J. Gen. Appl. Microbiol., 58, 339‒347 (2012)

Short Communication

Covalently linked polyamines in the peptidoglycan of the anaerobes belonging to the order

Koei Hamana,1,* Takashi Itoh,2 Mitsuo Sakamoto,2 and Hidenori Hayashi 1

1 Faculty of Engineering, Maebashi Institute of Technology, Maebashi, Gunma 371‒0816, Japan 2 Japan Collection of Microorganisms, RIKEN BioResource Center (BRC), Wako, Saitama 351‒0198, Japan

(Received May 1, 2012; Accepted May 20, 2012)

Key Words—Acidaminococcaceae; peptidoglycan; polyamine; Selenomonadales;

In our studies on cellular polyamine analysis in bac- outer membrane of the envelope in typical Gram-neg- teria (the domain ) as a source of chemotaxo- ative taxa (Takatsuka and Kamio, 2004; Kamio et al., nomic information (Hamana, 1999a, 2002a; Hamana 2007). On the other hand, intracellular polyamine and Hosoya, 2006; Hamana and Matsuzaki, 1992; Ha- pools in these Gram-negative anaerobes were not re- mana et al., 2006; Hosoya et al., 2006), covalently fl ected in their covalently linked polyamine levels (Ha- linked putrescine, cadaverine, spermidine and/or ag- mana, 1999b; Hamana et al., 2002). The relation be- matine in cell wall peptidoglycan have been found in tween acid-extractable ionic-binding polyamines and Gram-negative anaerobes belonging to the 13 genera covalently linked polyamine components should be , , , Anaerovi- presented for the evaluation of bacterial polyamine brio, , , , distribution profi les as a chemotaxonomic marker. To , Propionispira, Selenomonas, , obtain additional distribution catalogues of covalently and (Hamana, 1999b; Hamana linked polyamines within the order Selenomonadales et al., 2002). It has been revealed that one of these consisting of the two families Acidaminococcaceae polyamines binds covalently to the α-carboxy group of and Veillonellaceae (NCBI website, 2012), polyamines the D-glutamic acid residue of a peptidoglycan in Sele- of 17 new including the fi ve newly validated nomonas ruminantium, and An- genera , , Mitsuokella, aerovibrio lipolytica (Hirao et al., 2000; Kamio, 1987; and Sporolituus in the order Selenom- Kamio and Nakamura, 1987; Kamio et al., 1981; Taka- onadales were analyzed. Polyamines in the acid-ex- tsuka and Kamio, 2004). These Gram-negative anaer- tracts from whole cells and in the acid-hydolysates of obes belonging to the order Selenomonadales (for- cell residues after the acid extraction, were determined merly the Sporomusa subbranch), phylogenetically after growth at different temperatures and in culture located in the phylum , a Gram-positive tax- media supplemented with diaminopropane, diamino- on (NCBI website, 2012), however, lack murein lipo- hexane, spermidine, spermine or agmatine. Seven protein which maintains the structural integrity of the Gram-positive anaerobes belonging to the order Clostridiales of the phylum Firmicutes were used as a related anaerobic bacterium. * Address reprint requests to: Dr. Koei Hamana, Faculty of Engineering, Maebashi Institute of Technology, Maebashi, Gun- The organisms were provided by the RIKEN BRC ma 371‒0816, Japan. through the National Bio-Resource Project of the E-mail: [email protected] MEXT, Japan and grown anaerobically at 25‒60°C in 340 HAMANA et al. Vol. 58

Fig. 1. HPLC analysis of the PCA extract (PCA) and the HCl-hydrolysate of the cell residue after the PCA extraction (HCl-Res) of Selenomonas fl ueggei JCM 8544 (1) and Veillonella ratti JCM 6512 (2) grown at 37°C in GAM medium in the absence (control) (A), presence of 5 mM agmatine (B), 5 mM diaminopropane (C) or 5 mM diaminohexane (D). Abbreviations for polyamines are shown in Table 1.

GAM Broth (GAM) (Nissui Pharmaceutical Co., Tokyo, website, 2012), using Anaerobic Jar KJ-1 (Tomy Seiko,

Japan), Modifi ed GAM Broth (MGAM) (Nissui), Tokyo) under H2 gas. Cells in the stationary phase polyamine-free /arginine-rich synthetic RPMI 1640 me- were harvested from a 50‒100 ml liquid culture by cen- dium (Nissui) (Hamana et al., 2007) supplemented trifugation at 3,000 × g and washed fi ve times with an with 0.03% L-cysteine and 0.03% sodium thioglycol- isotonic saline or PBS (Nissui Pharmaceutical Co.). late (1640-Cys-TG) and JCM media (listed on the JCM The cell pellets were homogenized in equal volumes 2012 Polyamines in Selenomonadales 341 Put polyamines e Covalently linked mol/g wet weight) μ , and covalently linked polyamines. Firmicutes Polyamines ( Polyamines Put Cad Spd Spm Agm Dap Dah of the phylum (Ref.) Fraction HCl-Res 0.26 0.74 0.03 0.00 0.05 0.00 0.00 HCl-Res 0.35 0.85HCl-Res 0.30 0.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00HCl-Res 0.00 0.17 0.85 0.02 0.00 0.06 0.00HCl-Res 0.00 0.26 0.74 0.03 0.00 0.05 0.00 0.00 HCl-Res 0.00HCl-Res 0.15 0.00 0.00HCl-Res 0.15 0.00 0.00 0.00 0.00 0.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.00 0.84 0.00 0.00 0.00 0.00 0.00 Clostridiales C) and ° ( Temp. GAMGAM 37GAMGAM (a) 37GAM 37 37GAM (a) (a) 37 (b) 37 (b) (b)GAMGAM 37GAM Put, Cad, Spd 30 PCA 30 0.00 (a)GAM 0.40 Put, Cad GAM (a) Cad, Spd 0.05 Put, Cad, Spd 0.00 30 0.00 30 0.00 Cad (a) 0.00 (a) Cad Put, Cad Put, Cad Cad Put, Cad MGAMMGAMMGAM 25 30 PCA 25 0.00 (a) PCA 0.04 0.12 0.90MGAM 0.00 0.03 0.00 0.05 25 0.00 0.00 0.00 0.00 PCA 0.00 Put, Cad, Spd MGAM 0.00 0.52MGAM 0.53 25 0.65 Put 0.00 25 PCA 0.05 0.00 PCA 0.60 0.00 0.45 0.60 0.85 0.45 0.01 Put, Cad 0.85 0.05 Put, Cad 0.00 0.00 0.05 0.00 0.00 0.00 Put, Cad Put, Cad JCM282JCM282 30JCM282 30 PCA 30 PCA 0.05 0.54 PCA 0.05 0.00 0.45 0.00 0.02 0.00 0.00 0.33 0.07 0.00 0.00 0.05 0.00 0.00 0.00 0.00 0.00 0.00 Cad 0.00 Cad Cad Selenomonadales ATCC1249 37 (a) Cad T T T T T T T T T T T T T T T T T T T T ATCC 25974 ATCC ) ATCC 25085 ATCC JCM 12564 ATCC 43704 ATCC T ATCC 51455 ATCC NCIMB12297 ATCC 25560 ATCC JCM 17568 Acid-extractable polyamines from cells and released by acid-hydrolysis cell residue after acid-extraction in th JCM 17567 ) JCM 6130 JCM 6129 GAM 30 (a) Put, Cad ATCC 33276 ATCC JCM 15077 ) JCM 10004 JCM 14723

ATCC 33332 ATCC JCM 1772 JCM 2054 ATCC 51177 ATCC JCM 12226 quercicolus

ATCC 51454 ATCC polytropa

fermentans

anaerobes belonging to the orders Organism Med. JCM 17566 acidaminophila Table 1. Table

cerevisiae cerevisiae elsdenii lypolytica

multiacidus) hypermegas)

burkinobensis lypolyticus glycerini acidaminophila hypermegale funiformis

quercicolus

longum jalaludinii multacida

frisingensis cerevissiiphilus

pneumosintes invisus succinatiphilus microaerophilus propionicifaciens

Veillonellaceae Acidaminococcaceae Selenomonadales Anaerovibrio Selenomonas Clostridium Anaeromusa Mitsuokella Dialister Dialister Megamonas Pectinatus Dialister (Bacteroides Dialister Dialister Megamonas Anaerovibrio Megasphaera Dendrosporobacter Desulfosporomusa Acetonema Anaerovibrio ( ( ( (Bacteroides Megasphaera Megasphaera Mitsuokella Pectinatus Anaerovibrio Acidaminococcus Family Family Order Family 342 HAMANA et al. Vol. 58 polyamines Covalently linked mol/g wet weight) μ Polyamines ( Polyamines Put Cad Spd Spm Agm Dap Dah (Ref.) Fraction HCl-Res 0.00HCl-Res 0.02 0.35 0.05 0.01 0.00 0.13 0.10 0.00 0.00HCl-Res 0.34 1.02 0.00 0.00 0.00 0.00 0.10 0.00 0.20 0.00 0.61 HCl-Res 0.02 1.40 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.15 0.85 0.01 0.00 0.00 0.00 0.00 HCl-Res 0.03 0.10 0.06 0.00 0.04 0.00 0.00 HCl-Res 0.08HCl-Res 0.07 0.02 0.16 0.02 0.00 0.20 0.78 0.00 0.00 0.70 0.00 0.00 0.00 HCl-Res 0.00HCl-Res 0.12 0.00 0.00 0.15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.01 0.00 0.10 0.00 0.58 0.06HCl-Res 0.00 0.18 0.02 0.15 0.00 0.38 0.10 0.00 HCl-Res 0.49 0.14 0.25 0.00 0.35 0.00HCl-Res 0.00 0.70 0.04 0.10 0.00 0.60 0.00 0.00 HCl-Res(b) 0.53 0.15 0.70 0.00 0.36 0.00 0.00 HCl-Res (b) 0.26 1.35 0.03 0.00 0.00 0.00 0.00 Table 1(Continued) Table C) ° ( Temp. Dah 37 PCA 0.00 0.00 Dah 0.20 25 0.00 0.00 PCA 0.00 0.67 0.00 0.00 Spd, Agm, Dah 0.13 0.00 0.03 0.00 0.60 Spd, Agm, Dah Spd 37 PCA 0.30 0.10 0.50 0.00 0.00 0.00 0.00 Cad, Spd Dap 37 PCA 0.20 0.00 Dap 0.51 25 0.00 0.30 PCA 0.27 0.00 0.05 0.01 Spd, Agm, Dap 0.55 0.00 0.03 0.17 0.00 Put, Spd, Agm, Dap Agm 37 PCA 0.41 0.02 Agm 0.22 25 0.00 0.42 PCA 0.00 0.00 0.30 Put, Cad, Spd, Agm 0.00 0.35 0.00 0.25 0.00 0.00 Put, Spd, Agm M M M M M M M GAMGAM 30GAM (a) 30GAMGAM (b) 30 30 (b) 25 PCA (b) 0.05 PCA 0.07 0.76 0.03 0.00 0.02 0.01 0.14 0.00 0.00 0.00 0.30 Put, Cad, Spd, Agm 0.00 0.00 Put, Cad, Spd, Agm GAMGAM Put, Cad, Spd GAM 30GAM 30 30 Cad (b) (b) 25 PCA (b) Cad, Spd 0.35 PCA 1.45 1.00 0.00 0.00 1.52 0.00 0.07 0.00 0.00 0.00 0.02 0.00 0.00 Put, Cad Cad Put, Cad, Spd Put, Cad MGAM 25 PCA 0.00 0.05 0.20 0.00 0.06 0.00 0.00 Cad JCM841 30 PCA 0.00 0.03 0.20 0.00 0.00 0.00 0.00 Put, Cad GAM (control) 25 PCA 0.28 0.01 0.80 0.01 0.04 0.00 0.00 Put, Spd, Agm GAM (control) 37 PCA 0.01 0.02 0.25 0.00 0.42 0.00 0.00 Spd, Agm GAM (control) 37 PCA 0.10 0.85 0.02 0.00 0.02 0.00 0.00 Cad GAM+5 m GAM+5 m GAM+5 m GAM+5 m GAM+5 m GAM+5 m GAM+5 m T T T T T T T T T JCM 6582 JCM 17185 JCM 8543 JCM 8544 JCM 8542 ATCC 33732 ATCC JCM 8545 JCM 15470 JCM 8546 Organism Med. vibrioides infelix dianae artemidis bovis ueggei fl noxia ruminantium

arboris

Selenomonas Selenomonas Selenomonas Propionispora Selenomonas Selenomonas Selenomonas Selenomonas Propionispira 2012 Polyamines in Selenomonadales 343 polyamines Covalently linked mol/g wet weight) μ Polyamines ( Polyamines Put Cad Spd Spm Agm Dap Dah (Ref.) Fraction HCl-Res 0.64 0.25 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.70 0.55HCl-Res 0.00 0.36 0.00 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.60 0.40 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.01 0.46 0.07 0.00 0.00 0.00 0.00 HCl-Res 0.80 0.20 0.02 0.00 0.00 0.00 0.00 HCl-Res 0.02 0.35 0.42 0.00 0.68 0.00 0.00 HCl-Res 0.88 0.35 0.02 0.00 0.02 0.00 0.00 HCl-Res 0.35HCl-Res 0.15 0.00 0.00HCl-Res 0.15 0.00 0.00 0.00HCl-Res 0.00 0.20 0.00 0.00 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.04 0.00 0.00 0.00 HCl-Res (b) 0.44 0.32 0.60 0.00 0.20 0.00 0.00 Table 1 (Continued) Table C) ° ( Temp. Spd 37 PCA 0.24 0.00 0.98 0.00 0.00 0.00 0.00 Put, Cad Agm 37 PCA 0.20 0.00 0.30 0.00 0.19 0.00 0.00 Put, Cad Spm 37 PCA 0.17 0.00 0.23 0.36 0.00 0.00 0.00 Put, Cad M M M GAMGAM 30 37 PCA (b) 0.00 PCA 0.00 1.40 0.00 0.00GAM 0.00GAM 0.00 0.40GAM 0.00GAM 0.00 30 0.00 0.10 30 Put, Cad, Spd, Agm 30 0.00 (a) 30 0.00 (a) (a) Cad, Spd, Agm (a)GAM 30 (a) Put, Cad Put, Cad Put, Cad Put, Cad Put, Cad MGAMMGAM 25 PCA 25 0.22 PCA 0.75 0.16 0.40 0.00 0.02 0.08 0.65 0.00 0.00 0.00 0.02 0.00 0.00 Put, Cad Put MGAMMGAM 25 37 PCA PCA 0.02 0.80 0.75 0.12 1.07 0.00 0.07 0.00 0.03 0.00 0.00 0.00 0.00 0.00 Put, Cad Cad JCM718 50 PCA 0.02 0.00 0.50 0.30 0.00 0.00 0.00 Cad, Spd ATCC1819ATCC1819ATCC1425 30ATCC1425 30ATCC1425 30 (a) 30 (a) 30 (a) (a) (a) Cad Put, Cad Put, Cad Put, Cad Put, Cad 1640-Cys-TG1640-Cys-TG 25 37 PCA PCA 0.00 0.77 0.00 0.03 0.40 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Cad Cad GAM (control) 37 PCA 0.16 0.04 0.27 0.00 0.00 0.00 0.00 Put, Cad GAM+5 m GAM+5 m GAM+5 m T T T T T T T T T T T T T T JCM 7528 GAM 30 (a) Cad T JCM 15556 ATCC 35900 ATCC ATCC 49682 ATCC JCM 6580 JCM 15641 ATCC 49684 ATCC ATCC 49683 ATCC ATCC 17744 ATCC JCM 15053 ATCC 35899 ATCC Organism Med. ATCC 33540 ATCC ATCC 17748 ATCC IID 5225 17747 ATCC JCM 6511 sputigena ruminantium

malonica ovata sphaeroides termitida acidovorans thermophilus

dispar atypica caviae criceti criceti criceti magna denticariosi

Veillonella Veillonella Sporomusa Sporomusa Sporomusa Sporomusa Sporolituus Sporomusa Veillonella Veillonella Veillonella Veillonella Veillonella Veillonella Selenomonas Selenomonas 344 HAMANA et al. Vol. 58 ― ― ― ― Put Put polyamines Covalently linked HCl-hydrolysate of the M Japan Collection of Microorganisms, rentheses. Covalently linked polyamines University of Tokyo, Tokyo, Japan; NCIMB, Tokyo, University of Tokyo, lyamines found in the media (c) were cited from mol/g wet weight) μ Polyamines ( Polyamines perchloric acid-extraction of the cells; HCl-Res, 6 M Put Cad Spd Spm Agm Dap Dah (Ref.) Fraction HCl-Res 0.65 0.00 0.00 0.00 0.00 0.00 0.02 HCl-Res 0.74 0.30 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.90 0.04 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.00HCl-Res 0.00 0.01 0.00HCl-Res 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.73 0.00 0.00 0.00 0.00 0.00 0.00 HCl-Res 0.77 0.00 0.00 0.00 0.02 0.00 0.00 type strain; PCA, 0.5 Table 1 (Continued) Table T C) ° ( Temp. Dah 37 PCA 0.55 0.00 0.27 0.03 0.00 0.00 0.75 Put Dap 37 PCA 0.97 0.00 0.51 0.04 0.00 0.40 0.00 Put Agm 37 PCA 0.60 0.00 0.25 0.02 0.65 0.00 0.00 Put M M M GAMGAM 30GAM (a) 30 30 (a) (a) GAM 30 (a)GAMGAM 30GAM 37 PCA 30 PCA 0.02 0.20 Put, Cad PCA 0.00 0.95 0.02 0.10 0.02 0.80 0.15 0.00 0.03 0.00 1.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Put, Cad MGAM 25 PCA 0.30 0.10 0.70 0.00 0.10 0.00 0.00 Put, Cad JCM784 30 PCA 0.00 0.00 0.72 0.00 0.00 0.00 0.00 ATCC1767ATCC1767 30 30 (a) (a) Cad Cad GAM (control) 37 PCA 1.21 0.01 0.72 0.04 0.00 0.00 0.00 Put GAM+5 m GAM+5 m GAM+5 m T T T T T T T T JCM 1765 T JCM 16185 T ATCC 49691 ATCC JCM 15094 T ATCC 49689 ATCC JCM 8139 T mol/L) (c) 0.15 0.10 0.05 0.01 0.01 0.00 0.00 ATCC 17743 ATCC mol/L) (c) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 μ acidiphilus μ JCM 15642

ATCC 17745 ATCC GAM 30 (a) Put, Cad ATCC 10790 ATCC Organism Med. aminiphila

IID 5231 ATCC 17746 ATCC JCM 6512 asaccharolyticus lacrimalis

paucivorans raffi nosivorans raffi

alcalescence)

rodentium parvula rogosae ratti ratti ratti parvula

Peptostreptococcaceae Clostridiales Veillonella Zymophilus Peptoniphilus Peptoniphilus Desulfosporosinus Veillonella Veillonella (Veillonella Veillonella Veillonella Veillonella Zymophilus Anaerosphaera Veillonella Put, putrescine; Cad, cadaverine; Spd, spermidine; Spm, spermine; Agm, agmatine; Dap, diaminopropane; Dah, diaminohexane; JCM, Order Family Liquid GAM medium ( RPMI 1640 medium ( cell residues after the PCA extraction. Med., culture medium. Temp., culture temperature. Former bacteria names are shown in pa culture temperature. Former cell residues after the PCA extraction. Med., culture medium. Temp., previously found in our references (Ref.) (a) and (b) were cited from Hamana (1999b) and Hamana et al. (2002), respectively. Po (a) and (b) were cited from Hamana (1999b) et al. (2002), respectively. previously found in our references (Ref.) Hamana et al. (2007). National Collections of Industrial, Foods and Marine Bacteria, Scotland, UK; National Collections of Industrial, Foods RIKEN, Wako, Saitama, Japan; ATCC, American Type Culture Collection, Manassas, VA, USA; IID, Institute of Medical Science, The Culture Collection, Manassas, VA, USA; IID, American Type Saitama, Japan; ATCC, RIKEN, Wako,

2012 Polyamines in Selenomonadales 345 of cold 1.0 M perchloric acid (PCA) by sonication in a in the acid-extract of the organism as well as other Branson Cell Disruptor 200. After fi ve repetitive of the species analyzed here. It has been known that cadav- 0.5 M PCA extraction by centrifugation at 10,000 × g, erine and putrescine are constitutively synthesized the packed cell residues were hydrolyzed with 6 M HCl from L-lysine and L-ornithine by lysine/ornithine decar- at 110°C for 20 h (Hamana, 1999b; Hamana et al., boxylase, respectively, in S. ruminantium (Takatsuka et 2002). Ionic-binding polyamines extracted into 0.5 M al., 1999). Agmatine is produced from L-arginine by PCA from the cells and covalently linked polyamines arginine decarboxylase and putrescine is also pro- released into 6 M HCl-hydrolysate from the cell resi- duced from agmatine via N-carbamoyl-putrescine in dues containing cell wall peptidoglycan, were ana- S. ruminantium (Liao et al., 2008). S. noxisa contained lyzed by high-performance liquid chromatography putrescine and cadaverine, S. dianae contained ca- (HPLC) on a Hitachi L6000 high-speed liquid chro- daverine and spermidine, and S. infelix contained pu- matograph using a column of cation-exchange resin trescine, cadaverine and spermidine as the covalently (Hamana, 1999b, 2002b; Hamana et al., 2002), as linked polyamines. Putrescine, cadaverine, spermi- shown in Fig. 1. This cell residues after the PCA extrac- dine and agmatine were found as the covalently linked tion used in this method agreed with the results of the polyamines in S. fl ueggei and S sputigena as shown in polyamine analysis of the isolated cell wall peptidogly- the present and previous studies. Our cellular poly- can fraction (Hamana et al., 2002). Diaminopropane･ amine analyses indicate that these decarboxylase ac- 2HCl, putrescine･2HCl, cadaverine･2HCl, diamino- tivities and agmatine pathway are widespread within hexane･ 2HCl, spermidine･3HCl, spermine･ 4HCl and the genus Selenomonas. agmatine･H2SO4 used as the standards for HPLC When the exogenous diamines, diaminopropane analysis and supplements for GAM culture medium and diaminohexane, were supplemented at 5 mM into were purchased from Sigma, USA. Detected polyamine the culture media for S. fl ueggei, the growth was inhib- components, expressed as μmol/g wet weight cells, ited signifi cantly by diaminohexane but not by diamin- and the covalently linked polyamines were found in opropane. A small amount of diaminopropane and the total 18 genera analyzed in the present and previ- large amount of diaminohexane were incorporated ous reports (Hamana, 1999b; Hamana et al., 2002), into the peptidoglycan fraction, HCl-Res, as well as the are shown in Table 1. PCA extract fraction (Fig. 1-(1) and Table 1). Covalent- In the 17 new species belonging to the family Veil- ly linked putrescine, cadaverine, agmatine and sper- lonellaceae analyzed here, signifi cant amounts of pu- midine were replaced signifi cantly with the supple- trescine, cadaverine, spermidine and/or agmatine mented diaminohexane. Covalently linked putrescine were found ubiquitously in their HCl-Res fractions as and cadaverine were replaced weekly with the supple- well as acid-extractable PCA fractions (Table 1). In mented diaminopropane. The supplement of agma- eight Selenomonas species, relative advantage among tine suppressed the growth slightly and increased the covalently linked polyamine components was varied ionic binding and covalently linked putrescine levels, in different cultures. Temperature dependent acid-ex- suggesting that putrescine is produced from agmatine tractable and covalently linked polyamine levels were as well as L-ornitine in the organism. Recently, incor- not observed under the growth at 20‒37°C. S. artemi- poration of exogenous diaminopropane and diamin- dis and S. bovis contained cadaverine alone as the hexane into the peptidoglycan has been reported in S. covalently linked polyamine. In S. ruminantium, a sig- ruminantium (Kojima et al., 2011). nifi cant amount of putrescine as well as cadaverine In 15 Veillonella species, putrescine, cadaverine and was found peptidoglycan fraction in some cultures. spermidine were found as the major cellular poly- Covalently linked agmatine was detected in the culture amines, whereas spermine and agmatine were spo- grown in 1640-Cys-TG medium rich in L-arginine (Table radically detected as a minor polyamine (Table 1). Pu- 1), favorable for bacterial agmatine production by argi- trescine alone or putrescine plus cadaverine were nine decarboxylase (Hamana et al., 2007). Minor cova- covalently linked to the peptidoglycan fraction in the lently linked spermidine was detected under the high all Veillonella spcies analyzed here (Table 1). When acid-extractable spermidine level as found by the sup- 5 mM spermine or agmatine were supplemented into plement of 5 mM spermidine into the medium (Table the cultivation of Veillonella criceti containing covalent- 1). A small amount of cellular spermine was detected ly linked putrescine and cadaverine, the polyamines 346 HAMANA et al. Vol. 58 appeared in the PCA but were not detected signifi cant- sphaera aminiphila, Peptoniphilus asaccharolyticus, ly in the HCl-Res (Table 1). When 5 mM diaminopro- Peptoniphilus lacrimalis and Desulfosporosinus acid- pane, diaminohexane or agmatine was addel to GAM iphilus located in the family Peptostreptococcaceae of medium for V. ratti containing putrescine alone as the the order Clostridiales, far from the order Selenom- covalently linked polyamine, the three polyamines onadales, shown in Table 1 as a reference. In the ther- were detected in the acid-extract of intracellular mophiles belonging to the order Clostridiales, Tepidi- polyamines (PCA) and none of the three polyamines microbium xylanilyticum grown at 60°C, Caldinitra- was detected in the HCl-Res (Fig. 1-(2) and Table 1). tiruptor microaerophilus grown at 65°C and Ther- Cellular spermidine found in the acid-extracts of the all maerobacter compost grown at 70°C containing of Veillonella species analyzed and the supplemented several acid-extractable tetra-amines, penta-amines spermidine in the culture of V. criceti was not incorpo- and hexa-amines, the amines were not found in their rated into the peptidoglycan as a covalently linked cell residues after the acid extraction (data not component. The growth of V. criceti and V. ratti was shown). not inhibited by the polyamine supplements. Six distribution profi les of polyamine components In the Gram-negative anaerobes belonging to other covalently linked to peptidoglycan, namely putrescine 16 genera (except for the two genera Selenomonas alone, cadaverine alone, putrescine plus cadaverine, and Veillonella) of the order Selenomonadales ana- cadaverine plus spermidine, putrescine plus cadaver- lyzed in our present and previous studies, putrescine ine plus spermidine, and putrescine plus cadaverine alone, cadaverine alone, putrescine plus cadaverine, plus spermidine plus agmatine, were found within the cadaverine plus spermidine, or putrescine plus cadav- total of the 17 species presented here and 36 species erine plus spermidine were observed as the covalently previously analyzed, of the order Selenomonadales. linked polyamines (Table 1). A minor occurrence of co- The covalently linked polyamines were distributed se- valently linked agmatine is possible in the Megamonas lectively in the order Selenomonadales consisting of and Mitsuokella species analyzed in the present study 31 genera (validated at the present time in the NCBI (Table 1). website, 2012), in which 18 genera were available in A major part of cellular cadaverine and minor part of our studies, save for as a chemotaxonomic property cellular spermidine remained in the cell residue (HCl- specifi c for the order within the domain Bacteria. How- Res) of moderately thermophilic Sporolituus thermo- ever, the distribution profi les of their covalently linked philus grown at 50°C, containing acid-extractable polyamine level did not directly correspond to their in- spermine as a major cellular polyamine (Table 1). The tracellular polyamine pools. The relation between ac- cellular occurrence of spermine, a tetra-amine, proba- id-extractable polyamine profi les and covalently linked bly correlate for its moderate thermophily, but the tet- polyamine profi les is also important for the chemotax- ra-amine was not found in the cell residue containing onomy of the order. peptidoglycan. Although polyamines in cell wall pepti- doglycan are essential for both cell surface integrity Acknowledgments and normal cell growth of Selenomonas species (Ka- mio et al., 1986, 2007; Takatsuka and Kamio, 2004; We would like to thank JCM for supplying anaerobes. Kojima et al., 2011), the occurrence of covalently linked References polyamines in the aerobes belonging to the order Selenomonadales would not be correlated directly to Hamana, K. (1999a) Polyamine distribution catalogues of their thermophily. 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