The clpP multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcus

Abstract

In the cyanobacterium Synechococcus sp. PCC 7942 the proteolytic subunit ClpP for the ATP-dependent Clp protease is encoded by a multigene family (clpP1-3). Whereas clpP1 is monocistronic, the other clpP genes are arranged as two bicistronic operons with other clp genes (i.e., clpP2-clpX and clpP3-clpR). The induction of ClpP2, -P3, -R and -X was investigated during several physiological stresses including heat, cold, high light, salt and oxidation. Like the previously described ClpP1, the other four Clp proteins are not heat-inducible but they do increase gradually during high light and cold. High salt and oxidative stresses failed to elicit marked increases in ClpP2, -P3, -R and ¿X protein, except for an initial rise in ClpP2 with salt treatment. Numerous attempts to genetically inactivate each clp gene resulted in viable transformants only for clpP2, indicating that ClpP3, -R and ¿X are essential for Synechococcus. Unlike the earlier described D clpP1 mutant, the D clpP2 strain exhibited no significant phenotypic changes from the wild type. Expression analysis of the clp genes showed that despite being bicistronically arranged, clpP2-clpX and clpR-clpP3 produce primarily four monocistronic transcripts, although minor polycistronic transcripts could be detected. Mapping of the 5¿-ends for clpX and clpP3 revealed promoters within the 3¿-region of clpP2 and clpR, respectively. Transcriptional and translational studies further showed differences in the expression and regulation between the various clp genes. Inactivation of clpP1 causes a significant decrease in ClpP2 protein concomitant to small increases in both ClpP3 and ClpR, whereas inactivation of clpP2 resulted in a large rise in clpP1 transcripts but a lesser increase in ClpP1 protein. Similar small increases in ClpP3, -R and ¿X proteins also occurred in D clpP2. These results demonstrate the regulatory complexity of the clpP multigene family and the essential nature of most Clp proteins in Synechococcus.