To ensure that the observed phenotypes were caused by the nonpolar deletion of prxs, the mutants with an intact MAI region were complemented with the wild-type prxs-hemagglutinin integrated into a large intergenic region, but expressed from its own promoter. Expression of the complemented Prxs was confirmed by a Western blot (Fig. S3). Complemented selleck kinase inhibitor cells restored the growth and magnetism to a level similar to that of the wild type (Fig. 2f and g). To observe whether Prxs would exert an effect
in the absence of oxygen, the growth and magnetosome synthesis of the isogenic mutants were analyzed under anaerobic conditions (Fig. 2c and d). In contrast to what occurred under aerobic conditions, neither the growth nor the synthesis
of the Cmag value was significantly affected by the absence of Prxs, although there was a slight decrease in the final cell density attained by strain AMB0104. These data highlight an important role for all three Prxs in protecting magnetotactic bacteria against oxidative stress in the presence of oxygen. Dapagliflozin manufacturer It has been observed that the MAI of spontaneous nonmagnetic mutants of M. gryphiswaldense exhibits extensive sequence polymorphism including the loss of key magnetosome genetic markers (Schubbe et al., 2003; Ullrich et al., 2005). Four different gene loci within the MAI region were found to be absent in the nonmagnetic prx mutant cells (Fig. 4a and b). To further analyze the effect of the absence of Prxs on the stability of MAI on a population level, we performed a real-time PCR analysis using primers specific for markers located inside and outside MAI to determine their presence quantitatively during subculture (Fig. 4c). In contrast to the wild type in which all the markers tested were maintained at the same level even after 30 rounds of subculture, mutants with the deletion of prx
displayed an accelerated loss of the MAI markers, with a reduction to 50–70% of the original level after 10 rounds of transfer. Prx1 seemed to exert a more dramatic effect on the stability of the MAI region, with about a 90% reduction in the detection level after 20 rounds of subculture. All mutants instead of the wild-type strain were negative for detection after 30 rounds of subculture, indicating that all Urease mutant cells in the culture had probably lost the MAI markers tested. Correspondingly, magnetic colonies in the wild-type subculture invariably accounted for the majority (>94%) of the population after 30 rounds of subculture, while prx mutants that still remained magnetotactic declined to 7% (AMB0101), 28% (AMB0102), and 22% (AMB0103) of subculture, respectively (Fig. 4d). These results imply that a selection against the stability of the MAI may occur due to the increased oxidative stress resulting from the deficiency of peroxiredoxins.