The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.