El paro campesino del Catatumbo: referente regional del PNACP de 2013

In a next step, the functionality of the nirA inducible systems of both strains had to be established. First, site-specific homologous recombination into the target genes was confirmed for both constructs by PCR analysis using isolated total DNA (Figure 20A and B). Cultures of transgenic strains were then grown under inducing (NO3-) and repressing (NH4+) conditions in

BG110 medium. At certain time points, samples were taken and after centrifugation frozen in

liquid nitrogen. After all samples from different time points had been collected,

Figure 20: NirA promoter controlled expression of pratA. (A) Genotyping of Big∆pratA∆slr0415::nirApratA

(M)and wild-type Big strain (WT) by PCR analysis. Primers Slr0415 fw and PratA re were used. Expected size: 1452 bp in mutant, no signal in wild-type. DNA marker is shown at the right. (B) Genotyping of Big∆pratA::nirApratA(1) (M) and wild-type Big strain (WT) by PCR analysis. Primers NirA re and PratA re were used. Expected size: 791 bp in mutant, no signal in wild-type. (C) Cultures of Synechocystis wild-type Big and Big∆pratA∆slr0415::nirApratA strains were grown in BG110 medium supplemented with 17.6 mM NaNO3 (O/N

green) or NH4Cl (O/N red) and incubated overnight. Crude soluble proteins (20 µg) were fractionated by SDS-

PAGE, and blots probed with antiserum against PratA. (D) Activation NH4+ to NO3-. Cultures of Synechocystis

wild-type Big and Big∆pratA::nirApratA(1) strains were grown in BG110 medium supplemented with 17.6 mM

NH4Cl to OD730 of 0.8-1 (0 h, red), washed and resuspended in BG110 supplemented with 17.6 mM NaNO3 (green).

Samples were taken at the time points indicated. The wild-type (WT) was incubated overnight either with NH4Cl or

NaNO3. Crude soluble proteins (20 µg) were extracted, fractionated by SDS-PAGE, and probed with antiserum

against PratA. (E) Inhibition NO3- to NH4+. Cultures of wild-type Big (WT) and Big∆pratA::nirApratA(1) strains

RESULTS

crude soluble proteins were extracted, fractionated by SDS-PAGE and accumulation of PratA protein subsequently examined by Western analysis. Figure 20C shows the result of a representative Big∆pratA∆slr0415::nirApratA strain. Cells treated with NaNO3 did not

accumulate PratA when compared to wild-type (Figure 20C, lanes 1 and 2). Similar to the NH4Cl treated cells and the HP∆pratA knockout strain, only the cross-reacting signal of the

PratA antiserum was observed (Figure 20C, lanes 1, 2 and 3). In all strains of Big∆pratA∆slr0415::nirApratA that had been tested, no accumulation of PratA protein was detectable (data not shown). This indicated that the construct was not functional in these strains, although the correct assembly and integration had been confirmed.

In contrast, when Big∆pratA::nirApratA(1) strain had been grown under inhibiting conditions (NH4Cl) and was then induced for several hours (NaNO3), a significant protein accumulation up

to wild-type levels was achieved (Figure 20D, compare lane 1 and 7). Accumulation of PratA was discernible from two hours after induction by nitrate (Figure 20D, compare lane 1 and 3). Taken together, these findings suggested that the nirA promoter could indeed induce PratA expression, but did not cause overexpression.

As mentioned before, the tight repression of the pratA gene would be crucial for visualization of the dynamic processes of membrane biogenesis in vivo, especially in a time-resolved manner. Instead, PratA was detectable even under repressing conditions (Figure 20D, lane 1). Although not at wild-type level, the remaining PratA molecules could severely interfere with the experiment. For this reason, additional experiments were performed that varied in the concentration of ammonium chloride used to repress the promoter (data not shown). Since PratA was detectable regardless of the ammonium concentration used, a different approach was performed. It was assumed, that growth of the culture under inhibiting conditions for several days could somehow have caused a reactivation of the nirA regulated pratA gene expression. Therefore, the Big∆pratA::nirApratA(1) strain had been grown under inducing conditions and was then inhibited for several hours (Figure 20E). After 8 hours of repression, a slight reduction of PratA protein level was observed (Figure 20E, lane 2), which was even more pronounced after 48 hours (Figure 20E, lane 4). Nevertheless, even by doubling the ammonium concentration to 35 mM for a short period, PratA protein was still detectable (Figure 20E, lane 3 and 5).

RESULTS

Figure 21: NirA promoter controlled expression of pratA in Big∆pratA background. (A) Genotyping of Big∆pratA::nirApratA(2) (M) and wild-type Big strain (WT) by PCR analysis. Primers NirA fw and PratA re were used. Expected size: 568 bp in mutant, no signal in wild-type. (B) Cultures of Synechocystis wild-type and Big∆pratA::nirApratA(2) strains were grown in BG110 medium supplemented with 17.6 mM NH4Cl to OD730 of

0.8-1 (0 h red), washed and resuspended in BG110 supplemented with 17.6 mM NaNO3 (green). Samples were

taken at the time points indicated. After 24 h cells were washed again, resuspended in BG110 supplemented with

17.6 mM NH4Cl and incubated overnight (O/N red). Soluble proteins (15 µg) were subjected to SDS-PAGE, and

blots probed with an antiserum against PratA.

The problem was finally solved by using the pBS_∆pratA::nirApratA construct that had previously been transformed into WT Big, for transformation into the Big∆pratA knockout strain. It was not clear whether homologous recombination into the pratA gene would work because it already contained the kanamycin resistance gene that had been used to create the knockout. However, after transformation dozens of colonies of Big∆pratA::nirApratA(2) strain were obtained. PCR analysis confirmed the integration of the inducible construct into the genomic DNA (Figure 21A). Further analysis suggested that in the new inducible strains, no PratA protein was present when cultures had been grown under repressing conditions (Figure 21B, lane 1). After changing to inducing conditions, protein accumulation was observed after 30 min (data not shown) with a maximum after overnight incubation (Figure 21B, lanes 2, 3 and 4). The PratA concentration could even be reduced again by changing to repressing conditions again, although some PratA protein was still available (Figure 21B, lane 5). A reason could be that the PratA stability is too high for the residual protein to have vanished completely after 12 hours.

RESULTS

4.2.4

Analysis of extra-chromosomal expression of N-terminal tagged