In order to investigate the relationship between host and symbiosis in the luminous marine fish, Physiculus japonicus, the bacteriallipopolysaccharides (LPS) of symbiotic luminous bacteria were compared serologically and electrophoretically.Five symbiotic luminous bacteria (PJ strains) were separately isolated from five individuals of this fish species caught at three points, off the coasts of Chiba, Nakaminato, and Oharai, LPS preparations were made from these bacteria by Westphal's phenol-water method and highly purified by repeated ultracentrifugation. These LPSs contained little or no 2-keto-3-deoxyoctonate and had powerful mitogenic activity.In sodium dodecylsulfate polyacrylamide gel electrophoresis, these PJ-I to -5 LPSs were separated by their electrophoretic patterns into three groups; the first group included PJ-I and PJ-4, the second group PJ-2 and PJ-3, and the third group PJ-5 alone. The results agreed with those of the double immunodiffusion test; precipitin lines completely coalesced within each group but not with other groups.In immunoelectrophoresis, one precipitin line was observed between anti PJ-2 LPS serum and PJ-5 LPS but the electrophoretic mobility of PJ-5 LPS was clearly different from that of the PJ-2 LPS group. Furthermore, in a 50% inhibition test with PJ-2 LPS by the passive hemolysis system, the doses ofPJ-2 LPS, PJ-3 LPS, and PJ-5 LPS required for 50% inhibition (ID5o) in this system were 0.25, 0.25, and 21.6 pg/ml for each alkali-treated LPS, respectively, and the ID50's of both PJ-I LPS and PJ-4 LPS were above 1,000 pg/ml. These results indicate that PJ-5 LPS has an antigenic determinant partially in common with LPS from the PJ-2 group but not with LPS from the PJ-I group and that the symbiotic luminous bacterium PJ-5 is more closely related to the PJ -2 group than to the PJ -I group.These results show that the species Physiculus japonicus is symbiotically associated with at least three immunologically different strains ofluminous marine bacteria in its specialized light organ. 847
A number of luminous animals and luminous bacteria inhabit the sea wherein some animals provide a place for luminous bacteria in their specialized light organs through specific symbiotic relationships; it has been reported that these relationships are mutually species-specific (3,4,14). However, more detailed information concerning these mutually symbiotic relationships is lacking because classifications of luminous marine bacteria have been performed so far only by their biochemical and morphological characteristics (1, 13). On the other hand, it is known that lipopolysaccharides (LPSs) localized in outer membranes of gram-negative bacterial cell walls consist of O-antigenic, core and lipid A regions wherein the O-antigenic region is specific for each bacterial species or strain. Therefore, serological types and chemical composition of the O-antigenic regions in these bacterial LPSs provide additional identifying information for taxonomy of gram-negative bacteria, in addition to their biochemical and morphological characteristics.We have tried grouping symbiotic luminous marine bacteria isolated from luminous marine animals from a serological point of view using their LPSs and reported that the species Photobacterium phosphoreum and Photobacterium leiognathi could be serologically separated into at least, respectively, five and two serotypes (7,8). In addition, we showed that the luminous marine fish, Physiculus japonicus, was symbiotically associated with at least three serotypes of the species P. phosphoreum in its symbiotic relationship as host (7). In the present study, we investigated LPSs from P. phosphoreum PJ-1 to -5 concerning the symbiotic relationship between P. japonicus (host) and its symbiotic luminous bacteria reported previously (7) from a chemical point of view and showed that for these P. phosphoreum strains, grouping by serological analysis of the LPSs agreed with that by chemical analysis.Symbiotic luminous bacteria, P. phosphoreum PJ-1 to -5, were isolated from light organs of P. japonicus 1 to 5. These photobacterial LPSs were extracted from whole cells of each strain by the hot phenol-water method of Westphal and Jann (18) previously (7). We first investigated the heterogeneity of LPSs from P. phosphoreum 75
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