Kenzaka Takehiko, Yamaguchi Nobuyasu, Tani Katsuji, Nasu Masao
Faculty of Pharmaceutical Sciences, Osaka University1-6 Yamada-oka, Suita, Osaka 565-0871Japan.
Microbiology (Reading). 1998 Aug;144 ( Pt 8):2085-2093. doi: 10.1099/00221287-144-8-2085.
An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Flavobacterium-Cytophaga; Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes; Vibrio-Aeromonas; Pseudomonas (rRNA I): the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. At the eutrophic site, 54-68% of all cells visualized 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 group-specific probes; the bacterial community structure was dominated by the Flavobacterium-Cytophaga group and Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes group. At the oligotrophic site, only 26-38% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.
一种改进的原位杂交技术，即使用2-羟基-3-萘甲酸2'-苯胺磷酸盐（HNPP）和固红TR的HNPP-FISH，被应用于分析河水中浮游细菌的群落结构。使用针对细菌域（EUB338）和五个细菌类群[黄杆菌-噬细胞菌；伯克霍尔德菌-假单胞菌（rRNA III）-正宗产碱菌；弧菌-气单胞菌；假单胞菌（rRNA I）：不动杆菌属]的寡核苷酸探针，来研究两个有机碳污染水平不同的位点的细菌群落结构。在富营养化位点，用4',6-二脒基-2-苯基吲哚（DAPI）染色可见的所有细胞中，有54%-68%能用探针EUB338检测到。在贫营养化位点的样本中，总细胞的39%-45%与EUB338杂交。在富营养化位点，约50%的总细胞能用五个类群特异性探针鉴定；细菌群落结构以黄杆菌-噬细胞菌类群和伯克霍尔德菌-假单胞菌（rRNA III）-正宗产碱菌类群为主。在贫营养化位点，只有26%-38%的总细胞能用五个类群特异性探针鉴定。尽管EUB338可检测细胞的百分比不同，但贫营养化位点的群落结构与富营养化位点相似。在任何一个位点的采样期间，群落结构都没有发现明显变化。改进后的HNPP-FISH技术应该是分析微生物群落组成的一个有用工具。
