ABSTRACT
σS is an alternative sigma factor, encoded by the rpoS gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σS general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σS levels transcriptionally, translationally, and posttranslationally in Escherichia coli It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σS synthesis, thus playing an important role in activation of the σS stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σS levels. In addition, based on effects of DksAN88I, a previously identified DksA variant with increased affinity for RNA polymerase (RNAP), we show that DksA can increase σS activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σS and also increase transcription from other promoters, including PdsrA and PiraP By improving the translation and stabilization of σS, as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation.IMPORTANCE Bacteria spend relatively little time in log phase outside the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σS Understanding the factors governing σS activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here, we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σS levels.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Pyrophosphatases/metabolism , RNA, Small Untranslated/metabolism , Sigma Factor/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Pyrophosphatases/genetics , RNA, Small Untranslated/genetics , Sigma Factor/genetics , Stress, PhysiologicalABSTRACT
Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F' plasmid, to upregulate the σ(E) extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σ(E) extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σ(E)-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.
Subject(s)
Conjugation, Genetic , Escherichia coli Proteins/metabolism , Escherichia coli/physiology , Sigma Factor/genetics , Transcription Factors/metabolism , Up-Regulation , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Operon , Promoter Regions, Genetic , Sigma Factor/metabolism , Stress, Physiological , Transcription Factors/genetics , Transcriptional ActivationABSTRACT
At specific times during bacterial growth, the transcription factor DksA and the unusual nucleotide regulator ppGpp work synergistically to inhibit some Escherichia coli promoters (e.g. rRNA promoters) and to stimulate others (e.g. promoters for amino-acid synthesis and transport). However, the mechanism of DksA action remains uncertain, in part because DksA does not function like conventional transcription factors. To gain insights into DksA function, we identified mutations in dksA that bypassed the requirement for ppGpp by selecting for growth of cells lacking ppGpp on minimal medium without amino acids. We show here that two substitutions in DksA, L15F and N88I, result in higher DksA activity both in vivo and in vitro, primarily by increasing the apparent affinity of DksA for RNA polymerase (RNAP). The mutant DksA proteins suggest potential roles for ppGpp in DksA function, identify potential surfaces on DksA crucial for RNAP binding, and provide tools for future studies to elucidate the mechanism of DksA action.