• International Journal of Industrial Entomology and Biomaterials
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• v.20 no.2
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• pp.107-114
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• 2010

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5'-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• Journal of the Korean Magnetic Resonance Society
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• v.11 no.2
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• pp.73-84
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• 2007

MTH1821 (UniProtKB/TrEMBL ID O27849) is a 96-residue hypothetical protein from the open reading frame of Methanobacterium thermoautotrophicum H one of the target organisms of structural genomics pilot project. Proteins which contain conserved sequence compared with MTH1821 have not been discovered yet and the functional and structural information for MTH1821 is not available. Here, we present the sequence-specific backbone resonance using multidimensional heteronuc1ear NMR spectroscopy and propose the secondary structure using GetSBY software. The backbone resonances of N, HN, $C_{\alpha}$, $C_{\beta}$, CO and $H_{\alpha}$ which are necessary for a prediction of secondary structure by GetSBY were assigned about 98% (557/568). The secondary structure of MTH1821 confirmed that it is comprised of four strand regions and two helical regions. This report will provide a valuable resource for the calculation solution structure of MTH1821 and for the other hypothetical protein that is targeted for structural-based functional discovery.

• Genomics & Informatics
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• v.20 no.4
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• pp.47.1-47.13
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• 2022

Klebsiella pneumoniae is a gram-negative bacterium that is known for causing infection in nosocomial settings. As reported by the World Health Organization, carbapenem-resistant Enterobacteriaceae, a category that includes K. pneumoniae, are classified as an urgent threat, and the greatest concern is that these bacterial pathogens may acquire genetic traits that make them resistant towards antibiotics. The last class of antibiotics, carbapenems, are not able to combat these bacterial pathogens, allowing them to clonally expand antibiotic-resistant strains. Most antibiotics target essential pathways of bacterial cells; however, these targets are no longer susceptible to antibiotics. Hence, in our study, we focused on a hypothetical protein in K. pneumoniae that contains a DNA methylation protein domain, suggesting a new potential site as a drug target. DNA methylation regulates the attenuation of bacterial virulence. We integrated computational-aided drug design by using a bioinformatics approach to perform subtractive genomics, virtual screening, and fingerprint similarity search. We identified a new potential drug, koenimbine, which could be a novel antibiotic.

• BMB Reports
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• v.38 no.6
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• pp.676-682
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• 2005

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of $NaBH_4$, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1288-1294
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• 2009

We have used mutational analysis to identity four genes, MXAN3553, MXAN3554, MXAN3555, and MXAN3556, constituting an operon that is essential for normal fruiting body development in Myxococcus xanthus. Deletion of MXAN3553, which encoded a hypothetical protein, resulted in delayed fruiting body development. MXAN3554 was predicted to encode a metallopeptidase, and its deletion caused fruiting body formation to fail. Inactivation of MXAN3555, which encoded a putative NtrC-type response regulator, resulted in delayed aggregation and a severe reduction in sporulation. Fruiting bodies also failed to develop with the deletion of MXAN3556, another gene encoding a hypothetical protein.

• Journal of Microbiology and Biotechnology
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• v.21 no.2
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• pp.136-146
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• 2011

This study provides first-hand proteomic data on the survival strategy of Anabaena sp. PCC 7120 when subjected to long-term iron-starvation conditions. 2D-gel electrophoresis followed by MALDI-TOF/MS analysis of iron-deficient Anabaena revealed significant and reproducible alterations in ten proteins, of which six are associated with photosynthesis and respiration, three with the antioxidative defense system, and the last, hypothetical protein all1861, conceivably connected with iron homeostasis. Iron-starved Anabaena registered a reduction in growth, photosynthetic pigments, PSI, PSII, whole-chain electron transport, carbon and nitrogen fixation, and ATP and NADPH content. The kinetics of hypothetical protein all1861 expression, with no change in expression until day 3, maximum expression on the $7^{th}$ day, and a decline in expression from the $15^{th}$ day onward, coupled with in silico analysis, suggested its role in iron sequestration and homeostasis. Interestingly, the up-regulated FBP-aldolase, Mn/Fe-SOD, and all1861 all appear to assist the survival of Anabeana subjected to iron-starvation conditions. Furthermore, the $N_2$-fixation capabilities of the iron-starved Anabaena encourage us to recommend its application as a biofertilizer, particularly in iron-limited paddy soils.

• The Plant Pathology Journal
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• v.32 no.2
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• pp.102-111
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• 2016

Olive culture is very important in the Mediterranean Basin. A severe outbreak of Olive Quick Decline Syndrome (OQDS) caused by Xylella fastidiosa infection was first noticed in 2013 on olive trees in the southern part of Apulia region (Lecce province, southern Italy). Studies were carried out for detection and diversity evaluation of the Apulian strain of Xylella fastidiosa. The presence of the pathogen in olive samples was detected by PCR amplifying the 16S rDNA, gyrase B subunit (gyrB) and HL hypothetical protein genes and single nucleotide polymorphisms (SNPs) assessment was performed to genotype X. fastidiosa. Twelve SNPs were recorded over gyrB and six SNPs were found for HL gene. Less variations were detected on 16S rDNA gene. Only gyrB and HL provided sufficient information for dividing the Apulian X. fastidiosa olive strains into subspecies. Using HL nucleotide sequences was possible to separate X. fastidiosa into subspecies pauca and fastidiosa. Whereas, nucleotide variation present on gyrB gene allowed separation of X. fastidiosa subsp. pauca from the other subspecies multiplex and fastidiosa. The X. fastidiosa strain from Apulia region was included into the subspecies pauca based on three genes phylogenetic analyses.

• Genomics & Informatics
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• v.20 no.4
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• pp.41.1-41.9
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• 2022

The pathogen Gallibacterium anatis has caused heavy economic losses for commercial poultry farms around the world. However, despite its importance, the functions of its hypothetical proteins (HPs) have been poorly characterized. The present study analyzed the functions and structures of HPs obtained from Gallibacterium anatis (NCTC11413) using various bioinformatics tools. Initially, all the functions of HPs were predicted using the VICMpred tool, and the physicochemical properties of the identified virulence proteins were then analyzed using Expasy's ProtParam server. A virulence protein (WP_013745346.1) that can act as a potential drug target was further analyzed for its secondary structure, followed by homology modeling and three-dimensional (3D) structure determination using the Swiss-Model and Phyre2 servers. The quality assessment and validation of the 3D model were conducted using ERRAT, Verify3D, and PROCHECK programs. The functional and phylogenetic analysis was conducted using ProFunc, STRING, KEGG servers, and MEGA software. The bioinformatics analysis revealed 201 HPs related to cellular processes (n = 119), metabolism (n = 61), virulence (n = 11), and information/storage molecules (n = 10). Among the virulence proteins, three were detected as drug targets and six as vaccine targets. The characterized virulence protein WP_013745346.1 is proven to be stable, a drug target, and an enzyme related to the citrate cycle in the present pathogen. This enzyme was also found to facilitate other metabolic pathways, the biosynthesis of secondary metabolites, and the biosynthesis of amino acids.

• Bulletin of the Korean Chemical Society
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• v.34 no.8
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• pp.2419-2424
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• 2013

Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.

• Journal of Life Science
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• v.27 no.9
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• pp.1040-1046
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• 2017

We describe the isolation and characterization of six different intestinal microorganisms from the digestive tract of the cricket Gryllus bimaculatus. Based on 16S rRNA gene sequences, we obtained six isolates belonging to four different genera: Staphylococcus, Bacillus, Citrobacter, and Proteus. All the isolates were resistant to ampicillin. Ampicillin is an irreversible inhibitor of the enzymeetranspeptidase, which is needed to make bacterial cell walls. None of the isolates were resistant to kanamycin, which binds to the 30S subunit of the bacterial ribosome and then inhibits total protein synthesis. Gram staining was conducted, in addition to morphological classification under a microscope. Four grampositive isolates and two gram-negative isolates were detected. The gram-positive isolates were GL1 (round shaped, 2 am in diameter), GL2 (rod shaped, $2.5{\mu}m$ in length), GL3 (rod shaped, $2{\mu}m$ in length), and GL4 (round shaped, $1.5{\mu}m$ in diameter). The gram-negative isolates were GL5 (rod shaped, $2{\mu}m$ in length) and GL6 (rod-shaped, $2.5{\mu}m$ in length). Notably, two of the isolates, GL2 and GL4, secreted specific extracellular proteins. These were determined by MALDI-TOF-MS spectral analysis to be a 87 kDa collagenase, 56 kDa hypothetical protein, and 200 kDa hypothetical protein. The six isolates in this study could be used for various biotechnological applications and pest management, both in the field and in greenhouse systems. In addition, it would be interesting to determine the relationship between these isolates and their host.