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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 ...

Inflammation in the vascular wall is important for development of atherosclerosis. We have shown previously that arachidonate 15-lipoxygenase type B (ALOX15B) is more highly expressed in human atherosclerotic lesions than in healthy arteries. This enzyme oxidizes fatty acids to substances that promote local inflammation and is expressed in lipid-loaded macrophages (foam cells) present in the atherosclerotic lesions. Here, we investigated the role of ALOX15B in foam cell formation in human primary macrophages and found that silencing of human ALOX15B decreased cellular lipid accumulation as well as proinflammatory cytokine secretion from macrophages. To investigate the role of ALOX15B in promoting the development of atherosclerosis in vivo, we used lentiviral shRNA silencing and bone marrow transplantation to knockdown mouse Alox15b gene expression in LDL-receptor-deficient (Ldlr −/−) mice. Knockdown of mouse Alox15b in vivo decreased plaque lipid content and markers of inflammation. In summary, we have shown that ALOX15B influences progression of atherosclerosis, indicating that this enzyme has an active proatherogenic role.

When Escherichia coli cells enter stationary phase due to carbon starvation the synthesis of ribosomal proteins is rapidly repressed. In a ⌬relA ⌬spoT mutant, defective in the production of the alarmone guanosine tetraphosphate (ppGpp), this regulation of the levels of the protein synthesizing system is abolished. Using a proteomic approach we demonstrate that the production of the vast majority of detected E. coli proteins are decontrolled during carbon starvation in the ⌬relA ⌬spoT strain and that the starved cells behave as if they were growing exponentially. In addition we show that the inhibition of ribosome synthesis by the stringent response can be qualitatively mimicked by artificially lowering the levels of the housekeeping factor, 70 . In other words, genes encoding the protein-synthesizing system are especially sensitive to reduced availability of 70 programmed RNA polymerase. This effect is not dependent on ppGpp since lowering the levels of 70 gives a similar but less pronounced effect in a ppGpp 0 strain. The data is discussed in view of the models advocating for a passive control of gene expression during stringency based on alterations in RNA polymerase availability.The alarmone guanosine tetraphosphate (ppGpp) 1 of the stringent response network in Escherichia coli affects ribosome production by specifically lowering the transcription of ribosomal RNA (rRNA) (1) and some of the genes encoding ribosomal proteins (2-4). The production of ribosomal proteins is also post-transcriptionally feedback-regulated to match the rRNA production (reviewed in 5). Two different ppGpp synthetases (PS) exist in E. coli, the ribosome-associated PS I, encoded by relA, and the cytoplasmic PS II (6), encoded by spoT (7,8). The protein PS II is also responsible for ppGpp hydrolysis (1).Increased levels of ppGpp not only down-regulate ribosome production but also induce transcription from several promoters (9 -14). As suggested by Schreiber et al. (9), the E. coli promoters can be divided into three groups depending on their response to ppGpp: promoters specifically induced or repressed by the stringent response and promoters that are unaffected by the rise in ppGpp levels. Promoters dependent on the housekeeping factor, 70 , exist in at least two of these groups; one group is positively regulated by ppGpp, e.g. PuspA (11) and Phis (8), whereas the other group is repressed during stringency, e.g. rrnP1. Attempts to explain this dual effect of ppGpp have involved considerations of RNAP (RNA polymerase) availability and intrinsic differences in the kinetic properties of the promoters affected (15). Several lines of evidence suggest that the availability of transcriptional and/or translational machinery play a role in global gene regulation in concert with classical activators and repressors (13-17). One well known example of this type of regulation, where the level of RNAP is involved, is factor competition in both Bacillus subtilis (18) and E. coli (19,20).Whether or not RNAP availability is involved in the actual m...

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.