16S rDNA restriction fragment length polymorphism analysis of psychrotrophic vibrios from Japanese coastal water

Restriction fragment length polymorphism (RFLP) analysis was carried out for 136 natural isolates belonging to the family Vibrionaceae. These were collected from inshore areas of Japan, mainly in winter. Twenty-eight 16S rDNA genotypes were obtained by digestion with four restriction endonucleases (HhaI, DdeI, RsaI, and Sau3AI). To estimate the genetic relationships, 53 informative fragments were scored by their presence or absence. A dendrogram was constructed using the unweighted pair group method with the arithmetic averages algorithm. Five RFLP groups (groups I to V) were obtained. Group I corresponded to Vibrio splendidus-like strains. It was confirmed that this group was not only found in Otsuchi Bay, but also in broad coastal areas of Japan. Group II strains were not identified as previously known Vibrio species. Group III strains were regarded as members of the Vibrio main group, which is a major phylogenetic group deduced from 16S rRNA gene analysis in the family Vibrionaceae. The RFLP profile indicated that Group IV strains were closely related to V. hollisae. Group V strains showed RFLP patterns which have not been observed previously. From the clustering analysis, it was concluded that group V strains were not Vibrio species. Most of the isolates studied were not identified as previously described species. It suggests that many psychrotrophic vibrios in cold marine environments remain as unknown species.