Carbonylation is currently used as a marker for irreversible protein oxidative damage. Several studies indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts. In this study, we observed that in Escherichia coli, more than 95% of the total carbonyl content consisted of insoluble protein and most were cytosolic proteins. We thereby demonstrate that, in vivo, carbonylated proteins are detectable mainly in an aggregate state. Finally, we show that detectable carbonylated proteins are not degraded in vivo. Here we propose that some carbonylated proteins escape degradation in vivo by forming carbonylated protein aggregates and thus becoming nondegradable. In light of these findings, we provide evidence that the accumulation of nondegradable carbonylated protein presented in an aggregate state contributes to the increases in carbonyl content observed during senescence.Proteins can become modified by a large number of reactions involving reactive oxygen species. Among these modifications, carbonylation has attracted a great deal of attention due to its irreversible and irreparable nature. Carbonyl derivatives are formed by a direct metal-catalyzed oxidative attack on the amino acid side chains of proline, arginine, lysine, and threonine (2). With the development of sensitive immunochemical methods for the detection of protein carbonyls, the presence of such groups has been extensively used as a marker of reactive oxygen species-mediated protein oxidation (17) and associated with a large number of age-related disorders, including Parkinson's disease, Alzheimer's disease, and cancer (5, 17). While carbonylated proteins are considered soluble in healthy cells, a decrease in proteolysis has been suggested to provoke increases in levels of carbonylated protein which may form aggregates during aging or disease (5,(12)(13)(14). Interestingly, in starvation, aging, or disease states, only some proteins appear more prone to carbonylation (3,11,17,24). Finally, in vivo studies using exponentially grown Escherichia coli cells or other organisms indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts (10, 14-16, 18, 21). Moreover, several groups have postulated that carbonylation may act as a tag for degradation (10,15,21).Here, contrary to observations made previously by Dukan et al. (10) and other groups, we show, using E. coli exponentialor stationary-phase cells, that carbonylated proteins are mainly cytosolic and that most of them are detectable in an aggregate state that does not degrade with time. As a consequence, we propose that increases in carbonyl content observed during bacterial senescence could be due at least in part to the accumulation of nondegradable carbonylated proteins presented in an aggregate state. MATERIALS AND METHODSBacterial strain and medium. E. coli MG1655 was grown aerobically or anaerobically in liquid Luria-Bertani (LB) medium in a rotary shaker at 37°C and 200 rpm.Protein preparation. Exponential (optical...

Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and "healthy" Escherichia coli cells. We observed overrepresentation of "aberrant proteins," as well as substrates of the major conserved chaperone DnaK (Hsp70) and the protease ClpXP (a serine protease), in the aggregates. In addition, the protein aggregates appeared to interact with chaperones known to be involved in the aggregate repair pathway, including ClpB, GroEL, GroES, and DnaK. Finally, we showed that the levels of reactive oxygen species and unfolded or misfolded proteins determine the levels of protein aggregates. Our results led us to speculate that protein aggregates may function as a temporary "trash organelle" for cellular detoxification.Formation or accumulation of aggregated proteins, which are present in all organisms, has become an important area of intensive research, mainly due to observations that there is a link with many disorders, including aging or neurodegenerative diseases (16).Major substrates for aggregation are unfolded or misfolded proteins. Indeed, misfolded proteins inappropriately expose hydrophobic surfaces normally buried in the protein's interior, leading to nonnative conformations able to interact and form aggregates. In cells, protein folding may fail because of amino acid misincorporation. Moreover, DnaK/Hsp70 and GroEL/ Hsp60 chaperone protein substrates, as well as partially degraded proteins, are more prone to unfolding and thus to aggregation (15).A common theory is that protein aggregation may be the consequence of a failed quality control mechanism normally charged with repairing or removing the misfolded or unfolded proteins. Indeed, to maintain functional proteins, the cells contain chaperones or proteases that assist proper protein folding and disaggregation of aggregates (17). For instance, Hsp104/ClpB has the capacity to rescue unfolded or misfolded proteins from an aggregated state in cooperation with the Hsp70/DnaK chaperone system (9). However, as recently shown in Huntington's disease, protein aggregates could serve as "temporary storage zones" within cells in order to maintain their function and integrity for prolonged periods of time (1, 13).A common opinion is that in healthy cells, the levels of formation and accumulation of protein aggregates are extremely low, although protein unfolding occurs constantly. Indeed, several groups have observed a virtual absence of protein aggregates in healthy cells (2, 5, 15), although it is possible that insufficient material was used for detection.Here we isolated and characterized protein aggregates in exponentially growing "healthy" Escherichia coli cells using classical procedures to extract E. coli insoluble cell fractions and protein aggregates. Our results led us to speculate that the...

In previous experiments we were able to separate, using a nondestructive separation technique, culturable and nonculturable bacteria, from a Luria-Bertani (LB) medium culture of Escherichia coli incubated for 48 h. We observed in the nonculturable bacterial population an increase in oxidative damage and up-induction of most defenses against reactive oxygen species (ROS), along with a decrease in cytoplasmic superoxide dismutases. In this study, using the same separation technique, we separated into two subpopulations a 10-h LB medium culture containing only culturable bacteria. For the first time, we succeeded in associating physical separation with physiological differences. Although the levels of defense against ROS (RpoS, RpoH, OxyR, and SoxRS regulons) and oxidative damage (carbonyl contents) were apparently the same, we found that bacteria in one subpopulation were more sensitive to LB medium starvation and to various stresses, such as phosphate buffer starvation, heat shock, and hydrogen peroxide exposure. Based on these results, we suggest that these physiological differences reflect uncharacterized bacterial modifications which do not directly involve defenses against ROS.Biological aging could be defined as the gradual decline in the capacity of an organism to resist stress, damage, and disease. In 1956, Denham Harman (10) postulated that this ubiquitous progressive decay in the functional capacity of aging eukaryotes is a consequence of the accumulation of oxidative damage caused by reactive oxygen species (ROS) (12); this was called the free radical theory (10). A small percentage of oxygen is chemically reduced by addition of single electrons, and the products are sequentially converted into ROS, including the superoxide anion, hydrogen peroxide, and the hydroxyl radical (8). ROS have been shown to cause molecular damage relatively indiscriminately to proteins, lipids, and nucleic acids (3, 9).In cells of prokaryotes, such as Escherichia coli, entering a nonproliferating state (stationary phase) due to nutrient depletion, the bacteria gradually lose the ability to divide and reproduce (21). Similar to eukaryotes, the life span of a starved bacterium appears to be limited by the cell's ability to combat ROS. Indeed, Dukan and Nyström demonstrated the existence of an accumulation of oxidized proteins during starvation of an E. coli population (6). Moreover, the life span of growtharrested wild-type E. coli can be increased Ͼ100% by omitting oxygen during stasis (7). This process has been referred to as conditional senescence elicited by growth arrest (17). Given that one of the criteria for defining senescence is an increase in the mortality rate over time (12), it appears that prokaryotes such as E. coli also senesce (20). More recently, using an ultracentrifugation separation technique, we (4) isolated a nonculturable subpopulation from a Luria-Bertani (LB) medium culture of E. coli incubated for 48 h. We suggested that the main reason for the loss of culturability observed after 48 h was a decrease ...

In Bacillus subtilis, carbon catabolite repression (CCR) of catabolic genes is mediated by ATP-dependent phosphorylation of HPr and Crh. Here we show that the different efficiencies with which these two proteins contribute to CCR may be due to the drastic differences in their synthesis rates under conditions that cause CCR.In Bacillus subtilis, carbon catabolite repression (CCR) of many catabolic genes is mediated by ATP-dependent phosphorylation of Ser-46 of HPr and of its homologue Crh (4). Although these two proteins exhibit high sequence identity (45%) and are both efficiently phosphorylated by the HPr kinase/phosphorylase, their contributions to CCR differ (4). P-Ser-Crh can only partly substitute for P-Ser-HPr in CCR, whereas P-Ser-HPr can completely substitute for P-Ser-Crh in this signal transduction pathway. In order to understand the different behaviors of these two proteins, we compared the expression levels of the corresponding genes, crh and ptsH (encoding HPr), in the presence of different carbon sources by using transcriptional and translational lacZ reporter gene fusions. MATERIALS AND METHODSPlasmids, bacterial strains, and growth conditions. The plasmids and the bacterial strains used in this study are listed in Table 1. Escherichia coli DH5␣ was used as a general cloning host. Plasmid DNAs of the pMutin4 derivatives were prepared from E. coli BMH71-18 (recA ϩ ) prior to transformation into B. subtilis. E. coli, and B. subtilis strains were routinely grown in Luria-Bertani broth supplemented with the appropriate antibiotics when necessary (ampicillin at 100 g/ml for E. coli and chloramphenicol at 5 g/ml and erythromycin at 0.3 g/ml for B. subtilis). Standard procedures were used to transform E. coli (15) and B. subtilis (7). Sequencing of PCR-derived DNA fragments in the final plasmid constructs was carried out by Genome Express (Meylan, France).Construction of transcriptional and translational fusions of crh and ptsH to lacZ. To construct the fusion of lacZ to crh-5Ј, the 5Ј region of crh (Ϫ120 to ϩ90) was amplified by using the primers BG9 (crh [Ϫ120 to Ϫ103]) and BG10 (crh [ϩ90 to ϩ73]), digested at the HindIII and BamHI sites within the primers, and inserted between these sites in pMutin4, resulting in plasmid pBGM6. To construct the fusion of lacZ to ptsH-5Ј, the 5Ј region of ptsH (Ϫ276 to ϩ90) was amplified by using the primers LF1 (ptsH [Ϫ276 to Ϫ258]) and LF2 (ptsH [ϩ90 to ϩ73]), digested at the NotI and HindIII sites within the primers, and inserted between these sites in pMutin4, resulting in plasmid pLF2. Plasmid pBGM8 carrying a ⌽(crh-lacZ)(Hyb) fusion gene was constructed by a three-fragment ligation. The entire crh gene with its ribosome binding site (RBS) was amplified by using the primers BG13 (crh [Ϫ20 to Ϫ3]) and BG14 (crh [ϩ255 to ϩ238]) and digested at the HindIII and XhoI sites within the primers. In parallel, the 5Ј part of lacZ was amplified from pMutin4 as a template by using the primers BG15 (pMutin2 [382 to 399]) and BG16 (pMutin2 [698 to 681]) and digested with XhoI (...