• Journal of Microbiology and Biotechnology
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• v.29 no.6
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• pp.887-896
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• 2019

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based pathogen identification relies on the ribosomal protein spectra provided in the proprietary database. Although these mass spectra can discern various pathogens at species level, the spectra-based method still has limitations in identifying closely-related microbial species. In this study, to overcome the limits of the current MALDI-TOF MS identification method using ribosomal protein spectra, we applied MALDI-TOF MS of low-mass profiling to the identification of two genetically related Bacillus species, the food-borne pathogen Bacillus cereus, and the insect pathogen Bacillus thuringiensis. The mass spectra of small molecules from 17 type strains of two bacilli were compared to the morphological, biochemical, and genetic identification methods of pathogens. The specific mass peaks in the low-mass range (m/z 500-3,000) successfully identified various closely-related strains belonging to these two reference species. The intensity profiles of the MALDI-TOF mass spectra clearly revealed the differences between the two genetically-related species at strain level. We suggest that small molecules with low molecular weight, 714.2 and 906.5 m/z can be potential mass biomarkers used for reliable identification of B. cereus and B. thuringiensis.

• Molecules and Cells
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• v.44 no.12
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• pp.911-919
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• 2021

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M₁ progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

• BMB Reports
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• v.44 no.8
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• pp.529-534
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• 2011

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.

• Journal of Life Science
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• v.12 no.2
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• pp.47-52
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• 2002

Saccharomyces cerevisiae KNU5377 (KNU5377) and S. cerevisiae ATCC24858 (ATCC24858) were exposed to $H_2SO_4$ as a stress, which was added at various concentrations to a YPD media. The growth of KNU5377 was reduced to approximately 60% in the YPD media containing 40 nm sulfuric acid when compared to the non-stressed condition. When their growth was monitored during an overnight culture, two strains, KNU5377 and ATCC24858, could not grow when exposed to over 50 mM of sulfuric acid. After a short exposure to this acid for 1 h, KNU5377 exhibited stronger resistance against $H_2SO_4$ than ATCC24858. The neutral trehalase activity of KNU5377 unchanged despite under various concentrations of $H_2SO_4$. In contrast, It at of ATCC24858 was much low at higher $H_2SO_4$concentrations. Trehalose, a non-reducing disaccharide, was maximally accumulated after a short exposure to 60 nm $H_2SO_4$ for KNU5377, but it was reduced under more severe stressful conditions. These results suggest that KNU5377 should modulate the trehalose concentrations under the severe stress condition of high sulfuric acid concentrations. The most highly induced protein in the KNU5377 exposed to sulfuric acid was found to be an approximately 23 kDa protein, which was revealed to be the 605 large subunit ribosomal protein, Ll3 by FASTA search results.

• Journal of Animal Science and Technology
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• v.63 no.5
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• pp.1126-1141
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• 2021

Recent evidence has shown that methionine (Met) supplementation can improve milk protein synthesis under hyperthermia (which reduces milk production). To explore the mechanism by which milk protein synthesis is affected by Met supplementation under hyperthermia, mammary alveolar (MAC-T) cells were incubated at a hyperthermic temperature of 42℃ for 6 h in media with different concentrations of Met. While the control group (CON) contained a normal amino acid concentration profile (60 ㎍/mL of Met), the three treatment groups were supplemented with Met at concentrations of 10 ㎍/mL (MET70, 70 ㎍/mL of Met), 20 ㎍/mL (MET80, 80 ㎍/mL of Met), and 30 ㎍/mL (MET90,90 ㎍/mL of Met). Our results show that additional Met supplementation increases the mRNA and protein levels of BCL2 (B-cell lymphoma-2, an anti-apoptosis agent), and decreases the mRNA and protein levels of BAX (Bcl-2-associated X protein, a pro-apoptosis agent), especially at an additional supplementary concentration of 20 ㎍/mL (group Met80). Supplementation with higher concentrations of Met decreased the mRNA levels of Caspase-3 and Caspase-9, and increased protein levels of heat shock protein (HSP70). The total protein levels of the mechanistic target of rapamycin (mTOR) and the mTOR signalling pathway-related proteins, AKT, ribosomal protein S6 kinase B1 (RPS6KB1), and ribosomal protein S6 (RPS6), increased with increasing Met supplementation, and peaked at 80 ㎍/mL Met (group Met80). In addition, we also found that additional Met supplementation upregulated the gene expression of αS1-casein (CSN1S1), β-casein (CSN2), and the amino acid transporter genes SLC38A2, SLC38A3 which are known to be mTOR targets. Additional Met supplementation, however, had no effect on the gene expression of κ-casein (CSN3) and solute carrier family 34 member 2 (SLC34A2). Our results suggest that additional Met supplementation with 20 ㎍/mL may promote the synthesis of milk proteins in bovine mammary epithelial cells under hyperthermia by inhibiting apoptosis, activating the AKT-mTOR-RPS6KB1 signalling pathway, and regulating the entry of amino acids into these cells.

• Journal of Life Science
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• v.27 no.6
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• pp.694-701
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• 2017

Mycoplasma genitalium represents the smallest genome among mono-cultivable prokaryotes. To discover and compare the orthologs (conservative genes) among M. genitalium and 14 prokaryotes that are uncultivable and have less orthologs than M. genitalium, COG (clusters of orthologous groups of protein) analyses were applied. The analyzed prokaryotes were M. genitalium, one hyperthermophilic exosymbiotic archaeon Nanoarchaeum equitans, four intracellular plant pathogenic eubacteria of Candidatus Phytoplasma genus, and nine endosymbiotic eubacteria of phloem- and xylem-feeding insects. Among 367 orthologs of M. genitalium, 284 orthologs were conservative between M. genitalium and at least one other prokaryote. All 15 prokaryotes commonly have 29 orthologs, representing the significance of proteins in life. They belong to 25 translation-related, including 22 ribosomal proteins, 3 subunits of RNA polymerase, and 1 protein-folding-related. Among the 15 prokaryotes, 40 orthologs were only found in all four Candidatus Phytoplasma. The other nine Candidatus, all endosymbionts with insects, showed only a single common COG0539 (ribosomal protein S1), representing the diversity of orthologs among them. These results might provide clues to understand conservative genes in uncultivable prokaryotes, and may be helpful in industrial areas, such as handling prokaryotes producing amino acids and antibiotics, and as precursors of organic synthesis.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.219-222
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• 2000

Physiological changes of Escherichia coli during the fed-batch fermentation process were characterized in this study. Overall cellular protein samples prepared at the different stage of fermentation were separated by 2-dimensional gel electrophoresis (2-DE), and differently expressed 15 proteins, Phosphotransferase enzyme I, GroEL, Trigger factor, ${\beta}$ subunit of ATP synthase, Transcriptional regulator KDGR, Phosphoglycerate mutase 1, Inorganic pyrophosphatase, Serine Hydroxymethyl-transferase, ${\alpha}$ subunit of RNA polymerase, Elongation factor Tu, Elongation factor Ts, Tyrosine-tRNA ligase, DnaK suppressor protein, Transcriptional elongation factor, 30S ribosomal protein S6 were identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). When bacterial cells grow to high cell density, and IPTG-inducible heterologous protein is produced, expression level of overall cellular proteins was decreased. According to their functions in the cell, identified proteins were classified into three groups, proteins involved in transport process, small-molecule metabolism, and synthesis and modification of macromolecules.

• BMB Reports
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• v.38 no.2
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• pp.161-166
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• 2005

Functional protein of MB78 bacteriophage having apparent molecular weight of 22 kDa is expressed from 1.7 kb HindIII G fragment. The nucleotide sequence of this fragment showed two open reading frames of 222 and 196 codons in tail-to-tail orientation separated by a 62-nucleotide intercistronic region. The ORF of 22 kDa protein is present in opposite orientation, i.e. in the complementary strand, preceded by a strong ribosomal binding site and a promoter sequence. Another ORF started from the beginning of the fragment whose promoter region and translational start site lies in the 0.45 kb HincII U fragment which is located next to the HindIII G fragment, that has the sequence for DNA bending. 3' end of the fragment has high sequence homology to the EaA and EaI proteins of bacteriophage P22, a close relative of MB78 phage.

• Journal of Microbiology
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• v.42 no.3
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• pp.200-204
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• 2004

Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase. It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation. In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis. Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS. Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources. One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation. The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive.

• BMB Reports
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• v.36 no.4
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• pp.344-348
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• 2003

Protein kinase CKII is composed of two catalytic ($\alpha$ or $\alpha$') subunits and two regulatory ($\beta$) subunits. The $CKII{\beta}$ subunit is thought to mediate the tetramer formation and interact with other target proteins. However, its physiological function remains obscure. In this study, point mutants of $CKII{\beta}$ that are defective for the L41 binding were isolated by using the reverse two-hybrid system. A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of $CKII{\beta}$ are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for $CKII{\beta}$ homodimerization. Two point mutants, R75 and R83, of $CKII{\beta}$ interacted with L5, topoisomerase $II{\beta}$, and CKBBP1/SAG, but not with the wild-type $CKII{\beta}$. This indicates that $CKII{\beta}$ homodimerization is not a prerequisite for its binding to target proteins. These $CKII{\beta}$ point mutants may be useful in exploring the biochemical physiological functions of $CKII{\beta}$.