Escherichia coli genes regulated by cell-to-cell signaling

Abstract

Utilizing the bicistronic reporter transposon mini-Tn5 lacZ-tet/1, we have identified lacZ fusions to four Escherichia coli genes/operons that are strongly activated by the accumulation of self-produced extracellular signals. These fusions were designated cma9, cma48, cma113, and cma114 for conditioned medium activated. Each of the cma fusions was expressed in a growth phase-dependent manner, and the presence of conditioned medium from a stationary phase E. coli culture resulted in the premature activation of these fusions in cells at early to mid-logarithmic phase. The cma48 and cma114 fusions were dependent on RpoS for growth phase expression and response to extracellular factors. The extracellular factors that activated the cma9, cma48, and cma114 fusions were produced in both rich complex and defined minimal media. The cma fusions were shown to be within the cysK (cma9), astD (cma48), tnaB (cma113), and gabT (cma114) genes. These genes function in the uptake, synthesis, or degradation of amino acids that yield pyruvate and succinate.

Figure 1

Effect of conditioned medium on expression of the cma fusions. The accumulation of β-galactosidase from the cma fusions at various stages of growth is shown. Cells were grown in 0.5× LB (LB) or in conditioned medium from early stationary-phase cells (CM). Each point in growth represents an OD₆₀₀ of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, or 1.2. The β-galactosidase values are in Miller units and represent the average of duplicate samples from a typical experiment. Repeated experiments gave similar values.

Figure 2

Fractionation of the factors involved in cma activation. Conditioned medium prepared from M9 minimal medium + glycerol was fractionated on a Sephadex G-10 column as described in the text. Column fractions 11 and 12 represented the void volume, and the M9 salts eluted at fraction 22. For fractions 12–22, the same column fraction was used to assay each of the four cma fusions by mixing 1 ml of fraction with 2 ml of 0.5× LB. The activation of each fusion was determined by β-galactosidase expression in cells at early-logarithmic phase and was normalized to the levels of expression in the absence of factor.