0001). Because CsrA regulation of direct targets occurs post-transcriptionally, it is unlikely that CsrA controls the rate of luxR transcription directly. However, it is possible that CsrA might impact the stability of the luxR mRNA. Several factors are known to directly regulate luxR transcription, including LuxR itself (Dunlap & Ray, 1989; Shadel & Baldwin, 1991; Chatterjee

et al., 1996; Williams et al., 2008). Because LuxR levels are very low in a ∆litR strain, it is considered unlikely that the effect seen in a csrA overexpression strain BMS-777607 in vivo was because of LuxR autoregulation. Therefore, experiments were performed to probe for interactions between CsrA and the known LuxR regulator cAMP-CRP. Activation of the cAMP-CRP activator by CsrA would result in an increased luxR transcription rate. Quantitative RT-PCR was performed on cDNA samples obtained from ES114 (wild type) and DAPT PMF8 (∆litR) strains with pJW3 or pJW4 in 20 nM AHL to examine crp transcript levels. In contrast to the dependence of luxR level on CsrA expression, the quantity of crp transcript did not depend on the expression

level of csrA or on strain (P > 0.14) (data not shown). Finally, in an effort to rule out any influence of cAMP levels on the increase in luminescence seen between PMF8 (pJW4) and PMF8 (pJW3), the luminescence experiment (Fig. 3a and b) was repeated with 5 mM exogenous cAMP (Fig. 5a and b). If cya activity were in some way being positively affected Aldehyde dehydrogenase by CsrA, then addition of high levels of cAMP would be predicted to make luminescence output in PMF8 CsrA-independent. A relatively high concentration of cAMP was chosen because V. fischeri is capable of metabolizing cAMP, and it therefore needed to be provided in excess to ensure that there was enough to generate a response. When 5 mM cAMP was added to the growth medium, the luminescence levels did increase for both the wild-type and PMF8 strains

(compare Figs 3a and b–5a and b). However, the degree of change in luminescence between PMF8 (pJW3) and PMF8 (pJW4) was the same for each strain whether the concentration of cAMP was 0 (Fig. 3b) or 5 mM (Fig. 5b). Hence, it can be concluded that regulation of cAMP levels did not produce the CsrA-dependent observed effects on luxR transcription. All of the above experiments were performed simultaneously using both factorial design and standard laboratory design of at least two independent experiments with samples analyzed in triplicate. This enabled for a direct comparison of the analysis of the data via these two methods. Factorial design is a standard method of experimental design and data analysis (for example, see Box et al., 1978; Montgomery, 1997) widely used in agricultural and industrial research and development. It provides significant enhancement of statistical power vs. standard experimental designs, to identify subtle interactions between various regulatory elements.