Identification of a functionally important loop in Salmonella typhimurium ArnT.

Biochemistry
Nicholas A ImpellitteriCandice S Klug

Abstract

ArnT confers resistance to the antibiotic polymyxin in Salmonella typhimurium and Escherichia coli through the modification of lipid A, a major component of the outer surface of gram-negative bacteria. ArnT transfers a neutral aminoarabinose moiety onto the negative phosphate groups of lipid A, reducing the surface charge of the bacteria and preventing cationic peptides such as polymyxin from electrostatically recognizing and killing the bacteria. We previously reported the first expression, purification, and functional analysis of ArnT from S. typhimurium [Bretscher, L. E., Morrell, M. T., Funk, A. L., and Klug, C. S. (2006) Protein Expression Purif. 46, 33-39]. Our studies showed that ArnT is highly alpha-helical and described a new in vivo functional growth assay. Here, we use the cysteine-specific mPEG-mal to demonstrate that all eight of the native cysteines in S. typhimurium ArnT are in the reduced form and not involved in disulfide bonds and show that the cysteine-free protein is structurally and functionally intact as characterized by circular dichroism and the in vivo growth assay. Following this initial characterization, in vivo expression and function profiles were surveyed for 31 consecutive mutations within a putat...Continue Reading

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Citations

Sep 26, 2019·Annals of the New York Academy of Sciences·Anandhi Anandan, Alice Vrielink
Jun 25, 2019·Chembiochem : a European Journal of Chemical Biology·Charlotte OlagnonPaul Kosma

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