Heterogeneity in expression of the Escherichia coli colicin K activity gene cka is controlled by the SOS system and stochastic factors

Abstract

Phenotypic diversity provides populations of prokaryotic and eukaryotic organisms with the Xexibility required to adapt to and/or survive environmental perturbations. Consequently, there is much interest in unraveling the molecular mechanisms of heterogeneity. A classical example of heterogeneity in Escherichia coli is the subset (3%) of the population that expresses the colicin K activity gene (cka) upon nutrient starvation. Here, we report on the mechanism underlying this variable response. As colicin synthesis is regulated by the LexA protein, the central regulator of the SOS response, we focused on the role of LexA and the SOS system in the variable cka expression. Real-time RT-PCR showed that the SOS system, without exogenous DNA damage, induces moderate levels of cka expression. The use of cka-gfp fusions demonstrated that modiWcation of the conserved LexA boxes in the cka promoter region aVected LexA binding aYnity and the percentage of cka-gfp expressing cells in the population. A lexA-gfp fusion showed that the lexA gene is highly expressed in a subset of bacteria.Furthermore, cka-gfp fusions cloned into higher copy plasmid vectors increased the percentage of cka-gfp positive bacteria. Together, these results indicate that the bistability in cka expression in the bacterial population is determined by (1) basal SOS activity, (2) stochastic factors and possibly (3) the interplay of LexA dimers at cka operator. Other LexA regulated processes could exhibit similar regulation.

The role of SOS boxes in enteric bacteriocin regulation

“…Similarly, induced enteric bacteriocin promoters express low basal levels of expression and high response ratios in the current (Fig. 4) and comparable studies (Mrak et al, 2007;Norman et al, 2005;Prieto et al, 2004;Vankemmelbeke et al, 2005). Moreover, when the contributions of each of the LexA binding sites were compared, expression under a single LexA repressor resulted in higher basal levels and lower response ratios (Fig.…”

“…However, in the alternative single mutant (2/+), the A tract is not present and the TA sequence forms a rigid structure. This observation may explain the decreased levels of luminescence measured and is supported by Mrak and colleagues (Mrak et al, 2007), who showed that T substitutions in the A tract of the distal SOS box (the AAAA motif was altered to ATAT) result in a decrease in gene expression, signifying the importance of the A motif. Interestingly, the single SOS box found in the colicin Ib promoter lacks A or T tracts (Table 3), and is thus less bendable, which might explain the lower response ratio observed (Fig.…”

“…Expression levels induced by the distal site are more pronounced than those of the proximal site in the colicin K promoter (Mrak et al, 2007). Additionally, the dual promoter sites of colicin E7, which both have an A tract (Table 3), show similar expression levels (Lu & Chak, 1996).…”

“…In vivo studies of the promoter region of colicins E1 (Salles & Weinstock, 1989) and E7 (Lu & Chak, 1996) show that mutations in either LexA binding motif result in an increased level of colicin production, suggesting that the colicin operon contains two distinct SOS boxes. In contrast, mutations within the SOS boxes of colicin K result in reduced expression (Mrak et al, 2007).…”

“…In vitro studies of the promoter region of colicins E1 (Ebina et al, 1983), A (Lloubes et al, 1988(Lloubes et al, , 1993 and K (Mrak et al, 2007) reveal that LexA binds cooperatively to the SOS regulatory region. Site-directed mutagenesis of LexA binding sites reveals that the first operator (Table 1, solid underline) is more pronounced in LexA binding affinity than the second (Table 1, dotted underline) (Lloubes et al, 1993).…”