We previously reported ionic binding cellular polyamines extracted into 0.5 M perchrolic acid (PCA) and total polyamines detected in 6 M HCl-hydrolysate of the whole cells in various anaerobes with Gramnegative cell walls, belonging to the Sporomusa subbranch (Sporomusa-Pectinatus-Selenomonas phyletic group) (Hamana, 1999). The occurrence of covalently linked putrescine and/or cadaverine in cell wall, nonextractable with 0.5 M PCA from cells, was suggested within the major members of this subbranch, viz. the genera Acetonema, Sporomusa, Megasphaera, Selenomonas, Veillonella, Pectinatus, Dialister, Acidaminococcus, Zymophilus, and Anaerovibrio (Hamana, 1999). Furthermore, a part of cellular spermidine of Selenomonas dianae, Selenomonas infelix, and Acidaminococcus fermentans was not extracted with 0.5 M PCA from the cells, suggesting the occurrence of covalently linked spermidine in their cell walls (Hamana, 1999). In the present study, cell wall peptidoglycan was prepared from seven Selenomonas species, Acidaminococcus fermentance, Anaerovibrio lipolyticus (formerly Anaerovibrio lipolytica) and two newly validated species, Anaeromusa acidaminophila (formerly Selenomonas acidaminophila) (Baena et al., 1999) and Dendrosporobacter quercicolus (formerly Clostridium quercicolum) (Stompl et al., 2000) phylogenetically located in the Sporomusa subbranch.The organisms were grown anaerobically in the anaerobic culture medium, GAM broth (Nissui Pharmaceutical Co., Tokyo, Japan), at 37°C. Peptidoglycan was prepared by two procedures, SDS-Trypsin method and SDS-Amylase method, according to Braun and Sieglin (1970) and Kamio et al. (1981b), respectively. Hydrolysis of the peptidoglycan fraction with 6 M HCl at 110°C for 20 h was carried out before high performance liquid chromatography (HPLC) on an Hitachi L6000 high-speed liquid chromatograph as described previously (Hamana, 1994). Agmatine was identified by alkaline hydrolysis with 1 M NaOH at 110°C for 20 h to convert agmatine into putrescine. PCA extractable polyamines from whole cells and polyamines in the 6 M HCl-hydrolysates of cellresidues (after five-fold extractions with 0.5 M PCA until polyamines were not detected in the PCA extracts) (Hamana, 1999), were also determined in the organisms.PCA-soluble cellular polyamines (PCA) and released polyamines by the acid hydrolysis from the cell residue after the PCA-extract (HCl-Res) estimated per wet cell weight are shown in
