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Cellular localization of choline-utilization proteins in
using novel fluorescent reporter systems
Lookup NU author(s)
Dr Ling Juan Wu
Professor Jeff Errington
Professor Waldemar Vollmer
Dr Jan-Willem Veening
Eberhardt A, Wu LJ, Errington J, Vollmer W, Veening J-W
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The molecular mechanisms underlying cell growth, cell division and pathogenesis in
are still not fully understood. Single-cell methodologies are potentially of great value to investigate
cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the
chromosome. We have also established a zinc-inducible system for the fine control of
-fusion gene expression and for protein depletion experiments in
. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of
teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.
Wiley-Blackwell Publishing Ltd.
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