Thermally aggregated, endogenous proteins of Escherichia coli form a distinct fraction, denoted S, which is separable by sucrose-density-gradient centrifugation. It was shown earlier that DnaK, DnaJ, IbpA and IbpB heat-shock proteins are associated with the S fraction. Comparison of the rise and decay of the S fraction in mutants defective for heat-shock proteases Lon (La), Clp, HtrA (DegP, Do) and in wild-type strains made studies of proteolysis and the function of the heat-shock response possible in vivo. Different timing and the extent of action of particular proteases was revealed by the initial size and decay kinetics of the S fraction. The proteases Lon, Clp, and HtrA all participated in removal of the aggregated proteins. Mutation in the gene encoding ClpB caused the most prominent effect (47% stabilization of the S fraction). The correlation between the disappearance of the S fraction and proteolytic activity was supported by the result of the in vitro reaction. Approximately one third of the isolated S fraction was converted to trichloroacetic acid-soluble products by the purified HtrA protease. Mg2+ ions stimulated the reaction, in contrast to the reaction of the HtrA protease with casein. The digestion of the aggregated proteins, unlike the digestion of casein, by HtrA protease in vitro was inhibited by added DnaJ, which might reflect protection of the aggregated proteins in vivo by DnaJ from excessive degradation. One might expect that such an activity of DnaJ would promote denatured protein renaturation versus proteolysis. Moreover, among the aggregated proteins that are discernible by electrophoresis, none could be identified as being more susceptible than any other to HtrA degradation. The separation pattern of these proteins before and after the in vitro digestion did not show a difference corresponding to the loss of about 30% of constituting proteins. This was interpreted as recognition by the HtrA protease of a state of protein denaturation rather than specific amino acid sequences in particular proteins. We conclude that the fraction consisting of proteins heat-aggregated in vivo (i.e. the S fraction) contains endogenous substrates for the heat-shock proteases tested. Their use for in vitro reaction reveals information that is in some respects different from that obtained with exogenous substrates such as casein.

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The roles of the Escherichia coli IbpA and IbpB chaperones in protection of heat-denatured proteins against irreversible aggregation in vivo were investigated. Overproduction of IbpA and IbpB resulted in stabilization of the denatured and reversibly aggregated proteins (the S fraction), which could be isolated from E. coli cells by sucrose gradient centrifugation. This finding is in agreement with the present model of the small heat-shock proteins' function, based mainly on in vitro studies. Deletion of the ibpAB operon resulted in almost twofold increase in protein aggregation and in inactivation of an enzyme (fructose-1,6-biphosphate aldolase) in cells incubated at 50 SC for 4 h, decreased efficiency of the removal of protein aggregates formed during prolonged incubation at 50 SC and affected cell viability at this temperature. IbpA/B proteins were not needed for removal of protein aggregates or for the enzyme protection/renaturation in cells heat shocked at 50 SC for 15 min. These results show that the IbpA/B proteins are required upon an extreme, long-term heat shock. Overproduction of IbpA but not IbpB caused an increase of the level of β-lactamase precursor, which was localized in the S fraction, together with the IbpA protein, which suggests that the unfolded precursor binds to IbpA but not to IbpB. Although in the wild-type cells both E. coli small heatshock proteins are known to localize in the S fraction, only 2 % of total IbpB co-localized with the aggregated proteins in the absence of IbpA, while in the absence of IbpB, the majority of IbpA was present in the aggregates fraction.

Heat shock induces protein aggregation in Escherichia coli and E. coli (lambda cl857). The aggregates (S fraction) appear 15 min post-induction and are separable from membranes by sucrose density-gradient centrifugation. The S fraction quickly disappears in wild type strains but persists in rpoH mutant with concomitant quick inner membrane destruction. We propose that: (1) the disappearance of the S fraction reflects a rpoH-dependent processing, (2) the membrane destruction explains the lethality of the rpoH mutation at elevated temperatures; and (3) the protection of the inner membrane integrity is an important physiological function of the heat-shock response. We assume that the S fraction of aggregated proteins represents the signal inducing the heat-shock response. The prophage thermo-induction results in an increase (35 min post-induction) in the A fraction resembling that of the adhesion zones of the membranes. This fraction is greater than the corresponding fraction from uninduced cells. The increase is mediated by the lambda late genes, since it is absent in the induced E. coli (lambda cl857 Qam21). Since heat shock is widely used for induction of the lambda promoters in expression vectors it is possible that the formation of the protein aggregates (though transient in WT strains) and/or the fragility of membranes in rpoH mutants may be the cause of poor expression of cloned genes or may lead to mistaken localization of their expression products.

The radioactively labeled proteins synthesised in Escherichia coli minicells infected by bacteriophage lambda R and lambda R+ were compared by polyacrylamide gel electrophoresis. lambda R mutants, which have lost the ability to lyse host cells, lack a polypeptide of molecular weight 17.5 KD corresponding to the molecular weight of murein transglycosylase - a bacteriolytic enzyme from lambda lysates which we have described previously. It has been shown by direct comparison using radio-labeled enzyme that transglycosylase comigrates with the R gene product. The enzyme was undetectable in induced cultures of E. coli W3350 su degrees (lambda cI857 Ram 5) and C600 (lambda cI857 acR301), while it was present in a lambda Rz mutant lysate. We conclude that the transglycosylase is the R gene product.