Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones trigger factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by overexpression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroEL or SecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that overexpressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that overexpression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK. The relevance for such a convergence of networks of chaperones and proteases acting directly on EF-Tu to modulate the intracellular rate of protein synthesis in response to protein aggregation is discussed. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.