RIBOSOMAL-RNA-TARGETED FLUORESCENT IN-SITU HYBRIDIZATION ANALYSIS OF BACTERIAL COMMUNITY STRUCTURE IN RIVER WATER

An improved in situ hybridization technique, HNPP-FISH, using 2-hydrox y-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR w as applied to analyse the community structure of planktonic bacteria i n river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Flavobacterium-Cytophaga; Burkhol deria-Pseudomonas (rRNA III)-authentic Alcaligenes; Vibrio-Aeromonas; Pseudomonas (rRNA I); the genus Acinetobacter] were used to investigat e the bacterial community structure at two sites differing in organic carbon pollution level. At the eutrophic site. 54-68% of all cells vis ualized by staining with DAPI (4',6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 39 -45% of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five gr oup-specific probes; the bacterial community structure was dominated b y the Flavobacterium-Cytophaga group and Burkholderia-Pseudomonas (rRN A Ill)-authentic Alcaligenes group. At the oligotrophic site, only 26- 38% of the total cells were identified with the five group-specific pr obes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectab le cells differed. No appreciable change was found in the community st ructure during the sampling period at either site. The improved HNPP-F ISH technique should be a useful tool for the analysis of microbial co mmunity composition.