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Polyamine distribution profiles of new members of the phylum Bacteroidetes.

Cellular polyamine profiles as a phenotypic chemotaxonomic marker within the phylum Bacteroidetes, which is divided into the three classes (orders) Bacteroidetes (Bacteroidales), Flavobacteria (Flavobacteriales) and Sphingobacteria (Sphingobacteriales) have been studied (Hamana and Matsuzaki, 1990, 1991; Hamana and Nakagawa, 2001a, b; Hamana and Niitsu, 2001; Hamana et al., 1995; Hosoya and Hamana, 2003, 2004). Our previous analyses revealed that most of the strains examined in the classes Bacteroidetes and Flavobacteria contained two triamines, spermidine and homospermidine, respectively, as major polyamine components; however, the polyamine profiles over the two classes remain to be elucidated with sizable number of species. On the other hand, the polyamine profiles in the class Sphingobacteria are variable depend on genera or families. In the present study, 105 new strains belonging to the phylum Bacteroidetes were analyzed to survey the distribution of spermidine, homospermidine and other polyamines to evaluate their chemotaxonomic significance. The strains examined were cultured anaerobically at optimum growth temperature (30‒37°C) in GAM Broth (Nissui Pharmaceutical Co., Tokyo, Japan), using Anaerobic Jar KJ-1 (Tomy Seiko, Tokyo) (Hamana et al., 1995), or aerobically at 25‒30°C in Marine Broth (DIFCO, MI, USA), Marine Agar, Trypticase Soy Broth (BBL, MD, USA), polyamine-free Synthetic 199 Medium dissolved in seawater (199-SW) and various media listed in ATCC, JCM and NBRC (ATCC website, 2008; JCM website, 2008; NBRC website, 2008). Cells in the stationary growing phase were harvested from a 50‒100 ml liquid culture by centrifugation at 3,000×g and washed with an isotonic saline or PBS (Nissui Pharmaceutical Co.). The anaerobic strains grown on bloodagar plates were raked from the plates. The cell pellets were homogenized in equal volumes of cold 1.0 M perchloric acid (PCA). Polyamines extracted into 0.5 M PCA were analyzed by high performance liquid chromatography (HPLC) on a Hitachi L6000 high-speed liquid chromatograph using a column of cation-exchange resin (Hamana, 2002; Hamana et al., 1995). When a low polyamine level was detected in the PCA extracts, the PCA extracts from a large-scale culture (1 L) were subjected to a cation-exchange Dowex-50W column (Hamana, 2002), and then the concentrated polyamine fractions were analyzed by HPLC. Class Bacteroidetes (Order Bacteroidales): The class Bacteroidetes is phylogenetically divided into four families, Bacteroidaceae, Porphylomonadaceae, Prevotellaceae and Rikenellaceae (Boone and Castenholz, 2001; Sakamoto, 2007; NCBI website, 2008). Spermidine has been detected as the major polyamine J. Gen. Appl. Microbiol., 54, 229‒236 (2008)