Iron Response Regulator Protein IrrB in Magnetospirillum gryphiswaldense MSR-1 Helps Control the Iron/Oxygen Balance, Oxidative Stress Tolerance, and Magnetosome Formation

Abstract

cMagnetotactic bacteria are capable of forming nanosized, membrane-enclosed magnetosomes under iron-rich and oxygen-limited conditions. The complete genomic sequence of Magnetospirillum gryphiswaldense strain MSR-1 has been analyzed and found to contain five fur homologue genes whose protein products are predicted to be involved in iron homeostasis and the response to oxidative stress. Of these, only the MGMSRv2_3149 gene (irrB) was significantly downregulated under high-iron and low-oxygen conditions, during the transition of cell growth from the logarithmic to the stationary phase. The encoded protein, IrrB, containing the conserved HHH motif, was identified as an iron response regulator (Irr) protein belonging to the Fur superfamily. To investigate the function of IrrB, we constructed an irrB deletion mutant (⌬irrB). The levels of cell growth and magnetosome formation were lower in the ⌬irrB strain than in the wild type (WT) under both high-iron and low-iron conditions. The ⌬irrB strain also showed lower levels of iron uptake and H 2 O 2 tolerance than the WT. Quantitative real-time reverse transcription-PCR analysis indicated that the irrB mutation reduced the expression of numerous genes involved in iron transport, iron storage, heme biosynthesis, and Fe-S cluster assembly. Transcription studies of the other fur homologue genes in the ⌬irrB strain indicated complementary functions of the Fur proteins in MSR-1. IrrB appears to be directly responsible for iron metabolism and homeostasis and to be indirectly involved in magnetosome formation. We propose two IrrB-regulated networks (under high-and low-iron conditions) in MSR-1 cells that control the balance of iron and oxygen metabolism and account for the coexistence of five Fur homologues. Iron is an essential nutrient for most organisms. It is involved in crucial biological processes such as nitrogen fixation, oxygen transport, central metabolism, respiration, gene regulation, and DNA biosynthesis (1). Under aerobic conditions at a neutral pH, iron is metabolically unavailable because of its insoluble state (2, 3). In cells, ferrous iron (Fe 2ϩ ) and ferric iron (Fe 3ϩ ) function, respectively, as the electron donor and electron acceptor, maintaining a compatible redox potential for many biochemical reactions (3). Excess ferrous iron catalyzes the formation of reactive oxygen species (ROS) via the Fenton reaction, resulting in cell damage or death (4). Bacteria and other microorganisms have developed various systems to maintain iron homeostasis, the most studied of which is the ferric uptake regulator (Fur) system. In Escherichia coli and Pseudomonas aeruginosa, iron binds Fur so as to occupy its promoter and inhibit the expression of Fur-controlled genes (5, 6).Magnetospirillum gryphiswaldense MSR-1, a Gram-negative alphaproteobacterium, is capable of synthesizing unique intracellular magnetic nanoparticles composed of Fe 3 O 4 , termed magnetosomes (7). The biosynthesis of magnetosomes, whose membranebound magnetite nanocrystals are aligned in chain-like s...

Magnetic genes: Studying the genetics of biomineralization in magnetotactic bacteria

“…MS-1 also expresses feoB, indicating that iron uptake systems are conserved across species of MTB [75]. The iron response regulator IrrB was shown to be important for magnetosome formation in MSR-1, and deletion affected the transcription of several genes involved in the regulation of iron uptake [76]. These studies taken together indicate that iron availability is an important factor to consider in magnetosome formation and genetic regulation.…”

Novel Protein Mg2046 Regulates Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1 by Modulating a Proper Redox Status

“…To investigate the role of Mg2046 in MSR-1 cell growth and magnetite biomineralization, we constructed null mutant strain Δmg2046 using homologous double-crossover strategy as described previously (Wang et al, 2015; Figure 1B). mam / mms genes were detected by PCR, in consideration of their possible auto-deletion during double-crossover of the target gene (Figure 1C).…”

“…Selected genes and primer sequences are listed in Supplementary Table S1. Relative transcription levels of genes were determined by 2 -ΔΔCp method (Zhang et al, 2012; Wang et al, 2015).…”

Efficient Genome Editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 System for Analyzing Magnetotactic Behavior

“…An approximately 1.0 kb upstream and downstream region flanking of amb0994 , and the gentamycin resistance cassette from pUCGm were amplified using primers (0994-Left arm- XbaI - XmaI -F1, 0994-Left arm- BamHI -R2, 0994-Right arm- BamHI -F3, 0994-Right arm- SacII -R4, Gm-F and 3_Gm-R, Table S2 ). Amplified DNA fragments were ligated into the pMD18-T cloning vector (Takara, Japan) and cut by appropriate restriction enzymes; subsequently, they were cloned into the suicide vector pUX19 to form pUXsuc0994 (Wang et al, 2015a ). Second, Δ amb0994 was constructed by CRISPR-Cas9 system.…”