• Journal of Ecology and Environment
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• v.40 no.1
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• pp.75-83
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• 2016

Background: In order to evaluate the effect of pH, known as a critical factor for shaping the biogeographical microbial patterns in the studies by others, on the bacterial diversity, we selected two sites in a similar geographical location (site 1; north latitude 35.3, longitude 127.8, site 2; north latitude 35.2, longitude 129.2) and compared their soil bacterial diversity between them. The mountain soil at site 1 (Jiri National Park) represented naturally acidic but almost pollution free (pH 5.2) and that at site 2 was neutral but exposed to the pollutants due to the suburban location of a big city (pH 7.7). Methods: Metagenomic DNAs from soil bacteria were extracted and amplified by PCR with 27F/518R primers and pyrosequenced using Roche 454 GS FLX Titanium. Results: Bacterial phyla retrieved from the soil at site 1 were more diverse than those at site 2, and their bacterial compositions were quite different: Almost half of the phyla at site 1 were Proteobacteria (49 %), and the remaining phyla were attributed to 10 other phyla. By contrast, in the soil at site 2, four main phyla (Actinobacteria, Bacteroidetes, Proteobacteria, and Cyanobacteria) composed 94 %; the remainder was attributed to two other phyla. Furthermore, when bacterial composition was examined on the order level, only two Burkholderiales and Rhizobiales were found at both sites. So depending on pH, the bacterial community in soil at site 1 differed from that at site 2, and although the acidic soil of site 1 represented a non-optimal pH for bacterial growth, the bacterial diversity, evenness, and richness at this site were higher than those found in the neutral pH soil at site 2. Conclusions: These results and the indices regarding diversity, richness, and evenness examined in this study indicate that pH alone might not play a main role for bacterial diversity in soil.

• The Journal of the Korea Contents Association
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• v.20 no.8
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• pp.674-684
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• 2020

To investigate the differences of bacterial- and archaeal communities depending on kind of wastewater (municipal/livestock) and on treating conditions of basins, sludges were sampled from 10 basins of 3 municipal wastewater treatment plants(WWTP) with A2O and a activated sludge sample from a livestock WWTP. The metagenomic DNAs of the sludge samples were extracted and amplified with primers, 27F/518R for bacteria and Arch519F/A958R for archaea, and pyrosequenced with Roche 454 GS-FLX Titanium. As results, the bacterial communities in basins of municipal WWTPs were quite different from those of livestock WWTP, but within the same municipal WWTP their community structures were similar to each other regardless of different environmental conditions such as O₂. And their archaeal communities resulted from anaerobic·anoxic basins were clustered only within communities originated from the same WWTP. Furthermore Seo-bu WWTP with high bacterial diversity and species richness performed better N/P-removal compared to the orther WWTPs.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1835-1841
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• 2015

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.771-780
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• 2014

A putative lipolytic enzyme gene, named as est9x, was obtained from a marine microbial metagenome of the South China Sea. Sequence analysis showed that Est9X shares lower than 27% sequence identities with the characterized lipolytic enzymes, but possesses a catalytic triad highly conserved in lipolytic enzymes of the ${\alpha}/{\beta}$ hydrolase superfamily. By phylogenetic tree construction, Est9X was grouped into a new lipase/esterase family. To understand Est9X protein in depth, it was recombinantly expressed, purified, and biochemically characterized. Within potential hydrolytic activities, only lipase/esterase activity was detected for Est9X, confirming its identity as a lipolytic enzyme. When using p-nitrophenol esters with varying lengths of fatty acid as substrates, Est9X exhibited the highest activity to the C2 substrate, indicating it is an esterase. The optimal activity of Est9X occurred at a temperature of $65^{\cric}C$, and Est9X was pretty stable below the optimum temperature. Distinguished from other salt-tolerant esterases, Est9X's activity was tolerant to and even promoted by as high as 4 M NaCl. Our results imply that Est9X is a unique esterase and could be a potential candidate for industrial application under extreme conditions.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1517-1526
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• 2018

Investigating bacterial diversity and its metabolic capabilities is crucial for interpreting the ecological patterns in a desert environment and assessing the presence of exploitable microbial resources. In this study, we evaluated the spatial heterogeneity of physicochemical parameters, soil bacterial diversity and metabolic adaptation at meter scale. Soil samples were collected from two quadrats of a desert (Thar Desert, India) with a hot, arid climate, very little rainfall and extreme temperatures. Analysis of physico-chemical parameters and subsequent variance analysis (p-values < 0.05) revealed that sulfate, potassium and magnesium ions were the most variable between the quadrats. Microbial diversity of the two quadrats was studied using Illumina bar-coded sequencing by targeting V3-V4 regions of 16S rDNA. As for the results, 702504 high-quality sequence reads, assigned to 173 operational taxonomic units (OTUs) at species level, were examined. The most abundant phyla in both quadrats were Actinobacteria (38.72%), Proteobacteria (32.94%), and Acidobacteria (9.24%). At genus level, Gaiella represented highest prevalence, followed by Streptomyces, Solirubrobacter, Aciditerrimonas, Geminicoccus, Geodermatophilus, Microvirga, and Rubrobacter. Between the quadrats, significant difference (p-values < 0.05) was found in the abundance of Aciditerrimonas, Geodermatophilus, Geminicoccus, Ilumatobacter, Marmoricola, Nakamurella, and Solirubrobacter. Metabolic functional mapping revealed diverse biological activities, and was significantly correlated with physicochemical parameters. The results revealed spatial variation of ions, microbial abundance and functional attributes in the studied quadrats, and patchy nature in local scale. Interestingly, abundance of the biotechnologically important phylum Actinobacteria, with large proposition of unclassified species in the desert, suggested that this arid environment is a promising site for bioprospection.

• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1351-1358
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• 2010

The demand for ${\beta}$-glucosidases insensitive to product inhibition is increasing in modern biotechnology, for these enzymes would improve the process of saccharification of lignocellulosic materials. In this study, a ${\beta}$-glucosidase gene that encodes a 442-amino-acid protein was isolated from a marine microbial metagenomic library by functional screening and named as bgl1A. The protein was identified to be a member of the glycoside hydrolases 1 family, and was recombinantly expressed, purified, and biochemically characterized. The recombinant ${\beta}$-glucosidase, Bgl1A, exhibited a high level of stability in the presence of various cations and high concentrations of NaCl. Interestingly, it was activated by glucose at concentrations lower than 400 mM. With glucose further increasing, the enzyme activity of Bgl1A was gradually inhibited, but remained 50% of the original value in even as high as 1,000 mM glucose. These findings indicate that Bgl1A might be a potent candidate for industrial applications.

• KSBB Journal
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• v.28 no.5
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• pp.295-302
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• 2013

Indigo is utilized in various industries including textile dyeing, cosmetics, printing and medicinal products and its reduced form, leuco-indigo, is mainly used in these process. Chemical reducing agent (sodium dithionite, sodium sulfide, etc.) is preferred to use for the formation of leucoindigo in industry. In traditional indigo fermentation process, microorganisms can participate in the reduction of indigo and thus it has been known to reduce environmental pollution and noxious byproducts. However, in fermentation method using microorganisms it is difficult to standardize large scale production process due to low yield and reproducibility. In this study, we attempted to develop the indigo reduction process using microbial flora which was isolated from naturally fermented indigo vat or deduced by metagenomic approach. From the results of library analyses of PCR-amplified 16S rRNA genes from the traditional indigo fermentation vat sample (metagenome), it was confirmed that Alkalibacteriums (71%) was distinctly dominant in population. Some strains were identified after confirming that they become pure culture in nutrient media modified slightly. Four strains were separated in this process and each strain showed obvious reducing ability toward indigo in dyeing test. It is expected that the analyzed results will provide important data for standardizing the natural fermentation of indigo and investigating the mechanism of indigo reduction.

• Food Science of Animal Resources
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• v.37 no.6
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• pp.906-916
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• 2017

To investigate the effects of a single bacterium and a mixture of bacteria as probiotics in loperamide-treated animal models, loperamide (3 mg/kg) was administered to SD rats to induce constipation. The individual lactic acid bacterial doses, Enterococcus faecium (EF), Lactobacillus acidophilus (LA), Streptococcus thermophilus (ST), Bifidobacterium bifidum (BB), Bifidobacterium lactis (BL), Pediococcus pentosaceus (PP), and a mixture of the bacteria were orally administered to loperamide-induced constipated rats at a concentration of $10^8CFU/kg$ for 14 days. The weights and water contents of their stools were found to be significantly higher in PP, CKDB (mixture of 5 strains except PP), and CKDBP (CKDB+PP) groups than in the normal (constipation not induced) and the control (constipation-induced) groups (p<0.05). The intestinal transit ratio was significantly higher in all probiotic-treated groups than in the control group, and was the highest in the CKDBP group (p<0.05). The mucosal length and mucus secretion were significantly improved in all probiotic-treated-groups, as compared to that in the control group, and the CKDBP group was found to be the most effective according to immunohistochemistry (IHC) staining and total short chain fatty acid content analysis (p<0.05). Lastly, PP, CKDB, and CKDBP showed relatively higher Lactobacillus sp. ratios of 61.94%, 60.31% and 51.94%, respectively, compared to the other groups, based on metagenomic analysis.

• BMB Reports
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• v.44 no.1
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• pp.52-57
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• 2011

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and $37^{\circ}C$, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

• Journal of Microbiology
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• v.43 no.2
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• pp.172-178
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• 2005

To determine the prevalence and genotypes of $\beta$-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced $\beta$-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five ($167\%$) of thirty clones produced an extended-spectrum $\beta$-lactamase. 837- and 259-bp fragments specific to bla$_{TEM}$ genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM­1 was the most prevalent $\beta$-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type $\beta$-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type $\beta$-lactamases of the pre-antibiotic era indicates that TEM-type $\beta$-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.