatalytically self-process into α- and β-chains at a serine residue that generates the pyruvoyl cofactor. The pyruvoyl cofactor becomes the N terminus of the α-chain. The pylADC is found predominantly in the euryachaeota and is structurally and mechanistically similar to pyruvoyl-dependent histidine decarboxylase (21, 39). Some Chlamydia species use a very divergent pylADC as part of an acid resistance system (23, 54); however, the Chlamydia enzymes are unlikely to participate in polyamine biosynthesis. Like the PLP-dependent ADCs, the pylADCs are commonly found in polyamine-related gene clusters/operons (Fig. 5). Among the archaea, there are pylADC orthologues in 64 euryarchaeote genomes (Table 3) but in only one crenarchaeote (Thermofilum pendens Hrk 5 (YP_920276)). One euryarchaeote species, Methanosaeta thermophila PT, uses a typical AR-fold ADC (Figs. 2 and 3 and supplemental Fig. S1) and does not possess a pylADC orthologue. The pylADC appears to be the only route for putrescine biosynthesis in the anoxygenic, photosynthetic Chlorobi phylum (Table 3), and a subset of species of the Bacteroides and δ-Proteobacteria classes possess pylADC orthologues. One genome each in the Actinobacteria, ϵ-Proteobacteria, Planctomycetales, Elusimicrobia, and Synergistetes phyla and two in the Acidobacteria contain pylADC orthologues, but there is very shallow genome sampling of the Elusimicrobia and Synergistetes, so pylADC in principle could be more important in these poorly sampled phyla.

Most of the Crenarchaeota use an AdoMetDC paralogue to decarboxylate arginine for polyamine biosynthesis (22) and thus contain two AdoMetDC paralogues in each genome. This unusual method for arginine decarboxylation appears to be specific to the Crenarchaeota. Three species within the Crenarchaeota use diverged AR-fold ADCs although replete with the typical interdomain insertion (two are shown in Figs. 1 and 2 and supplemental Fig. S1). Both Desulfurococcus kamchatkensis 1221n and Staphylothermus hellenicus DSM 12718 of the Crenarchaeota possess an AR-fold ADC and only one AdoMetDC orthologue; however, Staphylothermus marinus F1 possesses two AdoMetDC paralogues and an AR-fold ADC.

The biosynthetic AAT-fold ADC is found almost exclusively in the Firmicutes, with 155 genomes containing an orthologue (Table 3). There are also orthologues in two Fusobacteria and two Mollicutes genomes and one Spirochaete. In contrast, the AR-fold ADC is excluded from the Firmicutes except for Clostridium cellulolyticum H10 and Clostridium papyrosolvens DSM 2782. The AR-fold ADC is most prevalent in the γ- and ϵ- and δ-Proteobacteria, the Cyanobacteria, the Bacteroides class of the Bacteroidetes phylum, the Verrucomicrobia, Deinococcus-Thermi, and Planctomycetales (Table 3). A smaller proportion of the α- and β-Proteobacteria also contain AR-fold ADC orthologues. As discussed above, the ancestral, shorter form of the AR-fold ADC lacking the interdomain insertion is limited to members of the Chloroflexi and Bacteroidetes. Almost all terrestrial plants contain long form AR-fold ADC orthologues, but they are absent in algae except for one example in Micromonas pusilla. The nucleus-encoded plant AR-fold ADC orthologues are derived from the cyanobacterial ancestor of the chloroplast (11, 55). Biosynthetic ADCs remain to be identified in other eukaryotes.

The clear discernable pattern in the phylogenetic distribution of the different ADC forms is that the AR-fold ADC is mostly absent from the Archaea, Firmicutes, and Actinobacteria, which are single-membraned. The Actinobacteria appear to lack any ADC orthologues (except for a pylADC orthologue in Beutenbergia cavernae DSM 12333 (YP_002880881)). The Firmicutes depend on the short AAT-fold biosynthetic ADC, and Archaea use mainly pyruvoyl-dependent ADCs, with the euryarchaeotes possessing the histidine decarboxylase-like pylADC, whereas the Crenarchaeota mostly use the AdoMetDC-derived ADC.

Arginine Decarboxylase Is the Dominant Route for Polyamine Biosynthesis in the Human Gut Microbiota

The genomes of the 55 most common bacterial species found in the human gut microbiota (43) were screened in silico for orthologues of the AR- and AAT-fold ODCs. No ODC orthologues from either fold were found in these species. In contrast, biosynthetic AR- and AAT-fold ADC genes were found in the majority of species, although 11 species appeared to be auxotrophic for polyamine biosynthesis (Table 4). The Firmicute species contained the AAT-fold biosynthetic ADC, whereas the Bacteroidetes contained the AR-fold ADC with the exception of Bacteroides capillosus and B. pectinophilus, which contain horizontally acquired AAT-fold ADCs. The Bacteroidetes species A. putredinis contains an ancestral short AR-fold ADC. Genes encoding AIH and NCAPH are always clustered except for the presence of isolated AIH-encoding genes in the firmicute Enterococcus faecalis and the actinobacterium Colinsella aerofaciens. At least in the case of E. faecalis, the AIH-encoding gene is involved in agmatine catabolism (56).