Figure 15.

Overexpression of the gcvB sRNA represses LpxC accumulation in the wild type. (a) Equivalent amounts of total cellular proteins were applied from the isogenic strain’s wild type with the vector alone (lane 1) and when the expression of gcvB was induced upon the addition of 100 and 300 μM IPTG (lanes 2 and 3, respectively). Proteins were resolved on a 12.5% SDS–PAGE and immunoblotted with anti-LpxC polyclonal antibodies. (b) Immunoblot analysis of the total cellular extracts obtained from ΔlapAB with the vector alone without the addition of IPTG (lane 1) and from ΔlapAB transformed with the plasmid with the inducible expression of gcvB sRNA (lanes 2 and 3) with the addition of the indicated amounts of IPTG. Cultures of ΔlapAB were grown in M9 minimal medium up to an OD₅₉₅ of 0.2 prior to the addition of IPTG, and allowed to grow for another 2 h. Equivalent amounts of proteins were resolved on a 12.5% SDS–PAGE and transferred using Western blotting. The immunoblots were treated with anti-LpxC polyclonal antibodies in order to detect LpxC. The arrow indicates the position of LpxC. (c) Exponentially grown cultures of the wild type and its deletion derivatives lacking sRNA encoding genes were serially spot diluted in LA agar, supplemented with or without varying concentrations of CHIR090, as indicated.