• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.663-669
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• 2000

The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with $\beta$-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur $\beta$-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1090-1094
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• 2008

In this article, we show that the orf slr1471 from Synechocystis sp. PCC 6803 codes for a functional member of the YidC/Alb3/Oxa1 protein family, and the encoded protein has a transmembrane topology with a common core structure. Using specific antibodies raised against the Synechocystis YidC homologous protein, we further show that the Synechocystis YidC protein appears to be predominantly localized in the cyanobacterial cytoplasmic membrane. The impact of the described findings for synthesis of membrane proteins and for protein sorting within cyanobacterial cells is discussed.

• YAKHAK HOEJI
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• v.52 no.3
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• pp.225-231
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• 2008

To clarify effects of the structural changes of Fur protein on the resistance to metronidazole (Mtz), the mutational analysis of structure and function of the protein in Helicobacter pylori (Hp) was undertaken. It was identified that some changes in Hp Fur protein resulted in increase of resistance to Mtz, and other changes resulted in decrease of resistance. Increase of Mtz resistance came from the enzyme's decreased ability of reducing prodrug Mtz to the form of bactericidal agent. Some sites that affects Mtz resistance (i) in Fur's N terminal extension, and (ii) in its central region, which links DNA binding and Fe-binding modules were identified. It was also found that the addition of FLAG tag to Fur's C terminus also significantly impairs Fur function.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1271-1278
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• 2012

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

• BMB Reports
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• v.39 no.6
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• pp.717-721
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• 2006

In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial pre-apo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.124-129
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• 2011

The role of the ferric uptake regulator (Fur) of Helicobacter pylori in the oxidative stress was investigated in this study. A fur knockout mutant of H. pylori was constructed by replacing the fur gene with an aphA (kanamycin resistant marker) gene. Photodynamic therapy using methylene blue (MB) and 660 nm light was chosen to induce oxidative stress. The bactericidal effect of photodynamic therapy (PDT) was compared between wild type H. pylori and fur knockout mutant H. pylori. The degree of oxidative damage of DNA was confirmed using alkaline gel electrophoresis and an assay of 8-hydroxy-2-deoxyguanosine (8-OHdG). In control groups, the number of viable cells was maintained constantly during experiment. After PDT, the mutant H. pylori showed 10,000 times decreased viable cell number compared with wild type H. pylori. Depending on the exposure time of 660 nm light, the 3-fold increase in the concentration of 8-OHdG was observed in mutant H. pylori. The results of this study showed that H. pylori's Fur protein may play a role in oxidative stress induced by PDT.

• Journal of Microbiology
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• v.45 no.6
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• pp.583-589
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• 2007

The promoter region of pyrC (dihydroorotase) gene of Escherichia coli was shown to have Fur protein binding properties by gel retardation assay. In vivo regulation of the pyrC expression was studied by measuring dihydroorotase activity and ${\beta}$-galactosidase level in the $fur^+$ and $fur^-$ genetic background. The expression of chromosomal dihydroorotase activity and ${\beta}$-galactosidase activity of pyrC-lacZ fusion plasmid was repressed to about 30% and 17%, respectively in the $fur^+$ strain compared to those in the $fur^-$ strain. Divalent ions such as $Fe^{2+}$ and $Zn^{2+}$ were not required for the repression. PyrC expression was also reduced to one half by 1 mM uracil. The effect of uracil was independent on the fur gene.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1452-1459
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• 2007

The Dps protein, which is overexpressed in harsh environments, is known to playa critical role in the protection of DNA against oxidative stresses. In this study, the roles of Fur in the expression of the dps gene in Salmonella and the protection mechanisms against oxidative stress in Salmonella cells preexposed to iron-stress were investigated. Two putative Fur boxes were predicted within the promoter region of the S. typhimurium dps gene. The profile of dps expression performed by the LacZ reporter assay revealed growth-phase dependency regardless of iron-status under the culture conditions. The fur mutant, $_X4659$, evidenced a reduced level of ${\beta}$-galactosidase as compared to the wild-type strain. The results observed after the measurement of the Dps protein in various Salmonella regulatory mutants were consistent with the results acquired in the reporter assay. This evidence suggested that Fur performs a function as a subsidiary regulator in the expression of dps. The survival ability of Salmonella strains after exposure to oxidative stress demonstrated that the Dps protein performs a pivotal function in the survival of stationary-phase S. typhimurium against oxidative stress. Salmonella cells grown in iron-restricted condition required Dps for full protection against oxidative stress. The CK24 (${\Delta}dps$) cells grown in iron-replete condition survived at a rate similar to that observed in the wild-type strain, thereby suggesting the induction of an unknown protection mechanism(s) other than Dps in this condition.

• Journal of Microbiology
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• v.41 no.2
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• pp.109-114
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• 2003

A promoter that is inducible by paraquat and menadione, the superoxide generators, independently of soxRS has been found in front of the sufABCDSE operon in Escherichia coli. Based on the observation that SufA is a holomog of IscA that functions in the assembly of iron sulfur cluster and the sufA promoter (sufAp) contains a putative Fur-binding consensus, we investigated whether this gene is regulated by Fur, a ferric uptake regulator, When examined in several sufAp-lacZ chromosomal fusion strains, sufAp was induced by EDTA, an iron chelator and a well-known Fur-inducer, The basal level of sufA expression increased dramatically in fur mutant, suggesting repression of sufAp by Fur. The derepression in fur mutant and EDTA-induction of sufA expression required nucleotides up to -61, where a putative Fur box is located. Purified Fur protein bound to the DNA fragment containing the putative Fur box between -35 and -10 promoter elements. The regulation by Fur and menadione induction of sufAp acted independently. The rpoS mutation increased sufA induction by menadione, suggesting that the stationary sigma factor RpoS acts negatively on sufA induction.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.187-194
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• 2007

In order to utilize different nitrogen sources and to survive in a situation of nitrogen limitation, microorganisms have developed sophisticated mechanisms to adapt their metabolism to a changing nitrogen supply. In this communication, the recent knowledge of nitrogen regulation in the amino acid producer Corynebacterium glutamicum is summarized. The core adaptations of C. glutamicum to nitrogen limitation on the level of transcription are controlled by the global regulator AmtR. Further components of the signal pathway are GlnK, a $P_{II}-type$ signal transduction protein, and GlnD. Mechanisms involved in nitrogen control in C. glutamicum regulating gene expression and protein activity are repression of transcription, protein-complex formation, protein modification by adenylylation, change of intracellular localization, and proteolysis.