Involvement of the selected genes in the colanic acid pathway and cloning under arabinose promoter.

<p>(A) The genes screened for histone resistance (bold pink) are represented in the scheme of the colanic acid pathway. The first steps of the colanic acid pathway consist on the production of activated sugar in the cytoplasm of the bacterium. Mannose-6-phosphate (Man-6-P) leads to the production to GDP-fucose by CpsG and CpsB. Others activated sugars, UDP-galactose (UDP-Gal), -glucosamine (-Glc) and –glucuronic acid (-GlcA), are produced also for other cellular components. The assembly of the first sugar unit, colanic acid (CA) unit, is assembled on the lipid carrier, called undecaprenyl pyrophosphate, by Wca proteins, such as WcaJ, WcaI and WcaA. Then the CA unit is exported and polymerized by the Wzc machinery, for the production of the exopolysaccharide. Another possibility is the addition of CA unit on the short form of the lipopolysaccharide (LPS) by an unknown mechanism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071097#pone.0071097-Meredith1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071097#pone.0071097-Stevenson1" target="_blank">[22]</a>. (B) The selected genes belonging to the colanic acid cluster were cloned under arabinose promoter on pBAD33 and pBAD18 vectors. Different constructs were obtained to address different gene combinations in the histone resistance and to refine the essential genes for the resistance. (C) The toxicity of the arabinose-induced genes was tested by following the bacterial population (CFU/ml). After 2 h of culture (indicated by an arrow and considered as time 0), L-arabinose was added at 0.4 (open triangle, full line), 0.04 (light gray triangle, dash line) and 0.004% (dark gray triangle, dot line) final concentration or not (black diamond, full line), and the culture was prolonged during 6 h.