Recently it has been shown that XylS dimers bind to DNA sequentially. The first monomer to bind is the one proximal to the RNAP binding site. This leads to [10DNA bending, which in turn enables the second monomer to bind, and indicates that XylS is dimerized prior to DNA binding . At typical cell-internal XylS-levels only 30-40% of the Pm promoter sequences are occupied in vitro and it has been proposed that complete occupancy cannot be achieved by XylS amounts which do not exceed its Selleck Seliciclib intracellular solubility . Vectors which
combine the XylS/Pm expression system with the broad-host-range mini-RK2 replicon [22, 23], in which XylS is expressed from
its natural Ps2 promoter, have been shown to be capable of producing recombinant RG-7388 mouse proteins at industrial levels in Escherichia coli[24, 25]. Expression levels of these vectors could be heavily increased by mutating different Aurora Kinase inhibitor DNA control elements of the expression cassette [10, 26, 27], and recently it has been demonstrated that they could be yet further improved when mutated DNA elements were combined . When induced expression levels are increased it leads, in most cases, to undesired high expression levels also in the absence of inducer. For the XylS/Pm expression system the background expression could be strongly reduced when the 5′-UTR flanking the Shine-Dalgarno site was mutagenized and this has been demonstrated to be useful for metabolic engineering purposes . With this approach an induction ratio of 260-fold could be reached, however, as a consequence induced expression levels were also reduced for these constructs. A possible alternative method of reducing uninduced expression could be to regulate the XylS expression level. Previous experiments have shown that strong XylS overexpression, as for example from the bacteriophage
T7 promoter or from Ps1, results in a complete loss of inducibility [21, 30]. Fusion of xylS to the Psal promoter, which can be activated by similar inducers as Pm, allowed simultaneous Endonuclease induction of XylS expression and XylS activation. Induction ratios that could be reached by this approach were about 180- to 240-fold . Here we report a more detailed study on the relationship between XylS expression levels and expression levels achieved from the Pm promoter, both under induced and uninduced conditions. Based on the outcomes of this study we propose a model that aims to explain the behaviour of XylS as a function of its concentration and its formation of monomers, dimers and higher order oligomers.