Topology and identification of critical residues of the O-acetyltransferase of serotype-converting bacteriophage, SF6, of Shigella flexneri

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Abstract

The modification of the LPS O-antigen, seen in the diverse serotypes of Shigella flexneri is brought about by the glucosyltransferases (Gtr) and the O-acetyltransferase (Oac). In this study, we establish the membrane topology of Oac using the dual reporter PhoA–LacZα. We have determined that Oac is an integral membrane protein with 10 transmembrane regions. The hydrophilic N- and C-termini are oriented in the cytoplasm. Functionally important cytoplasmic and periplasmic loops have also been identified. Furthermore, cytoplasmic residues R73 and R75R76 were found to be critical to Oac function.

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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.

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