Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from Myxococcus xanthus were established. Using these conditions, we analyzed protein patterns of heat-shocked M. xanthus cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase ␣ subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in M. xanthus (R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357-5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218-224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase ␣ subunit are heat shock inducible.All organisms respond and adapt to heat shock by inducing heat shock proteins (HSPs). A number of HSPs from bacteria to animals are well conserved (14,29). HSPs are also induced in response to carbon, nitrogen, or phosphate starvation in prokaryotes (19,20,21,24). In eukaryotes, HSP100, HSP90, HSP70, HSP60, and HSP40 function as molecular chaperones, and in prokaryotes, DnaK (a homologue of HSP70) (12), GroEL (HSP60) (26), DnaJ (HSP40) (12), and GroES (27) do so. In addition, ATP-dependent proteases such as ClpP and Lon are known to be HSPs (7, 10).Myxococcus xanthus is a gram-negative soil bacterium that feeds on microorganisms and organic debris (5, 6). Under nutrient starvation, M. xanthus cells aggregate by gliding motility and form multicellular fruiting bodies (FB) in which cells differentiate into spores. It has been shown that a number of developmental signals are coordinated during the differentiation process. Spores are metabolically dormant and resistant to desiccation, heat, and UV irradiation.The heat shock response of M. xanthus was previously investigated by labeling M. xanthus cells with [35 S] methionine during vegetative growth, glycerol-induced spore formation, and starvation-induced FB formation (17). During vegetative growth, 18 major HSPs with molecular masses of 91 to 14.5 kDa were found. When cells were heat shocked prior to starvation-induced FB and spore formation, FB and spore formation was accelerated with no effect on spore yield. During glycerol-induced spore formation, heat shock accelerated the rate of spore formation and enhanced the spore yield by fivefold (13).Here, we reinvestigated the heat shock response by twodimensional (2D) gel electrophoresis and N-terminal microsequencing of heat-shock-induced proteins in M. xanthus. We found that in addition to well-known molecular chaperones (DnaK, GroEL, a...