ALTERACIÓN DE LA CONVIVENCIA

The results presented up to this point have only been concerned with expression in relatively fast-growing cultures. Therefore, the effect of position within the chromosome on promoter activity was also analysed during stationary phase, by measuring increase in fluorescence on induction in E. coli strains carrying the lac28::egfp fusion at the different chromosomal locations shown in figure 3.5. Promoter activity was measured during the stationary phase of growth on M9 minimal salts medium supplemented with 250 μM IPTG. The higher concentration of IPTG was found to be required for induction during stationary phase, potentially due to the increased cell density of the cultures compared to that of mid-late exponential growth. Overnight cultures were split in two and the inducer added to one half of the culture to allow a direct comparison between culture with and without inducer after 5 hours induction. As in section 3.5, expression of egfp was derived from the fluorescence at 510 nm divided by OD620 of at least three separate experiments to give the mean and standard deviation. The starting E. coli K-12 MG1655 strain was found to auto-fluoresce at the higher cell densities required by this experiment, as shown in figure 3.20, therefore activity of un-induced cultures were subtracted from that of induced cultures for each separate experiment, to give induction of reporter expression, before calculation of mean and standard

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Figure 3.20: Promoter activity at the mntH and asl loci during stationary phase

Fluorescence/OD620 measured in strains BRY73 (mntH), BRY35 (asl), carrying lac28::egfp at the designated chromosomal locus, or the starting strain MG1655. Samples were taken during stationary phase (OD620 ~ 1.5) at 37 ˚C in M9 minimal medium supplemented with 250 μM IPTG, where stated. Overnight cultures were split into two aliquots prior to induction of one of these to allow direct comparison between induced and un-induced cultures. Data shown are averages of fluorescence/OD620 measurements from at least three independent experiments, and error bars show one standard deviation from the mean. Data show that fluorescence of the mntH and asl strains does vary slightly in the absence of inducer, but this is always lower than the starting MG1655 strain and is representative of all other strains used in this work. Data also show that fluorescence of the mntH and asl strains increased on addition of IPTG, however fluorescence of MG1655 decreased.

deviation. The autofluorescence of un-induced cultures did vary between strains, however this was always less than the starting MG1655 strain. Further to this, the fluorescence of strains carrying the lac28::egfp fusion always increased on average, whereas the fluorescence of the starting strain (MG1655) decreased on addition of IPTG (Figure 3.20). This suggests that variation of fluorescence in the un-induced culture was not due to leakage of the promoter (Figure 3.20). Subtraction of the background auto-fluorescence is the reason for the absolute level of fluorescence reported on the y-axis, of figure 3.21, being so much lower than that in figure 3.7 (exponential growth).

Expression of egfp during stationary phase was not only dependent upon regulation by the promoter region, but also upon position within the chromosome, as reported for exponential growth. Position-dependent modulation of promoter activity during stationary phase caused large variation of egfp expression by the lac28 fragment promoter across the chromosomal positions tested (Figure 3.21). The total variation of promoter activity was much less than the 310-fold differences found during exponential growth or even the 11-fold variation between non-tsEPOD loci. Also, the level of expression at all loci was very low compared to that during exponential growth, which was most likely due to the promoter being σ70-dependent (Figure 3.21; Figure 3.7).

The largest induction of fluorescence during stationary phase was at the mntH locus, which is positioned within the Left macrodomain. When positioned at the mntH locus, increase of fluorescence on inductrion was approximately 8-fold greater than that at the tam locus, at which the promoter activity was weakest (Figure 3.21; Figure 3.22). These results suggest that position within the E. coli chromosome does affect promoter activity during stationary phase, however the pattern of expression is significantly different compared to that during exponential growth and activity is generally much weaker.

NSR Right Ter Left NSL oriC Ori 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 ara dkgB yafT eaeH ycb tam rcs mntH yqe nupG pitB asl mel dif lac

Figure 3.21: Promoter activity at different chromosomal positions during stationary phase

Expression of the lac28::egfp fusion during stationary phase (OD620 ~ 1) at 37 ˚C in M9 minimal medium supplemented with 250 μM IPTG. Each point represents the fluorescence at 485 nm excitation, 510 nm emission, divided by the OD620, of the un-induced sample subtracted from the fluorescence/OD620 of the induced culture. Each point is the average of at least three independent experiments with the error bars showing one standard deviation from the mean. Position of each point on the x-axis indicates chromosomal position in Mb with respect to co-ordinate system origin and is to scale. The positions of the six macrodomains are represented by the grey shaded boxes with oriC and the dif indicated by vertical black lines.

0 0.01 0.02 0.03 0.04 0.05 tam rcs mntH Fluore s c e nce / OD 620 Chromosomal position

Figure 3.22: Promoter activity at the tam, rcs and mntH loci during stationary phase

Expression of egfp in the BRY33 (tam), BRY27 (rcs) and BRY73 (mntH) strains during stationary phase (OD620 ~ 1) at 37 ˚C in M9 minimal medium supplemented with 250 μM IPTG. Each point represents the fluorescence at 485 nm excitation, 510 nm emission, divided by OD620, of the un-induced sample subtracted from the fluorescence/OD620 of the induced culture. Each point is the average of at least three independent experiments with the error bars showing one standard deviation from the mean. Data show that promoter activity was strongest at the mntH locus, which was approximately 8-fold greater than the weakest activity at the tam locus. Expression of egfp at the mntH locus was also 2-fold stronger than that of the rcs locus, which also resides within the Left macrodomain