Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation.

The disaccharide trehalose is widely distributed in nature and can serve as a carbon reservoir, a signaling molecule for controlling glucose metabolism and a stress protectant. We demonstrated that in Escherichia coli ΔotsA cells, which are unable to synthesize trehalose, the aggregation of endogenous proteins during the stationary phase was increased in comparison to wild-type cells. The lack of trehalose synthesis boosted Nε-lysine acetylation of proteins, which in turn enhanced their hydrophobicity and aggregation. This increased Nε-lysine acetylation could result from carbon overflow and the accumulation of acetyl phosphate caused by the ΔotsA mutation. These findings provide a better understanding of the previously reported protective functions of trehalose in protein stabilization and the prevention of protein aggregation. Our results indicate that trehalose may participate in proteostasis not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation. We propose that trehalose protects E. coli against carbon stress - the synthesis and storage of trehalose can prevent carbon overflow, which otherwise is manifested by protein acetylation and aggregation.

Physiologically distinct subpopulations formed in Escherichia coli cultures in response to heat shock.

Bacteria can form heterogeneous populations containing phenotypic variants of genetically identical cells. The heterogeneity of populations can be considered a bet-hedging strategy allowing adaptation to unknown environmental changes - at least some individual subpopulations or cells might be able to withstand future adverse conditions. Using Percoll gradient centrifugation, we demonstrated that in an Escherichia coli culture exposed to heat shock at 50 °C, two physiologically distinct subpopulations were formed. A high-density subpopulation (HD50) demonstrated continued growth immediately after its transfer to LB medium, whereas the growth of a low-density subpopulation (LD50) was considerably postponed. The LD50 subpopulation contained mainly viable but non-culturable bacteria and exhibited higher tolerance to sublethal concentrations of antibiotics or H2O2 than HD50 cells. The levels of aggregated proteins and main molecular chaperones were comparable in both subpopulations; however, a decreased number of ribosomes and a significant increase in protein oxidation were observed in the LD50 subpopulation as compared with the HD50 subpopulation. Interestingly, under anaerobic heat stress, the formation of the HD50 subpopulation was decreased and culturability of the LD50 subpopulation was significantly increased. In both subpopulations the level of protein aggregates formed under anaerobic and aerobic heat stress was comparable. We concluded that the formation of protein aggregates was independent of oxidative damage induced by heat stress, and that oxidative stress and not protein aggregation limited growth and caused loss of LD50 culturability. Our results indicate that heat stress induces the formation of distinct subpopulations differing in their ability to grow under standard and stress conditions.

The effect of protein acetylation on the formation and processing of inclusion bodies and endogenous protein aggregates in Escherichia coli cells.

BACKGROUND: Acetylation of lysine residues is a reversible post-translational modification conserved from bacteria to humans. Several recent studies have revealed hundreds of lysine-acetylated proteins in various bacteria; however, the physiological role of these modifications remains largely unknown. Since lysine acetylation changes the size and charge of proteins and thereby may affect their conformation, we assumed that lysine acetylation can stimulate aggregation of proteins, especially for overproduced recombinant proteins that form inclusion bodies. RESULTS: To verify this assumption, we used Escherichia coli strains that overproduce aggregation-prone VP1GFP protein. We found that in ΔackA-pta cells, which display diminished protein acetylation, inclusion bodies were formed with a delay and processed faster than in the wild-type cells. Moreover, in ΔackA-pta cells, inclusion bodies exhibited significantly increased specific GFP fluorescence. In CobB deacetylase-deficient cells, in which protein acetylation was enhanced, the formation of inclusion bodies was increased and their processing was significantly inhibited. Similar results were obtained with regard to endogenous protein aggregates formed during the late stationary phase in ΔackA-pta and ΔcobB cells. CONCLUSIONS: Our studies revealed that protein acetylation affected the aggregation of endogenous E. coli proteins and the yield, solubility, and biological activity of a model recombinant protein. In general, decreased lysine acetylation inhibited the formation of protein aggregates, whereas increased lysine acetylation stabilized protein aggregates. These findings should be considered during the designing of efficient strategies for the production of recombinant proteins in E. coli cells.