Fifty nanograms of cDNA was the template for the RT-PCR, with primer concentrations of 250 μM. 2× SYBR Green master mix (Applied Biosystems) and H2O were added to a final reaction volume of 50 μL per well in a MicroAmp Optical 96-well reaction plate (Applied Biosystems). RG7204 purchase Thermal cycler settings were programmed for 52 °C for 2 min, 95 °C for 10 min, then 45 cycles of the following: 95 °C for 15 s, 51 °C for 15 s, and 60 °C for 1 min, which was the data collection point. Ideally, a csrA partial deletion strain would have been used for experiments as has been possible
in other systems (Liu et al., 1995; Lenz et al., 2005). However, repeated attempts failed to generate the desired construct. Therefore, an alternative strategy was employed to modulate CsrA levels whereby either csrA (pJW3) or csrB1 (pJW4) was overexpressed from a stable plasmid construct in two V. fischeri strains, ES114 (wild type) and PMF8 (ΔlitR). This approach was followed, because in factorial design just two selleck chemicals llc levels of each experimental factor are permitted and they should be as far apart from one another as possible. A 20 nM level of AHL was chosen for experiments because it permitted for detection of luminescence from ES114 strains without fully
saturating the system. The amount of csrA transcript was measured to ensure that there were significantly different levels expressed from pJW3 and pJW4. As anticipated, there were higher levels of csrA transcripts in cells overexpressing csrA (pJW3) in the presence Sclareol of 20 nM AHL in comparison with the cells overexpressing csrB1 (pJW4) (Fig. 2). Further, because CsrB1 post-translationally sequesters CsrA (Romeo, 1998; Timmermans & Van Melderen, 2010), the actual decrease in the cellular activity of CsrA in strains overexpressing csrB1 is likely greater than what is observed by simply measuring differences in csrA transcript levels. The V. fischeri ES114 (wild type) and PMF8 (ΔlitR) strains carrying pJW3, pJW4, or the control pVSV104 were next examined for luminescence expression. LitR was chosen as the quorum-sensing factor to be examined because of the fact that it is a critical link between the upstream
quorum-sensing regulatory network, and the downstream luminescence response regulated by LuxR (Fig. 1). The level of luminescence in the wild-type strain V. fischeri ES114 was independent of the expression level of CsrA (over the range studied) (Fig. 3a). In contrast, the ∆litR strain of V. fischeri (PMF8) produced the lowest level of luminescence when CsrA activity was depressed [strain PMF8 (pJW4)], an intermediate level for the control [strain PMF8 (pVSV104)], and the highest level when csrA was overexpressed (strain PMF8 (pJW3) (Fig. 3b). The results showed that there was a significant interaction between litR and the CsrA level (P < 0.0001). Thus, CsrA did not affect the luminescence level in V. fischeri ES114 (Fig. 3a), but in the absence of LitR luminescence was dependent on CsrA (Fig. 3b).