pTrc99A

pTrc99A-HasA(1-174)

pTrc99A-HasA(1-174)E148A, D167A

Supplementary figure 1: spectroscopic analysis of the content of E. coli MC4100 cells carrying pTrc99A or a derivative recombinant plasmid

Supplementary figure 2: Purification of (His)HasD by affinity chromatography

MW 1 2 3 4 5

(His)HasD55 kDa

Western-blot analysis with anti-His tag antibodies

1: whole cell membranes solubilized in 0.7% lauryl maltoside2: unbound fraction3: washed fraction with 20 mM imidazole4 and 5: eluted fraction with 250 mM imidazole

Degradation products

Supplementary figure 3: Secretion of HasA in cells expressing wild-type HasD or Walker B mutant HasDE474Q

HasA

HasA

(His)HasD

HasD WT E474Q

Coomassie-stainedCulture supernatants

WB His, HasACell pellets

MC4100 ara pBAD24-(His)HasD-HasE, pTrc99A-HasA