evolution and multiplicity of arginine decarboxylases in polyamine biosynthesis and essential role...

J. MichaelMurray, Nicola R. Stanley-Wall and Anthony Matthew Burrell, Colin C. Hanfrey, Ewan J. Biofilm Formation

Bacillus subtilisand Essential Role in Decarboxylases in Polyamine Biosynthesis Evolution and Multiplicity of ArginineMetabolism:

doi: 10.1074/jbc.M110.163154 originally published online September 27, 20102010, 285:39224-39238.J. Biol. Chem.

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Evolution and Multiplicity of Arginine Decarboxylases inPolyamine Biosynthesis and Essential Role in Bacillus subtilisBiofilm Formation□S

Received for publication, July 12, 2010, and in revised form, August 24, 2010 Published, JBC Papers in Press, September 27, 2010, DOI 10.1074/jbc.M110.163154

Matthew Burrell‡1, Colin C. Hanfrey‡, Ewan J. Murray§2, Nicola R. Stanley-Wall§3, and Anthony J. Michael‡¶4

From the ‡Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom, the §Division ofMolecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom, and the¶Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041

Arginine decarboxylases (ADCs; EC 4.1.1.19) from four dif-ferent protein fold families are important for polyamine bio-synthesis in bacteria, archaea, and plants. Biosynthetic alanineracemase fold (AR-fold) ADC is widespread in bacteria andplants. We report the discovery and characterization of an an-cestral form of the AR-fold ADC in the bacterial Chloroflexiand Bacteroidetes phyla. The ancestral AR-fold ADC lacks alarge insertion found in Escherichia coli and plant AR-foldADC and is more similar to the lysine biosynthetic enzymemeso-diaminopimelate decarboxylase, from which it hasevolved. An E. coli acid-inducible ADC belonging to the aspar-tate aminotransferase fold (AAT-fold) is involved in acid re-sistance but not polyamine biosynthesis. We report here thatthe acid-inducible AAT-fold ADC has evolved from a shorter,ancestral biosynthetic AAT-fold ADC by fusion of a responseregulator receiver domain protein to the N terminus. Ances-tral biosynthetic AAT-fold ADC appears to be limited to firmi-cute bacteria. The phylogenetic distribution of different formsof ADC distinguishes bacteria from archaea, euryarchaeotafrom crenarchaeota, double-membraned from single-mem-braned bacteria, and firmicutes from actinobacteria. Our find-ings extend to eight the different enzyme forms carrying outthe activity described by EC 4.1.1.19. ADC gene clustering re-veals that polyamine biosynthesis employs diverse and ex-changeable synthetic modules. We show that in Bacillus subti-lis, ADC and polyamines are essential for biofilm formation,and this appears to be an ancient, evolutionarily conservedfunction of polyamines in bacteria. Also of relevance to humanhealth, we found that arginine decarboxylation is the domi-nant pathway for polyamine biosynthesis in human gutmicrobiota.

The evolution of central metabolic pathways was one of thekey biochemical developments in the early stages of primor-dial life (1). Metabolic pathways evolve through mechanismssuch as gene duplication followed by neofunctionalization ofone of the copies to produce a new enzyme with an alteredsubstrate preference (2–4). Other mechanisms involved inthe evolution of metabolic pathways include gene fusions,which can be fusion of two paralogous genes to achieve geneelongation. An example is the formation of eukaryotic S-ad-enosylmethionine decarboxylase (AdoMetDC),5 a polyaminebiosynthetic enzyme, by fusion of two prokaryoticAdoMetDC genes (5). Gene fusions may bring together differ-ent protein functional modules such as regulatory and enzy-matic components and are often generated independently indifferent lineages by fusion of open reading frames withinoperons (6). New steps in metabolic pathways can be re-cruited from preexisting pathways in the same cell (the“patchwork” model of pathway evolution) (7) or from otherorganisms by horizontal or endosymbiotic gene transfer. Con-vergent evolution of the same enzymatic activity in differentorganisms by completely different, evolutionarily unrelatedproteins (recently described as non-homologous isofunctionalenzymes) (8) is a result of independent biochemical invention(9).The biosynthesis of polyamines is an excellent model for

studying processes of metabolic pathway evolution. Poly-amines are small organic polycations, usually linear di-, tri-,and tetra-amines, that are found in almost all cells. Synthesisof polyamines is achieved through biosynthetic modules: adiamine is produced directly or indirectly from an amino acid,and a triamine is produced from the diamine by the additionof an aminopropyl or aminobutyl group. Tetra-amines areproduced from triamines by aminopropyl or more rarely byaminobutyl group addition. The modular nature of polyamine□S The on-line version of this article (available at http://www.jbc.org) con-

tains supplemental Figs. S1–S5.1 Present address: MedImmune, Granta Park, Cambridge CB21 6GH, United

Kingdom.2 Present address: School of Molecular Medical Sciences, Centre for Biomo-

lecular Sciences, University of Nottingham, Nottingham NG7 2RD, UnitedKingdom.

3 Supported by Biotechnology and Biological Sciences Research Council UKGrants BB/C520404/1 and BB/E001572/1.

4 Supported by a Biotechnology and Biological Sciences Research CouncilUK CSG grant and Institute Development Fellowship BB/E024467/1. Towhom correspondence should be addressed: Dept. of Pharmacology,University of Texas Southwestern Medical Center, Dallas, TX 75390-9041.Tel.: 214-648-4170; E-mail: [email protected].

5 The abbreviations used are: AdoMetDC, S-adenosylmethionine decarboxy-lase; AAT-fold, aspartate aminotransferase fold; ADC, arginine decarboxy-lase; AIH, agmatine iminohydrolase; AR-fold, alanine racemase fold; AUH,agmatine ureohydrolase; CANSDC, carboxy(nor)spermidine decarboxy-lase; DAPDC, meso-diaminopimelate decarboxylase; LDC, lysine decar-boxylase; L/ODC, lysine/ornithine decarboxylase; NCPAH, N-car-bamoylputrescine amidohydrolase; ODC, ornithine decarboxylase; PLP,pyridoxal 5�-phosphate; pylADC, pyruvoyl-dependent arginine decar-boxylase; REC, receiver domain; aa, amino acids; CHES, 2-(cyclohexylami-no)ethanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 50, pp. 39224 –39238, December 10, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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biosynthesis facilitates horizontal and endosymbiotic transfer(10).By far, in all three domains of life, the most common dia-

mine is putrescine (1,4-diaminobutane), which can be used toform spermidine (by aminopropyl addition) or sym-homo-spermidine (by aminobutyl addition). Almost all eukaryotessynthesize putrescine directly from ornithine by the action ofornithine decarboxylase (ODC). However, plants possess anadditional, indirect pathway for putrescine biosynthesis fromarginine, through the action of arginine decarboxylase (ADC)(11). It is unclear which bacteria use the ADC route to synthe-size putrescine, and the relative importance of the ODC andADC routes in bacterial polyamine biosynthesis is also un-clear. The product of ADC, agmatine, may be converted di-rectly to putrescine by agmatine ureohydrolase (AUH) (12) orindirectly via N-carbamoylputrescine by agmatine deiminase/iminohydrolase (AIH) and N-carbamoylputrescine amidohy-drolase (NCPAH) (13).There are currently four known protein fold types contain-

ing an ADC. The alanine racemase fold (AR-fold) includessolved structures of the eukaryotic ODC from human andTrypanosoma brucei (14, 15); bifunctional lysine/ornithinedecarboxylase (L/ODC) from Vibrio vulnificus (16);meso-diaminopimelate decarboxylase (DAPDC) fromMethanocal-dococcus jannaschii,Mycobacterium tuberculosis, and Helico-bacter pylori (17–19); and carboxy(nor)spermidinedecarboxylase (CANSDC) and ADC (20). The AR-fold ADC issubstantially longer than the other members of the AR-foldbasic amino acid decarboxylases (16), due to a large insertionrelative to other members of the family. Acid-inducible ADCof Escherichia coli, which is not involved in polyamine biosyn-thesis, belongs to the aspartate aminotransferase fold (AAT-fold), which also includes the E. coli and Lactobacillus 30abiosynthetic ODC and also bacterial acid-inducible lysine de-carboxylases. A pyruvoyl-dependent ADC (pylADC) is pres-ent inM. jannaschii and most of the euryarchaeota (21),whereas Sulfolobus solfataricus and most of the crenarchaeotapossess an ADC that has recently evolved from the pyruvoyl-dependent AdoMetDC (22). An acid-dependent pylADC,probably not involved in polyamine biosynthesis, is present inChlamyophila species (23). In addition, large algal viruses,such as Paramecium bursaria chlorella virus-1, possess anAR-fold ADC that highly resembles and has recently evolvedfrom an AR-fold ODC (24, 25). Whatever the form of ADCenzyme encoded, in bacteria, ADC is encoded by the speAgene.Although polyamines are primordial constituents of life, it

has been difficult to elucidate their physiological roles. In eu-karyotes and archaea, spermidine is required for hypusineformation, which is essential for cell growth in eukaryotes andarchaea (26, 27). Furthermore, in archaea, it was shown re-cently that agmatine is required for an essential modificationof a cytidine nucleoside in a tRNA anticodon (to produce themodified nucleoside agmatidine) required for decoding theAUA triplet (28, 29). A number of studies have concludedthat the main physiological role of spermidine is in hypusineformation for eIF5A function, both in the yeast Saccharomy-ces cerevisiae (30, 31) and in mammalian cells (32, 33). Never-

theless, there are also studies demonstrating that polyaminesaffect cellular function independently of hypusine formation(34, 35). The essential requirements for spermidine and ag-matine in hypusine and agmatidine formation, respectively,obscure other core physiological functions of polyamines ineukaryotes and archaea. Thus, bacteria, which lack both hy-pusine and agmatidine modifications, should present a moretransparent model for determining physiological functions ofpolyamines. However, aerobic growth of E. coli is barely af-fected by complete polyamine depletion (36). In Yersinia pes-tis and Vibrio cholerae, depletion of either all polyamines or oftriamines, respectively, resulted in a slight reduction (�40%)of aerobic growth of planktonic cells (37, 38). In contrast,polyamine depletion abolished aerobic growth of Pseudomo-nas aeruginosa PAO1 cells (13) and significantly inhibitedgrowth of Rhizobium leguminosarum (10). Although poly-amine depletion had relatively little effect on growth of plank-tonic Y. pestis and V. cholerae cells, polyamine depletion inboth species abolished biofilm production (37, 38). Biofilmformation is thus a key physiological process where poly-amines are required independently of hypusine or agmatidineformation, at least in two species of �-Proteobacteria.Here we demonstrate that an ancestral form of the AR-fold

ADC exists in species of the Chloroflexi (green non-sulfurbacteria) and Bacteriodetes phyla. The ancestral AR-fold ADClacks the characteristic insertion of the longer AR-fold ADCs,which is found in most phyla of the double-membraned bac-teria. We also show that the firmicute AAT-fold biosyntheticADC is an ancestral form of the acid-inducible ADC found inE. coli and lacks the N-terminal wing domain necessary fordecamer formation. We found that the N-terminal wing do-main of the acid-inducible AAT-fold ADC is derived from aresponse regulator protein receiver (REC) domain that hasfused to the N terminus of a biosynthetic AAT-fold ADC-likeprotein. The Bacillus subtilis biosynthetic AAT-fold ADC isshown to be essential for biofilm formation, establishing thatpolyamines are involved in biofilm formation in single-mem-braned as well as doubled-membraned bacteria. This indi-cates that a role in biofilm formation may be an ancient physi-ological function of polyamines in bacteria. We map thephylogenetic distribution of the different ADC enzymes andshow that there is a clear division between double-mem-braned and single-membraned prokaryotes and between sin-gle-membraned bacteria and archaea. Finally, we show thatthe ADC route for putrescine biosynthesis is the dominantpathway for polyamine formation in the human gutmicrobiota.

EXPERIMENTAL PROCEDURES

Materials—All materials were of the highest grade avail-able and were purchased from Sigma unless otherwisestated. L-[1-14C]Ornithine hydrochloride (57.1 mCi/mmol),L-[U-14C]arginine monohydrochloride (346 mCi/mmol), andL-[U-14C]lysine monohydrochloride (320 mCi/mmol) werepurchased from PerkinElmer Life Sciences.Bacterial Strains and Growth Conditions—E. coli XL2 Blue

and BL21 (DE3) pLysS were purchased from Stratagene andNovagen, respectively. B. subtilis 168 was a kind gift from Dr.

Evolution of Arginine Decarboxylases

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Stephan Bornemann (John Innes Centre), and B. subtilisgenomic DNA was prepared using standard procedures. Theoriginal B. subtilis 168 �speA (BSIP 7010) and �yaaO knock-out strains were a kind gift of Drs. Agnieszka Sekowska andAntoine Danchin (Institut Pasteur; the �yaaO gene knock-out strain was originally made by Dr. Asai Kei). Strains ofB. subtilis were grown routinely in Luria-Bertani (39) medium(10 g/liter NaCl, 5 g/liter yeast extract, and 10 g/liter tryp-tone) 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 �M

CaCl2, 50 �M MnCl2, 50 �M FeCl3, 1 �M ZnCl2, 2 �M thia-mine, 0.5% glycerol, 0.5% glutamate) (41) was used for analy-sis of biofilm formation. The B. subtilis strains used in thisstudy for biofilm analysis were the strain 3610 (prototroph)obtained from the Bacillus Genetic Resource Center andstrain NRS3088 (3610 �speA (spc); created in this study).Phage transductions into the B. subtilis strain backgroundNCIB3610 were conducted as described previously (40).When required, antibiotics were used at the following con-centrations: erythromycin (1 �g/ml) with lincomycin (25 �g/ml) and spectinomycin (100 �g/ml). When required, poly-amines were added to the MSgg medium at a concentration of0.5 mM where specified.Biofilm Image Analysis—Analysis of biofilm formation was

performed essentially as described (41, 42). Strains of B. subti-lis to be tested were grown to mid-late exponential phase inLB liquid medium inoculated from a freshly streaked LB solidmedium plate. Ten �l of the undiluted culture was spottedonto an MSgg plate with 1.5% agar and incubated at 37 °C forthe time indicated. For pellicle analysis, 1.5 �l of the sameculture was added to 1.5 ml of MSgg medium in a 24-well tis-sue culture dish, placed at 37 °C for the time period indicated.Images were captured using a Leica MZ16 FA stereoscopeusing 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 fromgenomic DNA. Putative speAORFs from Clostridium difficile630 (YP_001087362), Chloroflexus auranticus J-10-fl(YP_001634722), and Gramella forsetii KT0803 (YP_863630)were synthesized by Genscript (Piscataway, NJ) with codonsoptimized for expression in E. coli. The speAORFs fromB. subtilis and C. difficile were subcloned into the BamHI andXhoI sites of pET21a, and those from Chloroflexus aurantia-cus and G. forsetti were subcloned into the NdeI and BamHIsites of pET15b.Protein Expression and Purification—For expression of pu-

tative decarboxylases, E. coli BL21 was transformed with pro-tein expression plasmids and grown to an A600 nm of 0.3 in LBliquid medium containing 100 �g/ml ampicillin at 37 °C. Pro-tein expression was induced with 0.4 mM isopropyl 1-thio-�-D-galactopyranoside, and cultures were incubated for a fur-ther 3 h at 37 °C. T7-tagged proteins were purified using theT7�Tag affinity purification kit (Novagen) according to themanufacturer’s instructions. For purification of His-taggedproteins, cells were resuspended in 20 mM sodium phosphate(pH 8.0) containing 500 mM NaCl, 20 mM imidazole, and0.02% (v/v) Brij35 before being broken by sonication. The cell

lysate was cleared by centrifugation and applied to aHiTrapTM chelating HP column (GE Healthcare) that hadbeen charged with Ni2� and equilibrated with the abovebuffer. The column was washed with the above buffer, andbound protein was eluted using a 0.02–1 M imidazole gradi-ent. Purified proteins were concentrated using Amicon Ul-tra-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-FluorTM reagent kit (Waters Corp.,Milford, MA) according to the manufacturer’s instructions.Labeling reactions contained 5 �l of stopped enzyme assayand 1.25 �M 1,7-diaminoheptane as an internal standard andwere heated to 55 °C for 10 min. Derivatized polyamine sam-ples were analyzed by HPLC using a reverse phase C18 col-umn (Luna 5 �, Phenomenex) on a Dionex Summit HPLCSystem. Polyamine separation was performed using 10 �l ofderivatized sample. The system was operated at 33 °C andequilibrated with Eluent A (70 mM acetic acid, 25 mM triethyl-amine, pH 4.82) at 1.2 ml/min. Elution was performed usingthe following linear gradients of Eluent B (80% acetonitrile):22% for 5 min, 39% for 12 min with 6% methanol, 33% for 30 swith 14% methanol, 10% for 6.5 min with 70% methanol, andfinally 100% for 21 min. Polyamines were monitored by fluo-rescence (Dionex RF 2000 detector) with a 248-nm excitationfilter and a 398-nm emission filter and identified by compari-son of retention times with known standards that were deri-vatized and analyzed in parallel with the enzyme assays.Enzyme Assays—Amino acid decarboxylase activity was

measured using a stopped 14CO2 release assay (44). Unlessotherwise stated, assays were buffered in 50 mM HEPES (pH7.5) containing 50 mM NaCl, 2 mM DTT, and 0.1 mM pyri-doxal 5�-phosphate (PLP) and contained 3.7 kBq L-[1-14C]or-nithine hydrochloride, L-[U-14C]arginine monohydrochloride,or L-[U-14C]lysine monohydrochloride, 0.04–10 mM unla-beled substrate, and 0.1–5 �g of purified protein. Reactionswere incubated at 30–80 °C for 10 min before being stoppedby the addition of 5% (v/v) trichloroacetic acid, and 14CO2release was quantified by liquid scintillation counting. Fordetermining the pH optima of enzymes, reactions were per-formed in the following series of buffers: 50 mM sodium ace-tate (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).

RESULTS

An Ancestral Form of the Alanine Racemase Fold of Biosyn-thetic Arginine Decarboxylase—The biosynthetic AR-foldADC has an interdomain insertion of between 90 and 105amino acids compared with other enzymes of the same struc-tural class (16). This insertion is located between the �/�-barrel N-terminal domain and the C-terminal �-barrel do-main, relative to the known structures of DAPDC, ODC,



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L/ODC, and CANSDC. Thus, AR-fold ADC proteins are usu-ally at least 100 amino acids bigger than other members of thefamily. The position of the interdomain insertion in the crys-tal structure of the V. vulnificus ADC monomer (20) is shownin Fig. 1A. A four-helix bundle structure is assumed by theinsertion peptide chain. We noted previously (16) that speciesof the Chloroflexi phylum do not possess orthologues ofADC, ODC, or CANSDC but do possess orthologues ofDAPDC. Closer inspection of Chloroflexi genomes revealed

that they contain genes encoding two paralogues of DAPDC-like sequences, relatively diverged from one another. Oneparalogue, although considerably shorter than ADC ortho-logues from other species due to lack of the interdomain in-sertion, exhibits closer similarity with the longer insertion-containing ADC orthologues than with DAPDC. BLASTPsequence comparison analysis revealed that members of theBacteroidetes phylum, mostly in the Sphingobacteria and Fla-vobacteria classes but also including Alistipes putredinis andAlistipes shahii of the Bacteroidia class, possess genes encod-ing short ADC-like orthologues lacking an interdomain inser-tion. The short putative ADCs from both the Chloroflexi andBacteroidetes phyla possess active site residues in the “speci-ficity helix” of the active site pocket that resemble the longform ADCs rather than DAPDC (20). Fig. 2 shows a neighborjoining tree of representative short and long ADC, DAPDC,ODC, and L/ODC proteins of the AR-fold. An alignment ofthe same sequences around the interdomain insertion regionof the long ADCs is shown in Fig. 3, and the full alignment isdisplayed in supplemental Fig. S1.Representative speA orthologues encoding short ADCs

lacking the interdomain insertion, from the Chloroflexi spe-cies C. aurantiacus J-10-fl (YP_001634722; 500 amino acids)and the Flavobacterium G. forsetii KT0803 (YP_863630; 467amino 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 andG. forsetii proteins decarboxylated arginine with specific ac-tivities of 1.5 and 0.39 �mol min�1 mg�1 protein, respec-

FIGURE 1. Structures of pyridoxal 5�-phosphate-dependent argininedecarboxylases. A, structure of the V. vulnificus AR-fold ADC monomer(Protein Data Bank entry 3N2O). The four-helix bundle insertion, which isabsent in the ancestral form, is depicted at the bottom of the structure intan, and the rest of the enzyme (which resembles ODC and DAPDC) isshown in dark blue for �-helices and light blue for �-strands. The pyridoxal5�-phosphate co-factor and agmatine are shown by colored spheres. A isbased on the structure determined by Deng et al. (20). B, structure of onepentamer layer of the decameric AAT-fold acid-inducible ADC of E. coli (Pro-tein Data Bank entry 2VYC). Each monomer is depicted in a different colorwith the N-terminal wing domains at the center of the doughnut and ren-dered a lighter colored version of the remainder of the corresponding mon-omer, the remainder being equivalent to the short biosynthetic AAT-foldADCs. B is based on the structure determined by Andrell et al. (51).

FIGURE 2. Neighbor joining tree of short and long form AR-fold ADC,bifunctional L/ODC, ODC, and DAPDC orthologues. Sequences werealigned using ClustalW. The tree was constructed using PAUP*. The num-bers represent percentage support derived from 1000 bootstraps. The align-ment used to construct the neighbor joining tree is displayed in supple-mental Fig. S1.



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tively, at pH 7.5 and 37 °C. The C. auranticus enzyme was alsoable to decarboxylate ornithine and lysine, at 1.4 and 1.9%,respectively, of its main ADC activity, and activity withmeso-diaminopimelate was less than 2% of that with arginine. De-pendence of the C. auranticus and G. forsetii ADC enzymeactivities on pH and temperature was determined (supple-mental Fig. S2). The enzymes differed substantially in theirtemperature 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 (supplemen-tal Fig. S2A). Attempts to determine the kinetic constants forC. auranticus ADC at 80 °C were unsuccessful due to enzymeinstability at this temperature. There was a large difference inthe pH optima of the two enzymes, with maximal activity forC. auranticus ADC at pH 6.5 and G. forsetii ADC at pH 8.5(supplemental Fig. S2B). Thermal stability of C. auranticusADC 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), indi-cating that C. auranticus ADC has high thermal stability.However, enzyme activity was almost completely abolishedwithin 5 min by incubating the enzyme at 80 °C (supplemen-tal Fig. S2C). Optimal growth temperature of C. auranticus isbetween 52 and 60 °C (46), whereas G. forsetii is a marine bac-terium found in surface waters (47). Kinetic constants for theC. auranticus and G. forsetii ADCs were determined at pH 6.5and 70 °C and at pH 8.5 and 30 °C, respectively (Table 1). Rel-ative kcat/Km values indicate that the C. auranticus ADC is�80-fold more efficient at decarboxylating arginine than theG. forsetii ADC. However, the long form AR-fold ADC fromV. vulnificus assayed previously with arginine at 37 °C exhib-ited a kcat of 20 s�1, a Km of 0.20 mM, and a kcat/Km value of

100,000 (16), indicating that the V. vulnificus ADC is �5.5-fold more efficient than the C. auranticus ADC. Assay of theG. forsetii ADC in the presence of a 5 mM concentration of theADC product agmatine revealed that the reaction productinhibited activity by �30%. To determine the affinity of theproduct/inhibitor for the two ADC enzymes, kinetic con-stants were calculated at various concentrations of agmatine,giving Ki values of 0.92 � 0.2 and 1.9 � 0.4 mM for C. auranti-cus and G. forsetii ADCs, respectively. Differences in Ki valuesfor agmatine were consistent with the substrate affinities ofthe two enzymes.The short form AR-fold ADC found in the Chloroflexi phy-

lum and Bacteroidetes phyla is probably the ancestral form ofthe AR-fold ADC enzyme. It is likely that the 90–105-aminoacid insertion in the long form ADC is a polarized transition(i.e. it is much less probable that the insertion was removedwithout trace to produce a short form ADC than that a preex-isting short form ADC acquired an insertion). Within theChloroflexi, agmatine is converted to putrescine by AIH/NCPAH. Within the Bacteroidetes, Sphingobacteria containonly AIH/NCPAH orthologues, whereas Flavobacteria pos-sess only AUH orthologues. There is one species of Sphingob-acteria encoding the long form ADC (Rhodothermus marinusDSM 4252; Figs. 2 and 3). An aberrant form of AR-fold ADCis encoded in Francisella species of the �-Proteobacteria; ithas an insertion of only 20 amino acids relative to the short

FIGURE 3. Regional sequence alignment of proteins represented in Fig. 1, flanking the long AR-fold ADC interdomain insertion. Ro.cas, Roseiflexuscastenholzii DSM 13941, Chloroflexi, Chloroflexales (YP_001432572) 528 aa; He.aur, Herpetosiphon aurantiacus ATCC 23779, Chloroflexi, Herpetosiphonales(YP_001547274) 576 aa; Ch.aur, C. aurantiacus J-10-fl, Chloroflexi, Chloroflexales (YP_001634722) 500 aa; Fl.jon, Flavobacterium johnsoniae UW101, Bacte-roidetes, Flavobacteria (YP_001195152) 467 aa; Gr.for, G. forsetii KT0803, Bacteroidetes, Flavobacteria (YP_863630) 467 aa; Cy.hut, Cytophaga hutchinsoniiATCC 33406, Bacteroidetes, Sphingobacteria (YP_678187) 430 aa; Fi.suc, Fibrobacter succinogenes subsp. succinogenes S85, Fibrobacteres (YP_003248595)632 aa; Ba.ova, Bacteroides ovatus ATCC 8483, Bacteroidetes, Bacteroidia (ZP_02064940) 630 aa; Bl.mar, Blastopirellula marina DSM 3645, Planctomycetes(ZP_01093671) 637 aa; So.usi, Solibacter usitatus Ellin6076, Acidobacteria (YP_828880) 637 aa; Bd.bac, Bdellovibrio bacteriovorus HD100, �-Proteobacteria,Bdellovibrionales (NP_967360) 662 aa; Cl.cel, C. cellulolyticum H10, Firmicutes (YP_002505457) 639 aa; De.ace, Denitrovibrio acetiphilus DSM 12809, Deferrib-acteres (ZP_03907069) 641 aa; Ne.men, Neisseria meningitidis Z2491, �-Proteobacteria (NP_284719) 630 aa; Es.col, E. coli str. K-12 substr. MG1655, �-Pro-teobacteria, Enterobacteriales (NP_417413) 658 aa; Ge.aur, Gemmatimonas aurantiaca T-27, Gemmatimonadetes (YP_002761563) 652 aa; Pa.aca,Parachlamydia acanthamoebae str. Hall’s coccus, Chlamydiae (ZP_06300561) 623 aa; Ma.sp., Magnetococcus sp. MC-1, �-Proteobacteria (YP_864648) 644 aa;Al.pro, �-proteobacterium HTCC2255, �-Proteobacteria, Rhodobacterales (ZP_01448245) 644 aa; Le.ara, Lentisphaera araneosa HTCC2155, Lentisphaerae(ZP_01873670) 631 aa; An.sp., Anaeromyxobacter sp. K, �-Proteobacteria, Myxococcales (ZP_02174099) 654 aa; Op.ter, Opitutus terrae PB90-1, Verrucomicro-bia (YP_001820343) 645 aa; Rh.mar, R. marinus DSM 4252, Bacteroidetes, Sphingobacteria (YP_003290689) 653 aa; De.rad, Deinococcus radiodurans R1,Deinococcus-Thermus, Deinococci (NP_293967) 662 aa; Th.the, Thermus thermophilus HB27, Deinococcus-Thermus, Deinococci (YP_005246) 630 aa; Pr.mar,Prochlorococcus marinus str. MIT 9211, Cyanobacteria, Prochlorales (YP_001549937) 648 aa; Pa.chr, Paulinella chromatophora, Eukaryota, Rhizaria, Cercozoa(ACB42807) 640 aa; An.var, A. variabilis ATCC 29413, Cyanobacteria, Nostocales (YP_323925) 671 aa; Or.sat, Oryza sativa (rice; japonica cultivar group) Eu-karyota, Archaeplastidia, Streptophyta (NP_001056695) 702 aa; Ar.tha, Arabidopsis thaliana, Eukaryota, Archaeplastidia, Streptophyta (AAB09723) 702 aa;Ph.pat, Physcomitrella patens subsp. patens, Eukaryota, Archaeplastidia, Streptophyta (XP_001754665) 668 aa; Mi.pus, M. pusilla CCMP1545, Eukaryota, Ar-chaeplastidia, Chlorophyta (EEH59440) 602 aa; Me.the, M. thermophila PT, Archaea, Euryarchaeota, Methanomicrobia (YP_843948) 643 aa; Su.sp., Sulfurovumsp. NBC37–1, �-Proteobacteria, Sulfurovum (YP_001358670) 626 aa; He.pyl, H. pylori Shi470, �-Proteobacteria, Campylobacterales (YP_001910502); 615 aa;Fr.tul, Francisella tularensis subsp. tularensis SCHU S4, �-Proteobacteria, Thiotrichales (YP_169472) 469 aa; St.mar, S. marinus F1, Archaea, Crenarchaeota,Thermoprotei, Desulfurococcales (YP_001040225) 589 aa; De.kam, D. kamchatkensis 1221n, Archaea, Crenarchaeota, Thermoprotei, Desulfurococcales(YP_002428344) 592 aa; Vi.vLO, V. vulnificus CMCP6, �-Proteobacteria, Vibrionales (NP_762948) 419 aa; Se.rLO, S. ruminantium, Firmicutes, Clostridia(BAA24923) 393 aa; Tr.bru, T. brucei, Eukaryota, Euglenozoa, (AAA30218) 445 aa; Mu.mus, Mus musculus, Eukaryota, Metazoa, Chordata (P00860) 461 aa; Sc.pom,Schizosaccharomyces pombe, Eukaryota, Fungi, Ascomycota (NP_594665) 432 aa; Da.str, Datura stramonium, Eukaryota, Archaeplastidia, Streptophyta(P50134) 431 aa; *PBCV-1, P. bursaria Chlorella virus 1, Phycodnaviridae, Chlorovirus (NP_048554) 372 aa; Ps.aer, P. aeruginosa PAO1, �-Proteobacteria,Pseudomonadales (NP_253209) 387 aa; Me.maz, Methanosarcina mazei Go1, Archaea, Euryarchaeota, Methanomicrobia (NP_635209) 390 aa; Me.jan, M. jann-aschii DSM 2661, Archaea, Euryarchaeota, Methanococci (NP_248090) 438 aa; Es.coD, E. coli str. K-12 substr. DH10B, �-Proteobacteria, Enterobacteriales(YP_001731726) 420 aa; My.tub, M. tuberculosis, Actinobacteria (AAA25361) 446 aa. *, the P. bursaria Chlorella virus 1 ADC has evolved relatively recentlyfrom an ornithine decarboxylase.

TABLE 1Kinetic constants for C. auranticus and G. forsetti ADCs

Enzyme pH Temperature kcat Km kcat/Km

°C s�1 mM M�1 s�1

C. auranticus ADC 6.5 70 11 � 0.4 0.59 � 0.07 18000 � 2000G. forsetii ADC 8.5 30 0.48 � 0.02 2.2 � 0.2 220 � 20



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ADCs but is more similar to the long form ADC. It is foundin a gene cluster containing an AIH gene disrupted by aframeshift (Fig. 4), with the implication that the ADC maynot function for putrescine biosynthesis in these species.Gene clusters containing long and short form AR-fold speAORFs together with other polyamine metabolic genes arecommon (Fig. 4).

Ancestral Biosynthetic Arginine Decarboxylases of the As-partate Aminotransferase Fold—The E. coli AAT-fold ADC isan acid-inducible enzyme with a monomer size of 756 aminoacids. This ADC activity is not involved in polyamine biosyn-thesis but in an acid resistance system. Five AAT-fold basicamino acid decarboxylases are encoded in the E. coli genome:the acid-inducible ADC, acid-inducible and constitutive

FIGURE 4. Polyamine-related gene clusters/operons containing long or short form AR-fold ADC orthologues. The dark green bar within the longerlight green ADC open reading frames represents the interdomain insertion in the long form AR-fold ADC. ADC open reading frames without the dark greenbar represent the short form AR-fold ADC orthologues. CASDH, carboxyspermidine dehydrogenase; CASDC, carboxyspermidine decarboxylase; hypo, hypo-thetical protein; potD, spermidine/putrescine substrate-binding protein; DHS, deoxyhypusine synthase; SpdSyn, spermidine synthase; AdoMetDC 1B, class 1B(similar to AdoMetDC of Themotoga maritima).



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LDCs, and acid-inducible and constitutive ODCs. Each decar-boxylase monomer is roughly of the same size: acid-inducibleADC, 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 similarityto the E. coli AAT-fold LDC was identified previously in thefirmicute bacterium B. subtilis (48). The biosynthetic AAT-fold ADC enzyme identity was established by disruption ofthe encoding speA gene and subsequent analysis of polyaminelevels in the gene knock-out strain (48). No biochemical char-acterization of the enzyme was performed in that study (48);therefore, we characterized the kinetic behavior of the B. sub-tilis 168 biosynthetic ADC (NP_389346), cloned fromgenomic DNA and expressed from pET21a in E. coli and puri-fied as an N-terminal T7-tagged fusion protein. The B. subtilisbiosynthetic ADC was found to have an optimal pH of 7.7 andoptimal temperature of �75 °C (supplemental Fig. S3), and at37 °C, the Km of the substrate arginine was 0.63 mM and thekcat was 0.21 s�1 (Table 2). Many of the firmicute bacteriacontain two AAT-fold basic amino acid decarboxylase paral-ogues. The second paralogue in B. subtilis, encoded by theyaaO gene, was shown previously to play no detectable role inpolyamine biosynthesis (48). There are two AAT-fold basicamino acid decarboxylase paralogues in the genome of C. dif-ficile 630 (YP_001087362 and YP_001090072). TheYP_001087362 open reading frame is clustered immediatelyadjacent to open reading frames orthologous to the polyaminebiosynthetic enzymes AdoMetDC, spermidine synthase, andAUH (Fig. 5). We therefore expressed YP_001087362 as anN-terminally T7-tagged protein in E. coli and purified the re-combinant enzyme. The C. difficile YP_001087362 protein is afunctional ADC (Table 2) but with relatively low efficiencycompared with the B. subtilis enzyme (Km � 3.3 mM, kcat �0.018 s�1 at 60 °C). Another AAT-fold ADC was recentlyidentified in the firmicute Selenomonas ruminantium (49). ItsKm for arginine was even higher than the C. difficile ADC, at5.6 mM, and the optimal temperature was 60 °C.

Biosynthetic AAT-fold ADCs from the Firmicutes are con-siderably shorter than the acid-inducible AAT-fold ADC fromE. coli and the biosynthetic AAT-fold ODC from Lactobacil-lus 30a (S. ruminantium ADC, 485 aa (BAD80720); B. subtilisADC, 490 aa (P21885); and C. difficile ADC, 491 aa(YP_001087362)). A crystal structure of the Lactobacillus 30aODC revealed that the functional unit is a dimer, with theactive sites formed across the interface between the twomonomers. The enzyme assembles into a dodecamer com-posed of six dimers (50). Similarly, the acid-inducible ADC ofE. coli is a decamer composed of five dimers (51). Monomerforms of both the E. coli acid-inducible ADC and the Lactoba-

cillus 30a biosynthetic ODC are composed of five structuraldomains: the wing, linker, PLP-binding region, the aspartateaminotransferase-like small domain, and the C terminus (50,51) (supplemental Fig. S4). The wing domain is required formultimeric assembly (Fig. 1B) and is also required for acidinduction of activity in the E. coli ADC (51). Lactobacillus 30abiosynthetic ODC is very similar in amino acid sequence toE. coli biosynthetic and acid-inducible ODCs, and probablythe Lactobacillus 30a ODC was acquired by horizontal trans-fer because only the Lactobacilli within the Firmicutes pos-sess the long form biosynthetic AAT-fold ODC. When thefirmicute B. subtilis, C. difficile, and S. ruminantium biosyn-thetic ADCs are aligned with the Lactobacillus 30a ODC andacid-inducible ADC of E. coli, it is clear that the firmicute bio-synthetic ADCs lack completely the wing domain of the E. coliand Lactobacillus 30a enzymes (supplemental Fig. S4). In ad-dition, the biosynthetic AAT-fold ADCs have small deletionsin each of the other four domains relative to the E. coli acid-inducible ADC and Lactobacillus 30a ODC enzymes (supple-mental Fig. S4).A sequence comparison analysis using BLASTP of either

the E. coli acid-inducible ADC or the Lactobacillus 30a ODCshows clearly that the wing domain of both proteins exhibitssequence homology to the signal receiver domains of re-sponse regulators, such as CheY (so-called REC domains). Analignment of the E. coli acid-inducible ADC wing domain withREC domains of diverse response regulator proteins is shownin supplemental Fig. S5. A number of aspartate residues areconserved between the ADC wing domain and the other RECdomains and may be involved in metal binding. Comparisonof the crystal structure of the acid-inducible ADC wing do-main with other structures, using the Vector AlignmentSearch Tool at the National Center for Biological Informa-tion, shows an even greater similarity than sequence-basedsearches to the structures of many diverse REC domain-con-taining proteins. It was shown previously that the wing do-main protrudes from the rest of the E. coli acid-inducibleADC protein structure (51). It is thus most likely that the ac-id-inducible ADC wing domain was acquired by fusion of aREC domain protein to the N terminus of a firmicute shortform biosynthetic AAT-fold ADC. Because the wing domainsare not found in other AAT-like proteins (51), it is very prob-able that the shorter biosynthetic ADCs found in the Firmi-cutes are ancestral to the longer acid-inducible ADC and theconstitutive and inducible LDCs and ODCs found in E. coli.Potential orthologues of the firmicute biosynthetic AAT-foldADCs are also encoded in Cyanobacteria, forming a distinctcyanobacterial clade. Genes for the corresponding enzymesfrom the filamentous cyanobacterium Anabaena variabilisATCC 29413 (YP_322672; 488 aa) and Nostoc punctiformePCC 73102 (YP_001864209; 504 aa) were synthesized withE. coli-optimized codons, expressed from pET21a in E. coli,and purified as T7-tagged proteins. Neither enzyme displayedany activity with arginine, ornithine, or lysine (results notshown), and they appear to be uninvolved in polyamine me-tabolism, similar to the AAT-fold ADC-like protein encodedby the yaaO gene of B. subtilis described above.

TABLE 2Kinetic constants for B. subtilis 168 and C. difficile 630 short AR-foldADCs

Enzyme pH Temperature Substrate kcat Km kcat/Km

°C s�1 mM M�1 s�1

B. subtilis 7.5 37 Arg 0.21 0.63 340B. subtilis 7.5 70 Arg 1.4 1.1 1300B. subtilis 7.5 70 Orn 0.04 7.5 5.6C. difficile 7.5 60 Arg 0.025 0.73 35



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The Biosynthetic AAT-fold ADC of B. subtilis Is Essential forBiofilm Development—A gene disruption mutant of theB. subtilis 168 biosynthetic ADC was made previously bySekowska et al. (48); however, the effect of the speA gene dele-tion on growth was not reported. We analyzed aerobicgrowth, in polyamine-free defined minimal medium, for boththe biosynthetic ADC gene deletion (�speA) strain made bySekowska et al. (48) and a deletion strain of the related yaaOgene (�yaaO). Deletion of either speA or yaaO had no dis-cernible effect on aerobic growth of planktonic cells in liquidmedium (Fig. 6, top), although polyamines were completelyabsent from the �speA cells (Fig. 6, bottom). This experiment

also confirmed that deletion of yaaO has no effect on poly-amine levels in �yaaO cells grown aerobically in polyamine-free medium. Polyamines are critical for biofilm formation inthe �-Proteobacteria Y. pestis and V. cholerae (37, 38, 52, 53).Therefore, the effect of disruption of speA on B. subtilis bio-film formation was examined. Phage transduction was used toreplace the speA (ADC) gene of B. subtilis NCIB3610 with the�speA region of B. subtilis 168 to facilitate biofilm analysis.The B. subtilis NCIB3610 strain forms complex and robustbiofilms in comparison with the laboratory isolate 168 (41).Polyamine depletion in the �speA gene knock-out strain re-sulted in abolition of biofilm formation using both complex

FIGURE 5. Polyamine-related gene clusters/operons containing biosynthetic AAT-fold ADC or pyruvoyl-dependent ADC orthologues. CASDH, car-boxyspermidine dehydrogenase; CASDC, carboxyspermidine decarboxylase; hypo, hypothetical protein; potD, spermidine/putrescine substrate-bindingprotein; DHS, deoxyhypusine synthase; SpdSyn, spermidine synthase; AdoMetDC 1B, class 1B (similar to AdoMetDC of Themotoga maritima); ADC, AAT-foldshort biosynthetic ADC; pADC, pyruvoyl-dependent ADC; DUF, domain of unknown function; AdoMetDC 1A, class 1A (similar to AdoMetDC of E. coli); ODC,AR-fold ornithine decarboxylase.



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colony architecture and pellicle formation as two independentmeasures of biofilm formation capability (Fig. 7). The negativeimpact on biofilm formation was fully reversed by the addi-tion of 0.5 mM agmatine or spermidine to the growth medium(Fig. 8). The addition of putrescine to the growth medium didnot restore biofilm formation, which may be due to lack ofuptake. Although biofilm formation in the B. subtilis �speANCIB3610 background was abolished (Fig. 7A), aerobicgrowth of planktonic cells of the same �speA NCIB3610strain in polyamine-free liquid growth medium was unaf-fected (Fig. 7B).The Phylogenetic Distribution of Polyamine Biosynthetic

ADCs—In addition to the AR-fold and AAT-fold pyridoxal5�-phosphate-dependent ADCs, there are two types of pyru-voyl-dependent ADCs found in archaea and some bacteria.Pyruvoyl-dependent decarboxylases autocatalytically self-process into �- and �-chains at a serine residue that generatesthe pyruvoyl cofactor. The pyruvoyl cofactor becomes the Nterminus of the �-chain. The pylADC is found predominantlyin the euryachaeota and is structurally and mechanisticallysimilar to pyruvoyl-dependent histidine decarboxylase (21,

39). Some Chlamydia species use a very divergent pylADC aspart of an acid resistance system (23, 54); however, theChlamydia enzymes are unlikely to participate in polyaminebiosynthesis. Like the PLP-dependent ADCs, the pylADCs arecommonly found in polyamine-related gene clusters/operons(Fig. 5). Among the archaea, there are pylADC orthologues in64 euryarchaeote genomes (Table 3) but in only one crenar-chaeote (Thermofilum pendens Hrk 5 (YP_920276)). One eur-yarchaeote species,Methanosaeta thermophila PT, uses a typ-ical AR-fold ADC (Figs. 2 and 3 and supplemental Fig. S1) anddoes not possess a pylADC orthologue. The pylADC appearsto be the only route for putrescine biosynthesis in the anoxy-genic, photosynthetic Chlorobi phylum (Table 3), and a sub-set of species of the Bacteroides and �-Proteobacteria classespossess pylADC orthologues. One genome each in the Acti-nobacteria, �-Proteobacteria, Planctomycetales, Elusimicro-bia, and Synergistetes phyla and two in the Acidobacteriacontain pylADC orthologues, but there is very shallow ge-nome sampling of the Elusimicrobia and Synergistetes, so py-

FIGURE 6. Growth and polyamine content of B. subtilis 168 wild type,and �speA and �yaaO gene disruption strains. Top, aerobic growth ofB. subtilis wild type, �speA, and �yaaO gene disruption strains in poly-amine-free defined minimal medium. Strains were grown overnight in Lu-ria-Bertani broth, washed twice in minimal salt medium (48), and diluted toan initial A600 nm (OD600 nm) of 0.02 in minimal salt medium. Cultures wereincubated at 37 °C on an orbital shaker (200 rpm). At 24-h intervals, cultureswere diluted 50-fold in fresh minimal salt medium. Data represent themeans of duplicate cultures � S.D. Bottom, HPLC chromatograms of poly-amines from B. subtilis cells grown in minimal salt medium. Data are fromthe same cultures used for the growth analyses. Cells from wild type and�yaaO strains were extracted with 10 �l of MOPS lysis buffer per mg of cellfresh weight; cells from the �speA strain were extracted with 2.5 �l of MOPSlysis buffer/mg of cell fresh weight. HPLC injection volumes were 5 �l for168 and yaaO and 10 �l for speA. R, fluorescent labeling dye; Spd, spermi-dine; IS, internal standard (1,7-diaminoheptane).

FIGURE 7. Differential effects of the B. subtilis �speA mutation on bio-film and planktonic cell growth. A, biofilm formation of B. subtilis NCIB3610 parental and �speA strains on solid MSgg medium. The top left imageshows an example of vigorous biofilm, and the bottom left image showsvigorous pellicle formation. B, aerobic growth in MSgg (polyamine-free)liquid medium.



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lADC in principle could be more important in these poorlysampled phyla.Most of the Crenarchaeota use an AdoMetDC paralogue to

decarboxylate arginine for polyamine biosynthesis (22) andthus contain two AdoMetDC paralogues in each genome.This unusual method for arginine decarboxylation appears tobe specific to the Crenarchaeota. Three species within theCrenarchaeota use diverged AR-fold ADCs although repletewith the typical interdomain insertion (two are shown in Figs.1 and 2 and supplemental Fig. S1). Both Desulfurococcus kam-chatkensis 1221n and Staphylothermus hellenicus DSM 12718of the Crenarchaeota possess an AR-fold ADC and only oneAdoMetDC orthologue; however, Staphylothermus marinusF1 possesses two AdoMetDC paralogues and an AR-foldADC.The biosynthetic AAT-fold ADC is found almost exclu-

sively in the Firmicutes, with 155 genomes containing an or-thologue (Table 3). There are also orthologues in two Fuso-bacteria and two Mollicutes genomes and one Spirochaete. Incontrast, the AR-fold ADC is excluded from the Firmicutesexcept for Clostridium cellulolyticum H10 and Clostridiumpapyrosolvens DSM 2782. The AR-fold ADC is most prevalentin the �- and �- and �-Proteobacteria, the Cyanobacteria, theBacteroides class of the Bacteroidetes phylum, the Verru-comicrobia, Deinococcus-Thermi, and Planctomycetales (Ta-ble 3). A smaller proportion of the �- and �-Proteobacteriaalso contain AR-fold ADC orthologues. As discussed above,the ancestral, shorter form of the AR-fold ADC lacking theinterdomain insertion is limited to members of the Chlo-roflexi and Bacteroidetes. Almost all terrestrial plants containlong form AR-fold ADC orthologues, but they are absent inalgae except for one example inMicromonas pusilla. The nu-cleus-encoded plant AR-fold ADC orthologues are derived

from the cyanobacterial ancestor of the chloroplast (11, 55).Biosynthetic ADCs remain to be identified in othereukaryotes.The clear discernable pattern in the phylogenetic distribu-

tion of the different ADC forms is that the AR-fold ADC ismostly absent from the Archaea, Firmicutes, and Actinobac-teria, which are single-membraned. The Actinobacteria ap-pear to lack any ADC orthologues (except for a pylADC or-thologue in Beutenbergia cavernae DSM 12333(YP_002880881)). The Firmicutes depend on the short AAT-fold biosynthetic ADC, and Archaea use mainly pyruvoyl-de-pendent ADCs, with the euryarchaeotes possessing the histi-dine decarboxylase-like pylADC, whereas the Crenarchaeotamostly use the AdoMetDC-derived ADC.Arginine Decarboxylase Is the Dominant Route for Poly-

amine Biosynthesis in the Human Gut Microbiota—The ge-nomes of the 55 most common bacterial species found in thehuman gut microbiota (43) were screened in silico for ortho-logues of the AR- and AAT-fold ODCs. No ODC orthologuesfrom either fold were found in these species. In contrast, bio-synthetic AR- and AAT-fold ADC genes were found in themajority of species, although 11 species appeared to be aux-otrophic for polyamine biosynthesis (Table 4). The Firmicutespecies contained the AAT-fold biosynthetic ADC, whereasthe Bacteroidetes contained the AR-fold ADC with the excep-tion of Bacteroides capillosus and B. pectinophilus, which con-tain horizontally acquired AAT-fold ADCs. The Bacteroidetesspecies A. putredinis contains an ancestral short AR-fold

FIGURE 8. Polyamine dependence of biofilm formation in B. subtilisNCIB 3610 parental and �speA strains. Effects of polyamines on biofilmrestoration in the �speA gene disruption mutant. Cells were grown onMSgg solid medium with the indicated concentration of polyamines.

TABLE 3Phylogenetic distribution of biosynthetic ADC orthologue families

Phylum/ClassLong

AR-foldShort

AR-foldShort

AAT-fold Pyruvoyl

Euryarchaeota 1 0 0 64Crenarchaeota 3 0 0 1aFirmicutes 2 0 155 10Actinobacteria 0 0 0 1�-Proteobacteria 6 0 0 0�-Proteobacteria 11 0 0 0�-Proteobacteria 206 1b 0 0�-Proteobacteria 22 0 0 11�-Proteobacteria 42 0 0 1cCyanobacteria 48 0 0 0Bacteroidetes Bacteroides 22 0 0 6Bacteroidetes Flavobacteria 0 21 0 0Bacteroidetes Sphingobacteria 1 8 0 0Chlorobi 0 0 0 17Chloroflexi 0 6 0 0Deinococcus-Thermi 6 0 0 0Verrucomicrobia 7 0 0 0Planctomycetales 5 0 0 1Acidobacteria 1 0 0 2Synergistetes 0 0 0 1Deferribacteres 2 0 0 0Gemmatimondales 1 0 0 0Fibrobacteres 1 0 0 0Lentisphaerae 1 0 0 0Elusimicrobia 0 0 0 1Spirochaetes 0 0 1 0Mollicutes 0 0 2 0Fusobacteria 0 0 2 0Chlamydias 1 0 0 12d

a Does not include the S-adenosylmethionine decarboxylase-like ADCorthologues.

b The Francisella ADC orthologue may be a highly diverged AR-fold long ADC.c This is a fusion with a phosphatidylserine decarboxylase (Sufurovum sp.NBC37-1 (YP_001358198) 613 aa).

d These are acid-inducible ADCs and are probably not involved in polyamine bio-synthesis.



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ADC. Genes encoding AIH and NCAPH are always clusteredexcept for the presence of isolated AIH-encoding genes in thefirmicute Enterococcus faecalis and the actinobacterium Co-linsella aerofaciens. At least in the case of E. faecalis, the AIH-encoding gene is involved in agmatine catabolism (56).

DISCUSSION

Arginine decarboxylation for polyamine biosynthesis hasreceived scant attention due to its absence from non-planteukaryotes. However, arginine decarboxylation in prokaryotesand plants provides a useful model for assessing the molecularprocesses shaping metabolic pathway evolution. Prominent

mechanisms involved in pathway evolution are gene duplica-tion and gene loss. The PLP-dependent AR-fold family of ba-sic amino acid decarboxylases most probably evolved by geneduplication from DAPDC, the last step in the lysine biosyn-thetic pathway. One of the AR-fold decarboxylases, the bi-functional L/ODC, is able to decarboxylate the product ofDAPDC (i.e. lysine) (16, 57). Another member of the AR-folddecarboxylases, CANSDC, is involved in extending the poly-amine pathway from a diamine to a triamine (norspermidineor spermidine). The ornithine-specific AR-fold decarboxylaseprovides a direct alternative to arginine decarboxylation forputrescine biosynthesis. Until the current study, it was

TABLE 4Arginine pathway polyamine biosynthetic genes in 55 frequently found genomes of the human gut microbiotaSpecies names are followed by B for Bacteroidales, F for Firmcutes, or A for Actinobacteria. The E. siraeum 70/3 genome is incomplete, and the ZP_02423330 gene isfound in a related strain. The genome of unknown sp. S3/4 is not available (NA).

AR-fold ADC AAT-fold ADC AUH AIH NCPAH

Bacteroides uniformis (B) ZP_02069527 Absent Absent ZP_02070604 ZP_02070605Alistipes putredinis (B) ZP_02424067a Absent ZP_02425128 Absent AbsentParabacteroides merdae (B) ZP_02034148 Absent Absent ZP_02032161 ZP_02032160Dorea longicatena (F) Absent ZP_01005018b Absent Absent AbsentRuminococcus bromii L2-63 (F) Absent CBL15803b Absent Absent AbsentBacteroides caccae (B) ZP_01961811 Absent Absent ZP_01958998 ZP_01958999Clostridium sp. SS2/1 (F) Absent ZP_02440164b Absent Absent AbsentBacteroides thetaiotaomicron VPI-5482 (B) NP_812306 Absent Absent NP_809789 NP_809788Eubacterium hallii (F) Absent ZP_03715546b Absent Absent AbsentRuminococcus torques L2–14 (F) Absent CBL25542 CBL25540 Absent AbsentUnknown sp. SS3 4 NA NA NA NA NARuminococcus sp. SR 1/5 (F) Absent CBL18584 CBL18586 Absent AbsentFaecalibacterium prausnitzii SL3/3 (F) Absent CBL02265 CBL02267 Absent AbsentRuminococcus lactaris (F) Absent ZP_03167723 ZP_03167721 Absent AbsentCollinsella aerofaciens (A) Absent Absent Absent ZP_01771916 AbsentDorea formicigenerans (F) Absent ZP_02235921b Absent Absent AbsentBacteroides vulgatus ATCC 8482 (B) YP_001298648 Absent Absent YP_001300474 YP_001300475Roseburia intestinalisM50/1 (F) Absent CBL08498 Absent CBL08502 CBL08503Bacteroides sp. 2_1_7 (B) Absent Absent Absent ZP_05285599 ZP_05285600Eubacterium siraeum 70/3 (F) Absent CBK97421 Absent ZP_02423330a CBK97306Parabacteroides distasonisATCC 8503 (B) Absent Absent Absent YP_001302561 YP_001302560Bacteroides sp. 9_1_42FAA (B) ZP_04539906 Absent Absent ZP_04540513 ZP_04540514Bacteroides ovatus (B) ZP_06616985 Absent Absent ZP_06616391 ZP_06616390Bacteroides sp. 4_3_47FAA (B) ZP_05253566 Absent Absent ZP_05254800 ZP_05254799Bacteroides sp. 2_2_4 (B) ZP_04550906 Absent Absent ZP_04548030 ZP_04548029Eubacterium rectaleM104/1 (F) Absent CBK93443 Absent CBK93439 CBK93438Bacteroides xylanisolvens XB1A (B) CBK67757 Absent Absent CBK65746 CBK65747Coprococcus comes (F) Absent ZP_03801366b Absent Absent AbsentBacteroides sp. D1 (B) ZP_04547174 Absent Absent ZP_04543700 ZP_04543701Bacteroides sp. D4 (B) ZP_04555452 Absent Absent ZP_04555788 ZP_04555787Eubacterium ventriosum (F) Absent ZP_02025094b Absent Absent AbsentBacteroides dorei (B) ZP_03300857 Absent Absent ZP_03301417 ZP_03301418Ruminococcus obeum A2–162 (F) Absent CBL22837 CBL22839 Absent AbsentSubdoligranulum variabile (F) Absent ZP_05981888 ZP_05981890 Absent AbsentBacteroides capillosus (B) Absent ZP_02036374 ZP_02036372 Absent AbsentStreptococcus thermophilus LMD-9 (F) Absent Absent Absent Absent AbsentClostridium leptum (F) Absent ZP_02080046 ZP_02080049 Absent AbsentHoldemania filiformis (F) Absent Absent Absent Absent AbsentBacteroides stercoris (B) ZP_02436242 Absent Absent ZP_02435205 ZP_02435204Coprococcus eutactus (F) Absent Absent Absent Absent AbsentClostridium sp. M62/1 (F) Absent ZP_06347603 ZP_06347601 Absent AbsentBacteroides eggerthii (B) ZP_03459356 ZP_03459720 Absent AbsentButyvibrio crossotus (F) Absent ZP_05792554 Absent ZP_05792550 ZP_05792549Bacteroides finegoldii (B) ZP_05416620 Absent Absent ZP_05415565 ZP_05415564Parabacteroides johnsonii (B) ZP_03477317 Absent Absent ZP_03476338 ZP_03476339Clostridium sp. L2-50 (F) Absent Absent Absent ZP_02075719 ZP_02075457Clostridium nexile (F) Absent ZP_03289957 ZP_03289955 Absent AbsentBacteroides pectinophilus (B) Absent ZP_03461361 Absent ZP_03461425 ZP_03461426Anaerotruncus colihominis (F) Absent Absent Absent Absent AbsentRuminococcus gnavus (F) Absent ZP_02043066 ZP_02043062 Absent AbsentBacteroides intestinalis (B) ZP_03013847 Absent Absent ZP_03014439 ZP_03014440Bacteroides fragilis 3_1_12 (B) ZP_05282264 Absent Absent ZP_05281501 ZP_05281500Clostridium asparagiforme (F) Absent Absent Absent Absent AbsentEnterococcus faecalis TX0104 (F) Absent Absent Absent ZP_03947749 AbsentClostridium scindens (F) Absent Absent Absent Absent AbsentBlautia hansenii (F) Absent ZP_05853960 ZP_05853962 Absent Absent

a Short ancestral form of AR-fold ADC.b Probably a yaaO orthologue.



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thought that ADC was anomolous within the AR-fold decar-boxylase family because it is considerably longer than theother members. This is primarily due to a single large inser-tion. The size of the insertion is conserved across prokaryoticphyla, but the amino acid sequence of the insertion is muchless conserved (shown in Fig. 3), suggesting a structural ratherthan catalytic role for the insertion domain. Recently, the in-sertion domain in the AR-fold ADC x-ray crystal structurehas been shown to be a four-helix bundle that is responsiblefor the AR-fold ADC being a tetramer rather than a dimer likethe rest of the AR-fold decarboxylase family (20). The shorterform of AR-fold ADC present in the Chloroflexi and Bacte-roidetes lacks the four-helix bundle domain insertion andthus represents an ancestral form of AR-fold ADC. It is thesame size as DAPDC and may represent a form intermediatebetween DAPDC and the AR-fold long ADC. The four-helixbundle is located precisely between the N-terminal �/� do-main and the C-terminal �-barrel domain. AR-fold decar-boxylases are obligate homodimers with two identical activesites, each formed from residues across the dimer interfacebetween the N-terminal �/� domain of one monomer andthe C-terminal �-barrel domain of the other. The positionof the four-helix bundle insertion is probably the only place inthe enzyme where an insertion would not disrupt enzymaticfunction. The related ODC enzyme can be split at this sameposition, and the resulting N- and C-terminal domains can beco-expressed and a functional ODC enzyme can be reconsti-tuted in vivo from the two separated domains (58). We do notknow whether the short ADC lacking the four-helix bundlewas widespread in bacteria and was then subsequently re-placed by the long form or whether the long form ADC aroseearly in bacterial evolution. The Chloroflexi and Sphingobac-teria/Flavobacteria are not thought to be phylogeneticallyclose. If there was not a widespread loss of the short formADC, then there was probably horizontal transfer of the shortform ADC between the two phyla. It appears that the longform ADC is present in all land plants and that this is a case ofendosymbiotic gene transfer from the cyanobacterial ancestorof the chloroplast.In the case of the pyridoxal 5�-phosphate-dependent AAT-

fold basic amino acid decarboxylases and in particular theacid-inducible ADC, gene fusion was a key mechanism in theevolution of biological function. The most plausible evolu-tionary scenario is that the short biosynthetic AAT-fold ADC,typically found in firmicutes, such as B. subtilis, C. difficile,and S. ruminantium, was the ancestral form of the acid-in-ducible AAT-fold ADC enzyme. In S. ruminantium, the shortform AAT-fold ADC is a dimer (49). A response regulatorreceiver domain protein was then fused to the N terminus endof the short biosynthetic AAT-fold ADC, thereby forming thewing domain of the acid-inducible AAT-fold ADC. The wingdomain is responsible for decamer formation (a pentamer ofhomodimers) of the acid-inducible ADC due to the role of thewing domain in forming interactions between the ho-modimers (51). In the E. coli acid-inducible ADC, which hasthe N-terminal wing domain, decamer formation is requiredfor enzyme activity in acidic conditions. At neutral pH, thenegative charge associated with the wing domain causes dis-

sociation of the decamer and loss of enzymatic activity. Incontrast, at acidic pH, the negative charge of the wing domainis neutralized, leading to decamer formation and catalyticactivation (51). Only the decamer form is active, and thedimer is inactive until decamer formation. The wing domaincan therefore be seen to be acting as a typical response regula-tor; the presence of the receiver domain in the acid-inducibleAAT-fold ADC confers environmental responsiveness (i.e.acid inducibility) to the output domain (i.e. the ADC activity).Besides the AR-fold and AAT-fold ADCs, there are also the

two pyruvoyl-dependent ADCs, the pylADC and theAdoMetDC-like ADC. Thus, there are at least four differentprotein folds and two different cofactors representing the cat-alytic activity EC 4.1.1.19 (ADC). There are three forms of theAR-fold ADC: the ancestral short form described herein, thelong form with four-helix bundle insertion, and the chlorovi-rus ADC which has evolved from the AR-fold ODC (24, 25).In addition, there are two forms of the AAT-fold ADC, thefirmicute biosynthetic ADC and the E. coli acid-inducibleADC; there are biosynthetic and acid-inducible forms ofthe pylADC (21, 23, 39) and the one known form of theAdoMetDC-like ADC. There are thus four protein folds andeight different enzyme forms represented by the descriptionEC 4.1.1.19. This is a rather extreme form of what have beenrecently described as, “non-homologous isofunctional en-zymes” (8), enzymes carrying out the same chemistry butfrom entirely different evolutionary origins. The multiplicityof ADC forms indicates the importance of arginine decarbox-ylation and polyamine biosynthesis for life because differentenzymatic solutions for arginine decarboxylation have arisenindependently multiple times during evolution.Polyamine-related gene clusters encoding either the AR-

fold, AAT-fold, or pyruvoyl-dependent ADCs are commonlyfound in prokaryotic genomes. These gene clusters exhibit aproperty that has been noted generally for gene clusters andoperons (i.e. there is a tendency for the genes of the cluster tobe arranged in the biochemical reaction order, known as co-linearity) (59, 60). In the ADC-containing gene clusters, thereis a marked tendency for the speAORF to be at the beginningof the cluster (Figs. 4 and 5). Another phenomenon that isrevealed in the ADC-containing gene clusters is the modular-ity of polyamine biosynthesis. Not only are there alternativeADC enzymes, but each form of speA involved in polyaminebiosynthesis can be clustered with either agmatine ureohy-drolase (agmatinase) or with agmatine deiminase and N-car-bamoylputrescine amidohydrolase, as part of a putrescinebiosynthetic module. The clusters may also include the car-boxy(nor)spermidine dehydrogenase/CANSDC pairs or theAdoMetDC/spermidine synthase pairs for the spermidinebiosynthetic module. Some of the clusters contain a deoxyhy-pusine synthase-like ORF that is probably an alternative ho-mospermidine synthase (10).The phylogenetic distribution of the different forms of

ADC establishes a distinct pattern. Both the short and longform AR-fold ADCs are limited to double-membraned bacte-ria except for two Clostridia species where the genes havebeen acquired by horizontal transfer. Biosynthetic AAT-foldADC is limited to the Firmicutes (single-membraned bacte-



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ria). Actinobacteria (single-membraned bacteria) do not con-tain ADC except for one genome. Pyruvoyl-dependent ADCis the predominant ADC of the Euryarchaeota (single-mem-braned), although it is found in some bacterial genomes aswell, and the AdoMetDC-like ADC is found only in the Cren-archaeota (single-membraned). Prominent hypotheses for theroot of the tree of life place the root near or in the Chloroflexi(61); between clades consisting of actinobacteria/double-membraned bacteria and firmicutes/archaea (62); or nowhere,due to insufficient support for a tree of life but with the majorprokaryotic division being between bacteria and archaea (63).The phylogenetic distribution of the different ADC formsrecapitulates the major divisions of life: bacteria, archaea, andeukaryotes. Except for plants, eukaryotes do not possess apolyamine biosynthetic ADC. Double-membraned bacteriaare distinguished from single-membraned bacteria by differ-ent ADC forms. In single-membraned bacteria, Firmicutes aredistinguished from Actinobacteria. The Euryarchaeotes aredistinguished from Crenarchaeotes, and the archaea are dis-tinguished from bacteria. Bacteria mainly use PLP-dependentADCs, and Archaea predominantly use pyruvoyl-dependentADCs. The alternative route for putrescine biosynthesis,ODC, is of limited phylogenetic distribution in prokaryotes,mainly in Proteobacteria, a few Actinobacteria, and Lactoba-cilli. Decarboxylation of arginine is the dominant mode ofpolyamine biosynthesis in bacteria and archaea. This domi-nance of the ADC pathway for polyamine biosynthesis in bac-teria is exemplified by the human gut microbiota, where ADCis the only route for polyamine biosynthesis among the 55most common gut microbiota species. Because human cellscontain only the ODC pathway for polyamine biosynthesis,pharmacological manipulation of ADC activity might permitreorganization of the gut microbiota population.Although polyamines are found in almost all bacteria, it is

not clear what the core physiological requirements for poly-amines are. We have shown that ADC and, therefore, poly-amines are essential for biofilm production in the single-membraned bacterium B. subtilis. It is already known thatpolyamines are required for biofilm production in the double-membraned bacteria Y. pestis and V. cholerae. However, in theY. pestis polyamine biosynthetic knock-out strain, putrescineand not spermidine restored biofilm formation, whereas inV. cholerae, sym-norspermidine but not spermidine was re-quired. In B. subtilis, spermidine is able to restore biofilm for-mation. Because 90% of spermidine is bound to RNA in E. coli(64), it is interesting to note that in B. subtilis biofilm forma-tion is dependent upon spermidine and is controlled by a reg-ulatory RNA mechanism (65).

Acknowledgment—We thank Meg Phillips (University of TexasSouthwestern Medical Center, Dallas, TX) for help with preparationof Fig. 1 and for constructive comments on the manuscript.

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evolution and multiplicity of arginine decarboxylases in polyamine biosynthesis and essential role...

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