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Supplementary figure 1: spectroscopic analysis of the content of E. coli MC4100 cells carrying pTrc99A or a derivative recombinant plasmid pTrc99A pTrc99A-HasA(1-174) pTrc99A-HasA(1-174)E148A, D167A 1

Supplementary figure 2: Purification of (His)HasD by affinity chromatography MW 1 2 3 4 5 Western-blot analysis with anti-His tag antibodies 1: whole cell membranes solubilized in 0.7% lauryl maltoside 2: unbound fraction 3: washed fraction with 20 mM imidazole 4 and 5: eluted fraction with 250 mM imidazole 55 kDa (His)HasD Degradation products 2

Supplementary figure 3: Secretion of HasA in cells expressing wild-type HasD or Walker B mutant HasDE474Q HasA (His)HasD HasD WT E474Q Coomassie-stained Culture supernatants WB His, HasA Cell pellets MC4100 ara pBAD24-(His)HasD-HasE, pTrc99A-HasA 3