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 ...