• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.134-139
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• 2004

Hybrid cluster protein (HCP) was investigated because of its unique iron-sulfur clusters, which have been found in bacteria and archaea. Here, HCP homologous proteins from the third domain, 'eukarya'(3 amitochondriate protozoans, Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis), were identified. All three amitochondriate protozoan HCPs (GlHCP, EhHCP, and TvHCP) belonged to Class I on the basis of two key characters, the cysteine spacing, Cys-(Xaa)₂Cys(Xaa)/sub 7-8/-Cys(Xaa)/sub 5/-Cys, and the absence of N-terminal deletion characteristic to the Class III. In phylogenetic analysis performed with amino acid sequences of 3 eukaryal, 5 bacterial, and 4 archaeal HCPs, the maximum likelihood (ML) tree indicated that TvHCP was clustered with Class I HCPs, whereas the other two HCPs (GlHCP and EhHCP) formed an independent clade with a high bootstrapping value (96%) not belonging to any previously recognized HCP class. In spite of the relatively lower bootstrapping value (61%), the position of the new eukaryal GlHCP-EhHCP clade was close to Class I, including the TvHCP, and Classes II and III were closely related with each other. The finding of eukaryal HCPs would help to understand the evolutionary history of HCP.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.34-45
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• 2012

A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2019-2029
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• 2016

Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two cysteine and two histidine residues ($Cys_2His_2$) coordinate to the zinc ion for the structural functions to generate a ${\beta}{\beta}{\alpha}$ fold, and this secondary structure supports specific interactions with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In this account, the structural similarity and differences of well-known $Cys_2His_2$-type zinc fingers such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology. These zinc finger proteins have different numbers of domains for their structural roles to maintain biological progress through transcriptional regulations from exogenous stresses. The superimposed structures of these finger domains provide interesting details when these fingers are applied to specific gene binding and editing. The structural information in this study will aid in the selection of unique types of zinc finger applications in vivo and in vitro approaches, because biophysical backgrounds including complex structures and binding affinities aid in the protein design area.

• Korean Journal of Microbiology
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• v.46 no.3
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• pp.286-290
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• 2010

C-1 compounds are observed in anaerobic sediment of high salt environments. Thus, surface sediments and waters from these environments are therefore potential habitats for aerobic methylotrophic microorganisms. The soil samples collected from saltern and tidal flat as inoculums and methanol as carbon and energy source was supplied. After subculture depending on the salt concentration, methanol oxidizing bacteria growth condition investigated, the results of methanol oxidizing bacteria can grow in salt conditions, and the maximum concentration was 20%. Analysis based on denaturing gradient gel electrophoresis of 16S rRNA genes indicates that Methelyophaga-like bacteria were dominants of methylotrophs in the enrichment culture. Quantitative PCR showed that archaeal cells were about 1-10% of bacterial cells. Additionally archaea were assumed not to be involved in methanol oxidation since bacterial antibiotics completely blocked the methanol oxidation. Our results suggest that Methelyophaga-like bacteria could be involved in C-1 compounds oxidation in hypersaline environments although those activities are sensitive to salinity above 20%.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1375-1382
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• 2016

The extremely halophilic archaeon Halobacterium noricense is a member of the genus Halobacterium. Strain CBA1132 (= KCCM 43183, JCM 31150) was isolated from solar salt. The genome of strain CBA1132 assembled with 4 contigs, including three rRNA genes, 44 tRNA genes, and 3,208 open reading frames. Strain CBA1132 had nine putative CRISPRs and the genome contained genes encoding metal resistance determinants: copper-translocating P-type ATPase (CtpA), arsenical pump-driving ATPase (ArsA), arsenate reductase (ArsC), and arsenical resistance operon repressor (ArsR). Strain CBA1132 was related to Halobacterium noricense, with 99.2% 16S rRNA gene sequence similarity. Based on the comparative genomic analysis, strain CBA1132 has distinctly evolved; moreover, essential genes related to nitrogen metabolism were only detected in the genome of strain CBA1132 among the reported genomes in the genus Halobacterium. This genome sequence of Halobacterium noricense CBA1132 may be of use in future molecular biological studies.

• Journal of Photoscience
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• v.9 no.3
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• pp.45-48
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• 2002

Microbial rhodopsins, photoactive 7-transmembrane helix proteins that use retinal as their chromophore, were observed initially in the Archaea and appeared to be restricted to extreme halophilic environments. Our understanding of the abundance and diversity of this family has been radically transformed by findings over the past three years. Genome sequencing of cultivated microbes as well as environmental genomics have unexpectedly revealed archaeal rhodopsin homologs in the other two domains of life as well, namely Bacteria and Eucarya. Organisms containing these homologs inhabit such diverse environments as salt flats, soil, freshwater, and surface and deep ocean waters, and they comprise a broad phylogenetic range of microbial life, including haloarchaea, proteobacteria, cyanobacteria, fungi, and algae. Analysis of the new microbial rhodopsins and their expression and structural and functional characterization reveal that they fulfill both ion transport and sensory functions in various organisms, and use a variety of signaling mechanisms. We have obtained the first crystallographic structure for a photosensory member of this family, the phototaxis receptor sensory rhodopsin II (SRII, also known as phoborhodopsin) that mediates blue-light avoidance by the haloarchaeon Natronobacterium pharaonis. The structure obtained from x-ray diffraction of 3D crystals prepared in a cubic lipid phase reveals key features responsible for its spectral tuning and its sensory function. The mechanism of SRII signaling fits a unified model for transport and signaling in this widespread family of phototransducers.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.181-191
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• 2021

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The BoxBehnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.1
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• pp.82-91
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• 2019

Objective: This experiment was conducted to compare the structure and composition of ruminal microorganisms in goats with high and low neutral detergent fibre (NDF) digestibility. Methods: Nineteen crossbred goats were used as experimental animals and fed the same total mixed rations during the 30-day pre-treatment and 6-day digestion trialperiods. All faeces were collected during the digestion period for measuring the NDF digestibility. Then, high and the low NDF digestibility individuals were chosen for the high NDF digestibility group (HFD) and low NDF digestibility group (LFD), respectively. Rumen contents were collected for total microbial DNA extraction. The V4 region of the bacterial 16S rRNA gene was amplified using universal primers of bacteria and sequenced using high-throughput sequencer. The sequences were mainly analysed by QIIME 1.8.0. Results: A total of 18,694 operational taxonomic units were obtained, within 81.98% belonged to bacteria, 6.64% belonged to archaea and 11.38% was unassigned microorganisms. Bacteroidetes, Firmicutes, and Proteobacteria were the predominant microbial phyla in both groups. At the genus level, the relative abundance of fifteen microorganisms were significantly higher (p<0.05) and six microorganisms were extremely significantly higher (p<0.01) in LFD than HFD. Overall, 176 core shared genera were identified in the two groups. The relative abundance of 2 phyla, 5 classes, 10 orders, 13 families and 15 genera had a negative correlation with NDF digestibility, but only the relative abundance of Pyramidobacter had a positive correlation with NDF digestibility. Conclusion: There were substantial differences in NDF digestibility among the individual goats, and the NDF digestibility had significant correlation with the relative abundance of some ruminal microorganisms.

• Microbiology and Biotechnology Letters
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• v.47 no.3
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• pp.354-358
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• 2019

In order to evaluate the migration of fungi into doenjang from its materials, meju and solar salt, microbial communities were analyzed using pyrosequencing. Dominant fungi of meju were Botrytis spp. (57.94%) and Dothiorella samentorum (24.08%). Unidentified fungal species (37.53%), unassigned species (32.60%) and several fungal species of small portion were identified in solar salt. In doenjang, Candida versatilis were predominantly detected (92.62%). Non-halophilic mold were dominantly identified from meju (low-salt fermented soybean), while halophilic bacteria and archaea for solar salt and salt-tolerance fungi such as C. versatilis for doenjang (high-salt fermented soybean) were frequently detected. These results implied that most predominant fungal species might not be migrated from meju and/or solar salt into doenjang.