Spermidine acetylation in response to a variety of stresses in Escherichia coli

Heat shock, cold shock, ethanol, and alkaline shift, but not hydrogen peroxide, stimulate the accumulation of monoacetylspermidine in Escherichia coli. Acetylation occurs with nearly equal frequencies at both the N1 and N8 positions of this ubiquitous polycation. Spermidine acetylation does not appear to be associated with known stress regulons, such as htpR, oxyR, and SOS. E. coli, capable of acetylating spermidine, constitutively express a spermidine acetyltransferase activity during all phases of growth, and this activity is unaffected by cold shock. A mutant strain, incapable of acetylating spermidine, does not express this enzyme activity but grows at an identical rate as the parent strain at 37 degrees C. These results demonstrate that the monoacetylation of spermidine in E. coli is regulated by some mechanism other than a stress-inducible acetyltransferase and is not essential for growth of these cells. They suggest that polyamine acetylation is involved in the responses of these organisms to a variety of chemical and physical stresses.     

Protein expression in response to folate stress in Escherichia coli.

Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the glycine cleavage enzyme complex.     

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.     

Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli

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In vivo acetylation of CheY, a response regulator in chemotaxis of Escherichia coli

CheY, the excitatory response regulator in the chemotaxis system of Escherichia coli, can be modulated by two covalent modifications: phosphorylation and acetylation. Both modifications have been detected in vitro only. The role of CheY acetylation is still obscure, although it is known to be involved in chemotaxis and to occur in vitro by two mechanisms—acetyl-CoA synthetase-catalyzed transfer of acetyl groups from acetate to CheY and autocatalyzed transfer from AcCoA. Here, we succeeded in detecting CheY acetylation in vivo by three means—Western blotting with a specific anti-acetyl-lysine antibody, mass spectrometry, and radiolabeling with [¹⁴C]acetate in the presence of protein-synthesis inhibitor. Unexpectedly, the level and rate of CheY acetylation in vivo were much higher than that in vitro. Thus, before any treatment, 9-13% of the lysine residues were found acetylated, depending on the growth phase, meaning that, on average, essentially every CheY molecule was acetylated in vivo. This high level was mainly the outcome of autoacetylation. Addition of acetate caused an incremental increase in the acetylation level, in which acetyl-CoA synthetase was involved too. These findings may have far-reaching implications for the structure-function relationship of CheY.     

Qualitative simulation of the carbon starvation response in Escherichia coli

In case of nutritional stress, like carbon starvation, Escherichia coli cells abandon their exponential-growth state to enter a more resistant, non-growth state called stationary phase. This growth-phase transition is controlled by a genetic regulatory network integrating various environmental signals. Although E. coli is a paradigm of the bacterial world, it is little understood how its response to carbon starvation conditions emerges from the interactions between the different components of the regulatory network. Using a qualitative method that is able to overcome the current lack of quantitative data on kinetic parameters and molecular concentrations, we model the carbon starvation response network and simulate the response of E. coli cells to carbon deprivation. This allows us to identify essential features of the transition between exponential and stationary phase and to make new predictions on the qualitative system behavior following a carbon upshift.     

N-terminal acetylation of ectopic recombinant proteins in Escherichia coli

N-terminal acetylation is a protein modification common in eukaryotes, but rare in prokaryotes. Here, we characterized five mammalian stathmin-like subdomains expressed in Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoESI Q-TOF tandem mass spectrometry. We revealed that RB3(SLD) and RB3'(SLD) are N(alpha)-acetylated, whereas SCG10(SLD) and SCLIP(SLD), although identical up to residue 6, are not, as well as stathmin. To assess the influence of the N-terminal sequences on N(alpha)-acetylation, we exchanged residues 7 and 8 between acetylated RB3(SLD) and unacetylated SCG10(SLD), and showed that it reversed the acetylation pattern. Our results demonstrate that ectopic recombinant proteins can be extensively N(alpha)-acetylated in E. coli, and that the rules governing N(alpha)-acetylation are complex and involve the N-terminal region, as in eukaryotes.     

Co-regulation of acetylation and phosphorylation of CheY, a response regulator in chemotaxis of Escherichia coli

CheY, a response regulator of the chemotaxis system in Escherichia coli, can be activated by either phosphorylation or acetylation to generate clockwise rotation of the flagellar motor. Both covalent modifications are involved in chemotaxis, but the function of the latter remains obscure. To understand why two different modifications apparently activate the same function of CheY, we studied the effect that each modification exerts on the other. The phosphodonors of CheY, the histidine kinase CheA and acetyl phosphate, each strongly inhibited both the autoacetylation of the acetylating enzyme, acetyl-CoA synthetase (Acs), and the acetylation of CheY. CheZ, the enzyme that enhances CheY dephosphorylation, had the opposite effect and enhanced Acs autoacetylation and CheY acetylation. These effects of the phosphodonors and CheZ were not caused by their respective activities. Rather, they were caused by their interactions with Acs and, possibly, with CheY. In addition, the presence of Acs elevated the phosphorylation levels of both CheA and CheY, and acetate repressed this stimulation. These observations suggest that CheY phosphorylation and acetylation are linked and co-regulated. We propose that the physiological role of these mutual effects is at two levels: linking chemotaxis to the metabolic state of the cell, and serving as a tuning mechanism that compensates for cell-to-cell variations in the concentrations of CheA and CheZ.     

Real-time detection of central carbon metabolism in living Escherichia coli and its response to perturbations

The direct tracking of cellular reactions in vivo has been facilitated with recent technologies that strongly enhance NMR signals in substrates of interest. This methodology can be used to assay intracellular reactions that occur within seconds to few minutes, as the NMR signal enhancement typically fades on this time scale. Here, we show that the enhancement of (13)C nuclear spin polarization in deuterated glucose allows to directly follow the flux of glucose signal through rather extended reaction networks of central carbon metabolism in living Escherichia coli. Alterations in central carbon metabolism depending on the growth phase or upon chemical perturbations are visualized with minimal data processing by instantaneous observation of cellular reactions.     

Cytokine response to Escherichia coli in gnotobiotic pigs

One-week-old-germ-free pigs were inoculated with 10(8) CFU of E.coli bacteria-either commensal 086 strain or virulent 055 strain for 1 d. Bacteria were counted in the small intestine, mesenteric lymph nodes, blood and lungs. The O55 strain reached higher levels in circulation and lungs. IL-8, IL-10 and TNF-alpha concentrations were determined by ELISA in plasma and intestinal washes . No difference in cytokine levels was found between control germ-free pigs and their counterparts associated with commensal O86 strain in spite of its high concentration in the gut and circulation.     

Protein export in Escherichia coli.

The export of protein from Escherichia coli has been studied by genetic, biochemical and biophysical techniques. These studies have defined a number of steps in the export pathway and have identified the cellular components required for the translocation process. New information is presented on the function of some of these components.     

Thiamine-Binding Protein of Escherichia coli

The ability to transport thiamine in Escherichia coli was reduced by osmotic shock treatment with a concomitant release of a thiamine-binding protein; its formation was repressed by thiamine added to the growth medium.