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- Iron Homeostasis in Brucella abortus: the Role of Bacterioferritin
- Marta A. Almirón , Rodolfo A. Ugalde
- J. Microbiol. 2010;48(5):668-673. Published online November 3, 2010
- DOI: https://doi.org/10.1007/s12275-010-0145-3
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- 14 Citations
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Abstract
- Brucella abortus is the etiological agent of bovine brucellosis, an infectious disease of humans and cattle. Its pathogenesis is mainly based on its ability to survive and multiply inside macrophages. It has been demonstrated that if B. abortus ferrochelatase cannot incorporate iron into protoporphyrin IX to synthesize heme, the intracellular replication and virulence in mice is highly attenuated. Therefore, it can be hypothesized that the unavailability of iron could lead to the same attenuation in B. abortus pathogenicity. Thus, the purpose of this work was to obtain a B. abortus derivative unable to keep an internal iron pool and test its ability to replicate under iron limitation. To achieve this, we searched for iron-storage proteins in the genome of brucellae and found bacterioferritin (Bfr) as the sole ferritin encoded. Then, a B. abortus bfr mutant was built up and its capacity to store iron and replicate under iron limitation was investigated. Results indicated that B. abortus Bfr accounts for 70% of the intracellular iron content. Under iron limitation, the bfr mutant suffered from enhanced iron restriction with respect to wild type according to its growth retardation pattern, enhanced sensitivity to oxidative stress, accelerated production of siderophores, and altered expression of membrane proteins. Nonetheless, the bfr mutant was able to adapt and replicate even inside eukaryotic cells, indicating that B. abortus responds to internal iron starvation before sensing external iron availability. This suggests an active role of Bfr in controlling iron homeostasis through the availability of Bfr-bound iron.
- Overexpression of Bacterioferritin Comigratory Protein (Bcp) Enhances Viability and Reduced Glutathione Level in the Fission Yeast Under Stress
- Ga-Young Kang , Eun-Hee Park , Kyunghoon Kim , Chang-Jin Lim
- J. Microbiol. 2009;47(1):60-67. Published online February 20, 2009
- DOI: https://doi.org/10.1007/s12275-008-0077-3
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- 6 Citations
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Abstract
- The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCP10. The bcp+ mRNA level in the pBCP10-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCP10 exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.
- Characterization, Cloning and Expression of the Ferritin Gene from the Korean Polychaete, Periserrula leucophryna
- Byeong Ryong Jeong , Su-Mi Chung , Nam Joo Baek , Kwang Bon Koo , Hyung Suk Baik , Han-Seung Joo , Chung-Soon Chang , Jang Won Choi
- J. Microbiol. 2006;44(1):54-63.
- DOI: https://doi.org/2336 [pii]
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Abstract
- Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5''-untranslated region of 128 bp, and a 3''-noncoding region of 456 bp. The 5''-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.
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