RelA and PopD act in the same pathway to regulate PopC secretion

In agreement with the faster and increased secretion of PopC in starving ΔpopD cells, our preliminary experiments suggest that p17 accumulated earlier in these cells than in WT and with maximum accumulation reached already after 6 hrs of starvation (Fig. 17E). In total, these findings strongly suggest that PopD inhibits PopC secretion in vegetative cells and is involved in the slow secretion of PopC during starvation.

To rule out that the increased accumulation of PopC in the supernatant of the ΔpopD mutant was caused by cell death/lysis, PopC secretion was determined in the ΔpopD, csgA, popC⁺, mutant, which has no growth defects (Table 11).

PopC secretion in this mutant was similar to that of the ΔpopD, csgA⁺, popC⁺ mutant (Fig. 17B) supporting the notion that PopD inhibits PopC secretion.

Finally, to rule out that the increased accumulation of PopC in the supernatant of the ΔpopD mutant was caused by increased PopC stability, PopC secretion in the ΔpopD mutant was determined in the absence of protease inhibitors. As shown in Fig. 17D, under these conditions PopC was neither detected in the ΔpopD mutant nor in WT suggesting that secreted PopC is not more stable in the ΔpopD mutant compared to WT.

Figure 18. RelA and PopD act in the same pathway to regulate PopC secretion.

(A) Cells of the indicated strains were exposed to starvation in the presence of protease inhibitors and cell free supernatants prepared. Supernatants from 10⁸ cells were analyzed by ELISA with anti-PopC antibodies. Level of PopC secreted by WT after 24 hours is indicated by the dashed line.

(B) PopC accumulation in total cell extracts. Cells were treated as described in (A) and total cell lysates prepared at the indicated time points. Protein isolated from 10⁸ cells was loaded per lane, separated by SDS-PAGE, and analyzed by immuno-blotting with anti-PopC antibodies. Position of PopC is indicated.

Note that the data for DK101, SA3439 are the same as shown in Fig. 2.8.

to starvation resulting in activation of PopC secretion. To test this hypothesis, we attempted to follow PopD protein accumulation during vegetative growth and starvation. In immuno-blots, we were repeatedly unable to detect PopD in total cell extracts as well as in the cell supernatants using two independently generated rabbit, polyclonal α-PopD antibodies or antibodies against the Strep-tag of a functional C-terminal Strep-Strep-tagged PopD fusion.

To determine the sensitivity, i.e. detection limit, of the α-PopD antibodies generated against full-length, soluble PopD-His6, we titrated the α-PopD antibodies against PopD-His6 purified from E. coli using immuno-blotting. As shown in Figure 19A, the α-PopD antibodies could detect 0.8 ng of purified PopD-His6 protein (corresponding to 280 × 10⁸ molecules). In total M. xanthus cell extract prepared from 10⁸cells, the α-PopD antibodies cannot detect PopD (data not shown) suggesting that either the number of PopD molecules per cell is <280 or that PopD is highly unstable. As described in section 2.2 our data suggest a PopC:PopD stoichiometry of 1:4 in the PopC/PopD complex.

Previously, we estimated that M. xanthus cells contain approximately 2000 PopC molecules. Based on our estimate of the stoichiometry of the PopC/PopD complex we would predict that M. xanthus contains 8000 PopD molecules per cell. Therefore, it is highly unlikely that inability to detect PopD in M. xanthus cell extracts is caused by low antibody sensitivity.

To address whether PopD is unstable in M. xanthus cell extracts, PopD-His6 purified from E. coli was added to total M. xanthus vegetative cell extracts and subsequently SDS-loading buffer was immediately added to stop potential proteolytic reactions. As shown in Fig. 19B, PopD-His6 is degraded when incubated with M. xanthus cell extract. At the same time, PopD-His6 was stable when incubated in buffer (Fig. 19B). Based on this experiment we suggest that PopD is rapidly degraded when M. xanthus cells extract are prepared and that this instability precludes the detection and analysis of PopD in M. xanthus cell extracts using immunological techniques.

Figure 2.19. PopD is unstable in M. xanthus cell extracts.

(A) Determination of the sensitivity, i.e. detection limit, of α-PopD antibodies generated against full-length PopD-His6. The indicated amounts of purified PopD-His6 was loaded on, separated by SDS-PAGE, and analysed by immuno-blotting with anti-PopD antibodies.

(B) Purified PopD-His6 is unstable in the presence of M. xanthus WT extract prepared from vegetative cells. The indicated amounts of PopD-His6 were added at room temperature to cell extracts from 10⁸ cells prepared from DK101 (WT) and SDS-loading buffer was immediately added to stop reactions. Equal volumes of the samples were analysed as in (A).

Therefore, to study the stability of PopD in response to starvation we used an in vitro approach. To this end, purified His6-PopC/PopD-S complex (see 3.1.2) and the control protein MalE purified from E. coli were incubated with total cell extracts prepared from vegetative or starving WT and ΔrelA M. xanthus cells.

Whereas MalE and PopC were stable in the presence of all four cell extracts, the PopD level specifically decreased in the presence of extract from starving

WT cells (Fig. 20). Notably, PopD levels did not decrease in the presence of extract from starving ΔrelA cells Thus, PopD is specifically degraded when incubated with cell extract from starving M. xanthus WT cells. These observations suggest that in response to starvation PopD is degraded and that this degradation depends on RelA.

Figure 20. PopD-S is rapidly degraded in WT cell extract from starving cells.

2 µg of purified His6-PopC/PopD-S complex or 1 µg of purified MalE protein were incubated with 7 µg of total cell extract of WT (DK101) or ΔrelA (MS1000) cells prepared from vegetative (0 hrs of starvation) or starving (2 hrs of starvation) cells. His6-PopC/PopD-S and MalE were similarly incubated in the absence of cell extract (-). Reaction mixtures were incubated at room temperature for 10 min. Proteins were separated by SDS-PAGE and analysed by immuno-blotting using antibodies against PopD, PopC and MalE as indicated. The immuno-blot with anti-PilC antibodies serves as a loading control for the four cell lysates.