Divergence of a Tandem Duplication of Manganese Superoxide Dismutase in Nosema bombycis

Manganese superoxide dismutase (Mn SOD ) is a key enzyme in the protection of cells from oxidative stress. A tandem duplication of the Mn SOD gene ( NbMn SOD 1 and NbMn SOD 2 ) in the genome of Nosema bombycis, a parasite of the silkworm Bombyx mori , was previously identified. Here, we compare the protein structures of NbMn SOD 1 and NbMn SOD 2 and characterize these two proteins in terms of cellular localization, timing of transcription, protein structure, and enzyme activity. Despite a similarity in the primary sequence of NbMn SOD 1 and NbMn SOD 2 , the latter shows a remarkable degree of amino acid sequence difference on the protein's surface and in the active site, where there is a substitution of a phenylalanine for a histidine in NbMn SOD 2 . Immuno‐electron microscopy demonstrates that NbMn SOD 1 is present in the cytosol of mature spores, whereas NbMn SOD 2 is localized on the polar tube and the spore wall. Immunofluorescence confirms the localization of NbMn SOD 2 on the polar tube of the germinated spore. Quantitative measurement of gene expression ( qRT ‐ PCR ) demonstrates production of both alleles during the first day of infection followed by a dramatic decrease during the second to fourth day of infection. From the fifth day onward, the two alleles show a complementary pattern of expression. The qRT ‐ PCR of the host manganese superoxide dismutase ( BmMn SOD ) shows a notable increase in transcription upon infection, leading to a three‐fold spike by the first day of infection, followed by a decrease in transcription. Measurement of overall Mn SOD activity shows a similar peak at day 1 followed by a decrease to a constant rate of enzyme activity. The differences in cellular localization and pattern of gene expression of NbMn SOD 2 compared to NbMn SOD 1 , as well as the differences in protein structure seen for NbMn SOD 2 compared to other microsporidial Mn SOD s, strongly suggest a unique, recently evolved role for NbMn SOD 2 .