J. Gen. Appl. Microbiol., 43, 49-59 (1997) Polyamine distribution patterns within the families Aeromonadaceae, Vibrionaceae, Pasteurellaceae, and Halomonadaceae, and related genera of the gamma subclass of the Proteobacteria Koei Hamana College of Medical Care and Technology, Gunma University, Maebashi 371, Japan (Received June 12, 1996; Accepted December 19,1996) Polyamines of the four families and the five related genera within the gamma subclass of the class Proteobacteria were analyzed by HPLC with the objective of developing a chemotaxonomic system. The production of putrescine, diaminopropane, cadaverine, and agmatine are not exactly correlated to the phylogenetic genospecies within 36 strains of the genus Aeromonas (the family Aeromon- adaceae) lacking in triamines. The occurrence of norspermidine was limited but not ubiquitous within the family Vibrionaceae, including 20 strains of Vibrio, Listonella, Photobacterium, and Salini- vibrio. Spermidine was not substituted for the absence of norspermidine in the family. Agmatine was detected only in Photobacterium. Salinivibrio and some strains of Vibrio were devoid of polyamines. Vibrio ("Moritella") marinus contained cadaverine. Within the family Pasteurellaceae, Haemophilus contained cadaverine only and Actinobacillus contained no polyamine. Halomonas, Chromohalobacter, and Zymobacter, belonging to the family Halomonadaceae, ubiquitously con- tained spermidine and sporadically cadaverine and agmatine. Shewanella contained putrescine and cadaverine; Alteromonas macleodii, putrescine, 2-hydroxyputrescine, cadaverine, 2-hydroxysper- midine, and spermidine; Pseudoalteromonas, putrescine, cadaverine, and spermidine; Marinobacter, spermidine; and Marinomonas, putrescine and spermidine. Their polyamine profiles serve as a chemotaxonomic marker within the gamma subclass. Key Words Aeromonadaceae; Halomonadaceae; norspermidine; Pasteurellaceae; polyamine; Pro- teobacteria; Vibrionaceae Studies on cellular polyamine distribution have al- Aeromonas was placed in the family Aeromon- ready provided some valuable chemotaxonomic infor- adaceae as the only genus (Colwell et al., 1986). The mation within the class Proteobacteria, and are usu- members of the genus Aeromonas were divided into ally characteristic for higher taxonomic rank such as at least 16 genospecies by DNA homology (Abbott et for the many members of the alpha subclass of the al., 1992; Ali et al., 1996; Altwegg et al., 1990; Carna- Proteobacteria, which feature homospermidine, or for han et al., 1991a, 1991b, 1991c; Collins et al., 1993; the organisms belonging to the beta subclass, which Esteve et al., 1995a, 1995b; Huys et al., 1994; Janda, feature 2-hydroxyputrescine (Auling et al., 1991; 1991; Joseph et al., 1991; Lucchini and Altwegg, Busse and Auling, 1988; Hamana and Matsuzaki, 1992; Martinez-Murcia et al., 1992a, 1992b; Sneath, 1992a, 1993; Yang et al., 1993). The four families, 1993), however, taxonomic conclusions are not com- Aeromonadaceae, Vibrionaceae, Pasteurellaceae, plete. We analyzed polyamines as the phenotypic and Enterobacteriaceae, are major members of the marker of 36 strains of Aeromonas, including 15 gamma subclass of the Proteobacteria and comprise genospecies, for chemotaxonomic interest. a cluster in the subclass. In the four families, determi- The phylogenetic classification of the genera Vibrio, nation of the polyamines of Enterobacteriaceae, in Photobacterium, and Listonella belonging to Vibri- which diaminopropane and acetyispermidine are onaceae is conflicting (Holmes, 1992; Kita-Tsukamoto widely spread, has been published (Hamana,1996). et al., 1993; Ruimy et al., 1994; Smith et al., 1991). Norspermidine has been found as a major polyamine Address reprint requests to: Dr. Koei Hamana, College of Medical in many Vibrio, Listonella, and Photobacterium Care and Technology, Gunma university, 3-39-15 Showa-machi, species, but some norspermidine-absent strains were Maebashi 371, Japan. reported within these three genera (Yamamoto et al., 50 HAMANA Vol. 43 1983, 1991). In this study, the polyamines of some contained 21 amino acids, 17 vitamins, 10 nu- new species of the above genera (Ishimaru et al., cleotides, sodium acetate, glucose, NaCI, KCI, CaCl2, 1995, 1996; Okuzumi et al., 1994), as well as the MgSO4, Na2HPO4, and Fe(NO3)3, but not ornithine, di- newly proposed genera Salinivibrio (Mellado et al., aminobutyric acid or polyamines. Putrescine, cadaver- 1996) and "Moritella" (Gauthier et al., 1995a) in Vibri- me, agmatine, spermidine, and spermine were de- onaceae, were analyzed in comparison with the tected in GAM, NB, BHIB, PY SW, and MB. The Acti- polyamine types found in other related marine AI- nobacillus and Haemophilus species were cultivated teromonas, Shewanella, Marinomonas, and Oceano- at 37°C and Shewanella benthica at 2°C, whereas the spirillum species, newly isolated Marinobacter hydro- others were grown at 20-30°C. Actinobacillus actino- carbonoclasticus (Gauthier et al., 1992) and the newly mycetecomitans was grown in the presence of 5% validated genus Pseudoalteromonas (Gauthier et al., CO2. 1995a) within the gamma subclass of the Proteobac- The pellets of cells harvested at the late-stationary teria. phase were homogenized in 0.5M perchloric acid The polyamines of some members of the genera (HCIO4) at 2°C. Polyamines were extracted into HCIO4 Pasteurella, Haemophilus, and Actinobacillus (Gau- and analyzed by high-performance liquid chromatog- thier et al., 1995b), which belong to the family Pas- raphy on a L600 high-speed liquid chromatograph teurellaceae in the gamma subclass of the class Pro- (Hitachi Co., Tokyo, Japan) as described previously teobacteria, were also determined. (Hamana and Matsuzaki, 1992b,1993). Phylogenetic analyses of the halophilic proteobacte- ria belonging to the genera Halomonas, Deleya, and Results and Discussion Halovibrio and two halophilic organisms, Volcaniella eurihalina and Paracoccus halodenitrificans, showed The major polyamines detected in the four families that these proteobacteria constitute another major (Aeromonadaceae, Vibrionaceae, Pasteurellaceae, family of the gamma subclass, Halomonadaceae and Halomonadaceae) and six related genera of ma- (Dobson et al., 1993; Mellado et al., 1995; Miller et al., rine bacteria (Shewanella, Pseudoalteromonas, AI- 1994). Unification of these genera into a single genus teromonas, Marinobacter, Marinomonas, and Oceano- (Halomonas), and placement of Chromohalobacter spirillum) are summarized into a schematic phyloge- marisomortui, and the newly isolated Zymobacter pal- netic tree in Fig. 1. The details of the polyamine com- mae (Okamoto et al., 1993) in the family, were pro- positions found in the four families and related genera posed (Dobson and Franzmann, 1996; Mellado et al., are shown separately in five tables, Tables 1-5, re- 1995). Although the major polyamine of some species spectively. of Halomonas and Deleya was spermidine in the pre- vious publication (Hamana and Matsuzaki, 1993), the Aeromonadaceae ( Table 1) polyamines of the new members of Halomonadaceae Ubiquitous distribution of putrescine and the ab- were analyzed in this study. sence of triamines such as spermidine and norspermi- dine were found in all strains of the Aeromonas Materials and Methods species tested in this study, as well as a report for some Aeromonas species by Yamamoto et al. (1991). Organisms were aerobically cultured in a synthetic When Aeromonas was grown in NB, the minor medium, 199 medium (199), pH 7.2 (Nissui Pharma- polyamines detected in the cultures was excluded (as ceutical Co., Tokyo, Japan); GAM medium (GAM), pH a contaminant) from the net cellular polyamine com- 7.0 (Nissui Pharmaceutical Co.); nutrient broth (NB), ponents. Variations in the distribution profiles of di- pH 7.0 (Nissui Pharmaceutical Co.); and brain heart aminopropane, cadaverine, histamine, and agmatine infusion broth (BHIB) supplemented with 5% Fildes were observed within the same species (genotype): Enrichment (Difco Lab., Detroit, MI, U.S.A.). PY putrescine type and putrescine-cadaverine type in medium (1% polypeptone, 0.2% yeast extract, and A, allosaccharophila (HG 14) and A. salmonicida (HG 0.1% MgSO4) dissolved in 75% seawater (PY SW), 3); and putrescine-cadaverine type and putrescine- the 199 medium dissolved in 75% seawater (199- cadaverine-agmatine type in A. schubertii (HG 12). SW), IFO 306 medium (List of Cultures, 10th ed., Diaminopropane and histamine were sporadically 1996, IFO), Marine broth 2216 (MB) (Difco Lab.), and spread in some genospecies. These findings indicate IAM B-15 and IAM B-84 media (Catalogue of Strains, that the appropriate decarboxylase activities to pro- 1st ed., 1993, IFO) were used for halophiles. The 199- duce diaminopropane, cadaverine, histamine, and ag- SW and PY SW supplemented with 10 mM L-2,4-di- matine are not exactly correlated to the phylogenetic aminobutyric acid, L-ornithine, 1,3-diaminopropane genospecies within Aeromonas, and that these amine and/or putrescine were also used. The 199 medium profiles are not useful as a phenotypic marker within 1997 Polyamines of Aeromonadaceae, Vibrionaceae, Pasteurellaceae, and Halomonadaceae 51 me was found but norspermidine was not detected. In this study, the absence of norspermidine in this organ- ism is clear. This polyamine profile for V. marinus ATCC 15381T supports the phylogenetic finding that V. marinus ATCC 15381T should be placed in a new separate genus,"Moritella," as "M. marinus" (Gauthier et al.,1995a). The newly isolated species