Cellular polyamines of 61 archaeal newly published strains were analyzed by HPLC to evaluate correlation of polyamine profiles to their extremophily and chemotaxonomy. A quaternary branched penta-amine, N⁴-bis( aminopropyl )spermidine, was found in hyperthermophilic Methanocaldococcus and Methanotorris belonging to the family Methanocaldococcaceae but not in the family Methanococcaceae nor any other methanogenic archaeal orders. Hyperthermophilic / extremely thermophilic Palaeococcus, Thermococcus and Pyrococcus of the order Thermococcales ubiquitously contained the branched penta-amine. Mesophilic, extreme halophiles including halo-alkaliphiles, belonging to the order Halobacteriales, contained a trace of spermidine, spermine and agmatine. In the phylum Crenarchaeota, extremely thermophilic Caldisphaera of the provisional order 'Caldisphaerales' contained norspermidine, spermidine and agmatine, and hyperthermophilic Pyrobaculum belonging to the order Thermoproteales contained norspermidine, spermidine, norspermine and spermine, but not any penta-amines. Within the order Desulfurococcales, Desulfurococcus and Ignisphaera contained spermidine, norspermidine, homospermidine, norspermine and spermine, whereas Aeropyrum contained caldopentamine and thermopentamine in addition to these triamines and tetra-amines.

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The product YkoN of the gene of unknown function, ykoN, of Bacillus subtilis Marburg has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and thus YkoN is expected to have a lipase or esterase activity. To characterize the expected enzyme activity the plasmid having a modified ykoN that include the sequence for His(x6) tag at its C-terminus of YkoN, which has 373 amino acid residues, was constructed. His-tagged YkoN protein of 39 kDa was induced in Escherichia coli BL21 (DE3) cells harboring chaperon plasmid pGro7 and purified to near homogeneity by using gel filtration and Ni-agarose. When p-nitrophenyl-esters of different fatty acid chain length were examined, the purified YkoN hydrolyzed the esters of fatty acid with short chain length (4-6 carbon atoms) preferentially. The esters of fatty acid with longer chain (C ≥ 10) were hydrolyzed inefficiently. The activity required no divalent cations and was not affected by addition of EDTA. The optimal pH for the activity was from pH 7.4 to pH 8.6. These results indicate that YkoN is a novel esterase which hydrolyzes the esters of fatty acid with short chain length.

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To understand the physiological role of the ykoN gene of Bacillus subtilis, the gene was expressed from the IPTG-inducible spac promoter in B. subtilis cells. When lipid composition of the cells induced for ykoN expression was examined, a new spot of phospholipid was found on the thin layer chromatogram. Induction of ykoN in Escherichia coli cells produced a similar new spot. The new spot was produced in mutant E. coli cells lacking cardiolipin or phosphatidylethanolamine, but not in the cells lacking phosphatidylglycerol. The result suggests an involvement of phosphatidylglycerol in production of the new spot. Examination of the lipid composition during the stages of B. subtilis growth revealed that the new phospholipid was produced four hours after cessation of logarithmic growth, consistent with an increase in the promoter activity of ykoN in the late stage in sporulation. YkoN has the pentapeptide lipase/esterase motif (Gly-X-Ser-X-Gly), and shows a lipolytic activity (Matsuura et al., in press). The mutant YkoN (S205A) in which the central serine residue of the motif is replaced with alanine produced the new phospholipid in the amount as small as only 1/10 of the wild type, suggesting that the lipolytic activity of YkoN is involved in the new phospholipid production.

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