• Korean Journal of Microbiology
• /
• v.40 no.4
• /
• pp.307-312
• /
• 2004

Diversity of the bacterial communities and the relation between community structure and components of waste­water were analyzed by 16S rRNA-based molecular techniques. Clone libraries of the 16S rDNAs from the sludges were constructed by PCR and cloning. The 1,151 clones from a sludge sample of sewage treatment plant were clustered into 699 RFLP phylotypes and the 1,228 clones from the wastewater disposal plant of chemical industry were clustered into 300 RFLP phylotypes. Shannon-Weiner diversity indices of two sampling sites were 8.7 and 6.1, indicating that the bacterial community structure of sewage treatment plant was more diverse than that of wastewater disposal plant of chemical industry. Forty clones belonging to predominant RFLP types were selected and sequenced. Seventy percent (28 clones) of the sequenced clones were related to the uncultured bacteria in public databases. The ${\beta}-Proteobacteria$ dominated in the bacterial communities of investigated two sludge samples. 16S rDNA sequences of the sewage treatment plant were similar to those of other activated sludges, while the bacterial community in wastewater disposal plant of chemical industry rep­resented the strains identified from high-temperature, anaerobic, hydrocarbon-rich, and sulfur-rich environ­ments. This result suggested that bacterial communities depended upon the components of wastewater.

• Journal of Life Science
• /
• v.22 no.2
• /
• pp.245-250
• /
• 2012

The 16S rDNA - RFLP types for six Vibrio species (V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus) including two core group members, V. alginolyticus and V. parahaemolyticu s, and Grimontia (Vibrio) hollisae were determined using PCR-RFLP analysis. Six tetrameric restriction enzymes (Alu I, Cfo I, Dde I, Hae III, Msp I, and Rsa I) were selected for RFLP analysis. V. alginolyticus, V. parahaemolyticus, and V. proteolyticus showed the same RFLP pattern following digestion with four of the six used restriction enzymes: CfoI, DdeI, MspI, and RsaI. Various restriction enzyme combinations generated digests recognizable as distinct RFLP types for each of the assayed Vibrio species. In particular, AluI single digestion produced species specific band patterns that enabled the differentiation between these Vibrio species. Dendrogram based on restriction patterns showed that two Vibrio core group members, V. alginolyticus and V. parahaemolyticus were closely related having a similarity over 90%. Although the observed RFLP pattern for Grimontia hollisae shared several common bands with other Vibrio spp., G. hollisae results were still clearly distinct from Vibrio spp. RFLP types for all restriction enzymes tested. If restriction enzymes are aptly selected, PCR-RFLP analysis is still a rapid and effective tool for differentiating Vibrio species.

• Korean Journal of Microbiology
• /
• v.40 no.3
• /
• pp.189-198
• /
• 2004

T-RFLP analysis and clone sequencing analysis based on bacterial 16S rDNA were conducted to assess bacterial community structure and diversity in two layers (0-1cm, 6-7cm depth) of the sediment from Janghwari intertidal flat in Ganghwa Island. The results of T-RFLP (terminal-restriction fragment length polymorphism) analysis using restriction enzyme HhaI showed that the T-RFs of various size ($60{\pm}2$) bp-($667{\pm}2$) bp) appeared evenly at the surface sediments but two T-RFs with 60(${\pm}2$)bp and 93 (${\pm}2$)bp predominated at 6-7cm depth. Analysis of partial sequences for 172 clones revealed that 98% of the clones were not matched with the sequences of cultured bacteria strains in the GenBank (${\geq}similarity$ 98%), and approximately 86% of them were classified as different phylotypes. Most clones belonged to $\alpha$-, $\gamma$-, and $\delta$-Proteobacteria, Acidobacteria/Holophaga and green nonsulfur bacteria group. Proteobacteria group occupied the highest proportion in both layers (69% at 0-1cm depth and 46% at 6-7cm depth). $\gamma$-Proteobacteria and $\delta$-Proteobacteria that are associated with oxidation and reduction of sulfur compounds were appeared to be dominant, and comprised 21.5% and 15.7% of total clones, respectively. Overall results indicated that extremely diverse bacterial groups were inhabiting in the sediment of Ganghwa intertidal flat, and bacterial communities associated with the behaviour of sulfur seemed to playa significant role in the biogeochemical environment in this anoxic sediment.

• Korean Journal of Microbiology
• /
• v.42 no.2
• /
• pp.103-110
• /
• 2006

T-RFLP analysis and clone sequencing analysis based on bacterial 16S rDNA were conducted to assess bacterial community structure and diversity in Zoysia japonica soil treated with liquid fertilizer containing amino acids(LFcAA) after spray with herbicide. The results of T-RFLP (terminal restriction fragment length poly-morphism) analysis using restriction enzyme Hae III showed that the T-RFs of various size appeared evenly in the 32 clones of KD3 and 38 clones of KD4 respectively that had been treated with liquid fertilizer containing amino acid(LFcAA) compared to 23 clones of KD2 hat had not been treated with LFcAA. The microbial com- munity structure in KD2 appeared less diverse than those in KD3 and KD4. Analysis of partial sequences for 110 clones from KDI (control), KD2 (non-treated), KD3 (LFcAA 1X), KD4 (LFcAA 2X), respectively, revealed that most bacteria were related with uncultured bacteria in a 16S rDNA sequence similarity range of 91-99% through blast search. Otherwise, the other clones were members of proteobacteria, Acidobacteria, Act-inobacteria, Sphingobacteria and Planctomyces groups. Especially in KD4, members of Alpha Proteobacteria, Rhizobiales, Sphigomonadales, Caulobacterales, Gamma Proteobacteria, the genus Pseudomonas, Betapro-teobacteria, Nitrosomonadales and genus Nitrosospira appeared to be dominant. In addition, Acidobacteria group, Actinobacteria group, Planctomycetacia and Sphingobacteria were also shown. The microbial com-munity structure in Z. japonica soil sprayed with herbicide was affected by LFcAA.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.2
• /
• pp.312-317
• /
• 2002

Indigenous lactobacilli were isolated from vaginas of Korean women for possible use in ecological treatment of bacterial vaginosis. Vaginal swab samples were obtained from a gynecological clinic and streaked on Rogosa SL agar plates to select the most predominant lactobacilli in each sample. The preliminary identification of the isolates as lactobacilli was based on microscopic observation of Gram-positive rod-shaped cell morphology. The initial characterization was performed on 108 isolates in terms of their cell surface hydrophobicity (CSH), antimicrobial activity, and hydrogen peroxide (H₂O₂) production capability, and 10 isolates were then selected for further molecular identification. For a rapid procedure to identify lactobacilli, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of the l6S rRNA genes were applied. The 10 selected lactobacilli and 9 different reference strains of Lactobacillus spp. were characterized by PCR-RFLP where the amplified l6S rDNA was digested with 7 different restriction endonucleases prior to analysis. DNA sequencing of the 16S rRNA gene of one particular isolate, KLB 46, that had been identified as L. crispatus by the PCR-RFLP analysis, further confirmed its identity as L. crispatus.

• Korean Journal of Microbiology
• /
• v.37 no.4
• /
• pp.265-272
• /
• 2001

The diversity of acid-tolerant epiphytic bacterial communities on deciduous oak tree (Quercus dentate Thunb.) leaves was examined both in the natural forest area with a clean air and in the industrial estate to assess effects of acidic depositions to the phyllosphere using 16S rDNA sequence data. A total of 444 acid-tolerant epiphytic bacterial clones were obtained, resulting in 17 phylotypes by performing a analysis of restriction fragment length polymorphism (RFLP) for PCR-amplified 16S rDNA products. A very low diversity of dominating acid tolerant bacterial communities in both areas was found, just 2 subphyla groups, $\gamma$-Proteobacteria and low-G+C gram-positive bacteria. As tree leaves grow older, diversities of acid-tolerant bacteria on them significantly increased. The community structure of acid-tolerant epiphytic bacteria consisted of Pseudomonas and Enterobacteriaceae groups in the $\gamma$-Proteobacteria subphylum, and Streptococcaceae and Staphylococcus groups in the low-G+C gram-positive bacteria subphylum. The direct influence of acidic depositions on bacterial phylogenetic composition could not be detected especially when higher taxonomic levels such as subphylum, but at narrower or finer levels it could be observed by a detection of Xanthomonadales group belonged to the $\gamma$-Proteobacteria only in the industrial area and of Acetobacteraceae group belonged to the $\alpha$-Proteobacteria. There remains that these specific acid-tolerant epiphytic bacterial groups could be used as indicators for assessing effects of acidic depositions on the phyllosphere.

• Parasites, Hosts and Diseases
• /
• v.34 no.2
• /
• pp.127-134
• /
• 1996

Twelve isolates of Accnthamoebc app. assigned to either A. castellanii or A. poIMphoSa, and type strains of A. culbefsoni, A. henIWi, A. pqkestinefiE, and A. astronyxi,s were examined by restriction fragment length polymorphism (RFLP) of a conserved region of small subunit ribosomal RMh gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellanii was 9.8% whereas that among the isolates assigned to A. polvphusn 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. costellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. poIWphosa was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. cnstellnnii and A. polwphasa (2.6%) which appeared between the Castellani (or CCAP 1501/12 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. ostronvxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphoga. It is suggested that taxonomic validity of the isolates assigned to either A. castellnnii or A. polyphoga should be reevaluated.

• Korean Journal of Microbiology
• /
• v.39 no.4
• /
• pp.272-276
• /
• 2003

Sapgyo Lake is an artificial freshwater reservoir which is located to the midwest of Korea and is the main water reservoir for industry and agriculture of the region. In this study we investigated environmental factors and the change of bacterial community with the influence of surrounding inflow water and the seasonal variation using the molecular ecological approach. Water samples were collected at front of the dike in May and August, 2001. Bacterial genomic DNAs were extracted directly and purified for the amplification of bacterial 16S rDNA. Clone libraries of the 16S rDNA were constructed using pGEM-T easy vector and RFLP analysis was performed to make a group as OTUs with 4 base recognizing enzymes (MspI and HaeIII). The estimated values of richness in August sample was higher than in May. Thirty-three of 153 clones in May and thirty-eight of 131 clones in August were sequenced from forward region of bacterial 16S rDNA for about 600~800 bp. Proteobacteria, Cytophaga, gram positive bacteria and Verrucomicrobia were observed both months. Especially, Planctomyces, cyanobacteria and chloroplast appeared in August when algal bloom occurred. On the whole investigation, Sapgyo lake showed a typical community structure of estuarine and was influenced by heterochthonous organic matters from the surrounding stream.

• Korean Journal of Soil Science and Fertilizer
• /
• v.44 no.1
• /
• pp.127-145
• /
• 2011

Various environmental ecosystems are valuable sources for microbial ecology studies, and their analyses using recently developed molecular ecological approaches have drawn significant attention within the scientific community. Changes in the microbial community structures due to various anthropogenic activities can be evaluated by various culture-independent methods e.g. ARISA, DGGE, SSCP, T-RFLP, clone library, pyrosequencing, etc. Direct amplification of total community DNA and amplification of most conserved region (16S rRNA) are common initial steps, followed by either fingerprinting or sequencing analysis. Fingerprinting methods are relatively quicker than sequencing analysis in evaluating the changes in the microbial community. Being an efficient, sensitive and time- and cost effective method, T-RFLP is regularly used by many researchers to access the microbial diversity. Among various fingerprinting methods T-RFLP became an important tool in studying the microbial community structure because of its sensitivity and reproducibility. In this present review, we will discuss the important developments in T-RFLP methodology to distinguish the total microbial diversity and community composition in the various ecosystems.

• Journal of Microbiology
• /
• v.43 no.1
• /
• pp.1-10
• /
• 2005

We isolated and cultured bacteria inhabiting solar saltern ponds in Taean-Gun, Chungnam Province, Korea. All of the isolated 64 strains were found to be moderately halophilic bacteria, growing in a salt range of 2-20 %, with an optimal concentration of 5% salt. Bacterial diversity among the isolated halophiles was evaluated via RFLP analyses of PCR-amplified 16S rDNAs, followed by phylogenetic analysis of the partial 16S rDNA sequences. The combination of restriction enzyme digestions with HaeIII, CfoI, MspI and RsaI generated 54 distinct patterns. A neighbor-joining tree of the partial 16S rDNA sequences resulted in the division of the 64 strains into 2 major groups, 45 strains of ${\gamma}-Proteobacteria$ (70.3%) and 19 strains of Firmicutes (29.7%). The ${\alpha}-Proteobacteria$ and Cytophaga-Flavobacterium-Bacterioides groups, which were repeatedly found to exist in thalassohaline environments, were not represented in our isolates. The ${\gamma}-Proteobacteria$ group consisted of several subgroups of the Vibrionaceae (37.5%), Pseudoalteromonadaceae (10.9%), Halomonadaceae (7.8%), Alteromonadaceae (7.8%), and Idiomarinaceae (6.3%). Members of Salinivibrio costicola (29.7%) were the most predominant species among all of the isolates, followed by Halobacillus treperi (12.5%). Additionally, three new species candidates were found, based on similarities of the 16S rDNA sequences to those of previously published species.