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Yang J.,China Agricultural University | Yang J.,France China Biomineralization and Nano structure Laboratory | Li S.,China Agricultural University | Li S.,France China Biomineralization and Nano structure Laboratory | And 9 more authors.
Frontiers in Microbiology | Year: 2013

Pure culture of magnetotactic bacteria with high magnetosome yield has been achieved for only a few strains. The major obstacles involve the nutritional requirements and culture conditions of the cells. To increase cell density and magnetosome production, it is necessary to elucidate the physiological characteristics of a particular strain during cell growth and develop an appropriate artificial control strategy. Large-scale culture of Magnetospirillum gryphiswaldense strain MSR-1 was successfully performed for 48 h in a 42-L autofermentor, and several key physiological parameters were measured in real time. Maximal values of cell density (OD565) (19.4) and cell yield (dry weight) (4.76 g/L) were attained at 40 h. The key time point for cell growth and magnetosome formation was found to be 18-20 h. At this point, cells entered the log phase of growth, the maximal values of Cmag (1.78), iron content (0.47%), and magnetosome number (26 ± 3 per cell) were observed, superoxide dismutase (SOD) activity began to decrease more rapidly, ATP content dropped to an extremely low level (0.17 fmol), and reducing power (NADH/NAD+ ratio) began to increase very rapidly. Excessive levels of dissolved oxygen (=20 ppb) and lactic acid in the medium caused notable cytotoxic effects after 20 h. Artificial control measures for fermentation must be based on realistic cell physiological conditions. At the key time point (18-20 h), cell density is high and magnetosomes have matured. The process of magnetosome synthesis involves a high consumption of ATP and reducing power, and the cells require replenishment of nutrients prior to the 18-20 h time point. Culture conditions that effectively minimize dissolved oxygen accumulation, lactic acid content, and reducing power at this point will enhance magnetosome yield without obvious inhibition of cell growth. © 2013 Yang, Li, Huang, Tang, Jiang, Zhang and Li. Source

Rong C.,China Agricultural University | Rong C.,CAS Institute of Microbiology | Zhang C.,China Agricultural University | Zhang C.,France China Biomineralization and Nano structure Laboratory | And 12 more authors.
Journal of Bacteriology | Year: 2012

Magnetotactic bacteria (MTB) synthesize unique organelles, the magnetosomes, which are intracellular nanometer-sized, membraneenveloped magnetite. The biomineralization of magnetosomes involves the uptake of large amounts of iron. However, the iron metabolism of MTBis not well understood. The genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense strain MSR-1 contains two ferrous iron transport genes, feoB1 and feoB2. The FeoB1 protein was reported to be responsible mainly for the transport of ferrous iron and to play an accessory role in magnetosome formation. To determine the role of feoB2, we constructed an feoB2 deletion mutant (MSR-1ΔfeoB2) and an feoB1 feoB2 double deletion mutant (MSR-1NfeoB). The single feoB2 mutation did not affect magnetite crystal biomineralization. MSR-1NfeoB had a significantly lower average magnetosome number per cell (∼65%) than MSR-1 ΔfeoB1, indicating that FeoB2 plays a role in magnetosome formation when the feoB1 gene is deleted. Our findings showed that FeoB1 has a greater ferrous iron transport ability than FeoB2 and revealed the differential roles of FeoB1 and FeoB2 in MSR-1 iron metabolism. Interestingly, compared to the wild type, the feoB mutants showed increased sensitivity to oxidative stress and lower activities of the enzymes superoxide dismutase and catalase, indicating that the FeoB proteins help protect bacterial cells from oxidative stress. © 2012, American Society for Microbiology. Source

Yang J.,China Agricultural University | Yang J.,France China Biomineralization and Nano structure Laboratory | Li S.,China Agricultural University | Li S.,France China Biomineralization and Nano structure Laboratory | And 10 more authors.
BMC Microbiology | Year: 2013

Background: Magnetotactic bacteria produce membrane-enveloped magnetite crystals (magnetosomes) whose formation is controlled primarily by a gene island termed the magnetosome island (MAI). Characterization of single gene and operon function in MAI has elucidated in part the genetic basis of magnetosome formation. The mamX gene, located in the mamXY operon, is highly conserved in the MAI of all Magnetospirillum strains studied to date. Little is known regarding the function of mamX in the process of biomineralization. Results: A mamX deletion mutant (mamX) and its complemented strain (CmamX) by conjugation in M. gryphiswaldense strain MSR-1 were constructed. There were no striking differences in cell growth among mamX, CmamX, and wild-type strain (WT). mamX displayed a much weaker magnetic response than WT. Transmission electron microscopy revealed the presence of irregular, superparamagnetic magnetite particles in mamX, in contrast to regular, single-domain particles in WT and CmamX. The phenotype of mamX was similar to that of an ftsZ-like deleted mutant and mamXY operon deleted mutant reported previously. Quantitative real-time RT-PCR (qPCR) results indicated that the deletion of mamX had differential effects on the transcription levels of the other three genes in the operon. Conclusions: The MamX protein plays an important role in controlling magnetosome size, maturation, and crystal form. The four MamXY proteins appear to have redundant functions involved in magnetosome formation. Our findings provide new insights into the coordinated function of MAI genes and operons in magnetosome formation. © 2013 Yang et al.; licensee BioMed Central Ltd. Source

Zhang C.,China Agricultural University | Zhang C.,France China Biomineralization and Nano structure Laboratory | Meng X.,China Agricultural University | Meng X.,Yangzhou University | And 12 more authors.
Journal of Bacteriology | Year: 2013

The bacterial strain Magnetospirillum gryphiswaldense MSR-1 does not produce siderophores, but it absorbs a large amount of ferric iron and synthesizes magnetosomes. We demonstrated previously the presence of six types of ferric reductase isozymes (termed FeR1 through FeR6) in MSR-1. Of these isozymes, FeR5 was the most abundant and FeR6 showed the highest ferric reductase activity. In the present study, we cloned the fer5 and fer6 genes from MSR-1 and expressed them separately in Escherichia coli. FeR5 and FeR6 were shown to be bifunctional enzymes through analysis of amino acid sequencehomologies, structural predictions (using data from GenBank), and detection of enzyme activities. FeR5 is a thioredoxin reductase and FeR6 is a flavin reductase, in addition to being ferric reductases. To elucidate the functions of the enzymes, we constructed two single-gene-deletion mutant strains (δfer5 and δfer6 mutants) and a double-gene-deletion mutant strain (δfer5 δfer6 [δfer5+6] mutant) along with its complemented strains (C5 and C6). An evaluationof phenotypicand physiological properties did not reveal significant differences between the wild-type and single-gene-deletion strains, whereas the double-gene-deletion strain showed reduced iron absorption and no magnetosome synthesis. Complementationof the double-gene-deletion strain using either fer5 or fer6 resulted in the partial recovery of magnetosome synthesis. Quantitative real-time PCR analysis of fer5 and fer6 transcriptional levels in the wild-type and complemented strains demonstrated consistent transcription of the two genes and confirmed that FeR5 and FeR6 are bifunctional enzymes that playcomplementary roles during the process of magnetosome synthesis in MSR-1. © 2013, American Society for Microbiology. Source

Qi L.,China Agricultural University | Qi L.,France China Biomineralization and Nano structure Laboratory | Li J.,China Agricultural University | Li J.,France China Biomineralization and Nano structure Laboratory | And 10 more authors.
PLoS ONE | Year: 2012

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe 3O 4). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense. © 2012 Qi et al. Source

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