Biochemical and Biophysical Research Communications
Topology and identification of critical residues of the O-acetyltransferase of serotype-converting bacteriophage, SF6, of Shigella flexneri
Section snippets
Materials and methods
Bacterial strains and culture conditions. Escherichia coli strain JM109 was used as a host strain for propagating all plasmids used in this study [9]. Functional expression of plasmid constructs was tested using S. flexneri serotype Y strain, SFL 124 [10]. Details of the strains created are described in Table 1 (Supplementary material). All cultures were grown at 37 °C aerobically in Luria–Bertani media with chloramphenicol (Cm—25 μg/ml final concentration). For the topology studies, dual
Topology analysis of Oac
Hydrophobicity plots of Oac formed the working model for designing experimental approaches to investigate the topology of the protein. The consensus of the computational predictions was that of Oac being an integral membrane protein, with nine transmembrane helices, the N-terminus oriented in the cytoplasm and the C-terminus oriented in the periplasm (Supplementary material, Fig. 1).
Experimentally, the topology of Oac was solved by constructing C-terminal fusions of Oac with PhoA-LacZα using
Acknowledgments
We thank Lily Shen of the Electron Microscopy Unit, Research School for Biological Sciences, ANU, for assistance with the microscopy. This work was funded by the National Health and Medical Research Council of Australia.
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Both authors have contributed equally to this work.