Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in a manner that depended on the formation of acetyl-phosphate (AcP) through glycolysis. Mutant cells unable to produce AcP had significantly reduced acetylation levels, while mutant cells unable to convert AcP to acetate had significantly elevated acetylation levels. We showed that AcP can chemically acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low level and is dynamically affected by metabolism and cell proliferation in a global, uniform manner.

The recent discovery that the protein DksA acts as a coregulator of genes controlled by ppGpp led us to investigate the similarities and differences between the relaxed phenotype of a ppGpp-deficient mutant and the phenotype of a strain lacking DksA. We demonstrate that the absence of DksA and ppGpp has similar effects on many of the observed phenotypes but that DksA and ppGpp also have independent and sometimes opposing roles in the cell. Specifically, we show that overexpression of DksA can compensate for the loss of ppGpp with respect to transcription of the promoters P uspA , P livJ , and P rrnBP1 as well as amino acid auxotrophy, cell-cell aggregation, motility, filamentation, and stationary phase morphology, suggesting that DksA can function without ppGpp in regulating gene expression. In addition, ppGpp and DksA have opposing effects on adhesion. In the course of our analysis, we also discovered new features of the relaxed mutant, namely, defects in cell-cell aggregation and motility.The ability of the cell to adapt to nutrient limitation depends on its capacity to change its gene expression immediately when nutrients are depleted. One important mechanism for regulating this change in gene expression is the stringent response, mediated by the alarmone ppGpp (6, 17). Two different ppGpp synthetases exist in Escherichia coli: the ribosome-associated RelA synthesizing ppGpp in response to amino acid starvation and the cytoplasmic SpoT synthesizing ppGpp in response to carbon starvation as well as most other starvations (6). SpoT is also responsible for ppGpp hydrolysis. During the stringent response the production of the protein-synthesizing system is repressed while some genes involved in stress resistance and survival are induced. Cells unable to produce ppGpp (deleted of relA and spoT, called ppGpp 0 ) are largely decontrolled upon entry to stationary phase and behave as if they were still growing exponentially (18). It has been suggested that this shift from growth mode to maintenance mode by ppGpp results in part from the regulation of the activity of alternative sigma factors (12,17).The array of phenotypes of a cell deficient in making ppGpp, the so-called relaxed phenotype, has been expanded, and it is obvious not only that gene regulation by ppGpp is important in response to starvation but that ppGpp also has a role in regulating traits involved in virulence (8) and other phenotypes both during growth and in stationary phase.DksA was first described as a multicopy suppressor of the temperature-sensitive growth and filamentation of a dnaK deletion mutant in E. coli (13). The protein DksA has since then been shown to influence the regulatory activities of ppGpp even though DksA in itself is not a typical regulatory protein.It is still under debate how DksA alters the activity of ppGpp (or vice versa). The levels of DksA in E. coli have been shown to be similar at all growth rates and in all growth phases tested (5, 22). DksA is structurally similar to the transcription factors GreA and GreB, although ...

Ribosomes are absolutely essential for growth but are, moreover, energetically costly to produce. Therefore, it is important to adjust the cellular ribosome levels according to the environmental conditions in order to obtain the highest possible growth rate while avoiding energy wastage on excess ribosome biosynthesis. Here we show, by three different methods, that the ribosomal RNA content of Escherichia coli is downregulated within minutes of the removal of an essential nutrient from the growth medium, or after transcription initiation is inhibited. The kinetics of the ribosomal RNA reduction vary depending on which nutrient the cells are starved for. The number of ribosomes per OD unit of cells is roughly halved after 80 min of starvation for isoleucine or phosphate, while the ribosome reduction is less extensive when the cells are starved for glucose. Collectively, the results presented here support the simple model proposed previously, which identifies the inactive ribosomal subunits as the substrates for degradation, since the most substantial rRNA degradation is observed under the starvation conditions that most directly affect the protein synthesis. K E Y W O R D Sbacterial stress response, Escherichia coli, nutrient starvation, ribosomal RNA, stable RNA degradation

Nutritionally induced changes in RNA polymerase availability have been hypothesized to be an evolutionary primeval mechanism for regulation of gene expression and several contrasting models have been proposed to explain how such 'passive' regulation might occur. We demonstrate here that ectopically elevating Escherichia coli RNA polymerase (Esigma(70)) levels causes an increased expression and promoter occupancy of ribosomal genes at the expense of stress-defense genes and amino acid biosynthetic operons. Phenotypically, cells overproducing Esigma(70) favours growth and reproduction at the expense of motility and damage protection; a response reminiscent of cells with no or diminished levels of the alarmone guanosine tetraphosphate (ppGpp). Consistently, we show that cells lacking ppGpp displayed markedly elevated levels of free Esigma(70) compared with wild-type cells and that the repression of ribosomal RNA expression and reduced growth rate of mutants with constitutively elevated levels of ppGpp can be suppressed by overproducing Esigma(70). We conclude that ppGpp modulates the levels of free Esigma(70) and that this is an integral part of the alarmone's means of regulating a trade-off between growth and maintenance.

Background: ppGpp/DksA regulate genes both positively and negatively during stringency. Results: Positive control by ppGpp/DksA is linked to RNA polymerase-promoter saturation kinetics dependent on the promoter's discriminator. Conclusion: Promoters harboring discriminators that make them easily saturated with RNA polymerase are controlled positively by ppGpp/DksA. Significance: The discriminator is a cis-acting element dictating the response of the promoter to alterations in ppGpp/DksA and RNA polymerase availability.