The resultant plasmids pAK3-0664, pAK3-2684, and pAK3-3876 were conjugated into Selleckchem Birinapant AMB0101, AMB0102, and AMB0103 mutant strains, respectively, to generate the complementary strains AMB0105, AMB0106, and AMB0107. Transmission electron microscopic (TEM) observations of AMB-1 cells and magnetosomes were performed with JEM-100CXII

at an acceleration voltage of 120 kV. The average magnetosome number per cell was determined directly by counting magnetosome particles in at least 130 individual cells. Strains of M. magneticum AMB-1, AMB0101, AMB0102, and AMB0103 were cultured to the stationary phase in a 300-mL sealed serum bottle containing 250 mL liquid medium before being transferred to initiate another round of subculture. This process was repeated at least 30 times. Cells were collected at the indicated rounds of subcultures

and genomic DNA was extracted using a Bacterial DNA Kit (Omega). Nonmagnetic cells were examined for the existence of a genomic MAI region by amplifying the four marker genes isocitrate dehydrogenase signaling pathway indicated in Fig. 4a using primers 16–19 listed in Table S1. Quantitative real-time PCR was performed in a Bio-Rad Sequence Detection System with a total volume of 20 μL, containing 250 nM of primers, 10 μL of SYBR Green PCR mix (Takara), 0.4 μL of ROX, 6.8 μL of ddH2O, and 2 μL DNA template under the following conditions: 3 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 15 s at 60 °C, and 15 s at 72 °C. All samples were also analyzed with primers specific for the 16S rRNA gene and this result was used as a ‘loading control’ to normalize results from Metalloexopeptidase the marker genes inside the genomic MAI (amb0956). The calibration standard curve of each

gene was run in triplicate from a series of 10-fold dilutions of genomic DNA of M. magneticum AMB-1. The ratio of signals from amb0956 and 16S rRNA gene starting the subculture was set to 100%, and other time points show the level relative to the starting point. Magnetospirillum magneticum AMB-1, AMB0101, AMB0102, and AMB0103 cells sampled at the indicated time intervals were in the meantime spread on enriched MSGM plates and incubated for 7 days. Each colony was transferred into a liquid culture in a 1.5-mL tube to test magnetic sensitivity by a bar magnet and microscope in an applied magnetic field. The percentage of magnetic-sensitive cells was calculated by counting at least 200 colonies. An extensive analysis of the genome of M. magneticum AMB-1 revealed the existence of three peroxiredoxin-like genes (Matsunaga et al., 2005). The gene encoding AhpC-like protein (amb0664, Prx1) is located upstream of two other genes encoding a hypothetical protein (amb0662) and a thioredoxin reductase (amb0663), respectively, with the downstream amb0665 transcribed in the opposite direction and amb0663 in the same orientation as amb0664 (Fig. S1).