The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp 0 ) and/or DksA (⌬dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp-and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp 0 ⌬dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

SummaryAntigen 43 (Ag43) is a self‐recognizing outer membrane protein of Escherichia coli expressed during intracellular growth and biofilm formation, suggesting a role in infection. The expression of agn43 is under phase variation control, meaning that there are regulatory mechanisms adjusting the percentage of agn43‐expressing cells in the population, in addition to mechanisms modulating the transcriptional expression level in each expressing cell. Phenotypic and transcriptional studies indicate that Ag43 expression is induced upon entry into the stationary phase in a ppGpp‐dependent and RpoS‐independent manner. The use of single‐cell approaches and phase variation deficient strains let to conclude that ppGpp stimulates agn43 promoter activity, rather than affecting the percentage of agn43‐expressing cells. The data highlight the relevance that promoter activity regulation may have, without any involvement of the phase variation state, in the final Ag43 expression output. The agn43 promoter of the MG1655 strain carries an AT‐rich discriminator between positions −10 and +1, which is highly conserved among the agn43 genes present in the different pathotypes of E. coli. Remarkably, the AT‐rich discriminator is required for the positive transcriptional control mediated by ppGpp.

ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection.