Abstract
Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the E. coli hns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex. Hence, these findings demonstrate that the hns promoter is subjected to an RNA polymerase-mediated down-regulation by increased intracellular levels of (p)ppGpp.
Highlights
The signaling molecules guanosine-tetraphosphate and guanosine-pentaphosphate, collectively named (p)ppGpp, are the main mediators of “stringent response,” a common phenomenon occurring when bacteria face environmental stresses [1,2,3]
The role of (p)ppGpp on hns expression was initially investigated by means of antibiotics as chloramphenicol (CAM) and kanamycin (Kan), which were known to affect the intracellular level of this unusual nucleotide
Northern analysis reveals that hns expression is ~ twofold higher in double mutant compared to wt cells in the exponential phase of growth (A600 < 1) and this difference gradually disappears as the cells approach the stationary phase (A600 > 1.5) (Figure 2B,D)
Summary
The signaling molecules guanosine-tetraphosphate (ppGpp) and guanosine-pentaphosphate (pppGpp), collectively named (p)ppGpp, are the main mediators of “stringent response,” a common phenomenon occurring when bacteria face environmental stresses [1,2,3]. Two enzymes are responsible for the production of (p)ppGpp, the major RelA synthase, and the SpoT degradase that displays both (p)ppGpp degradation and synthetic activity [4]. When bound to ribosome, the RelA enzyme senses through its autoinhibitory domain and is being activated by the entrance of uncharged tRNAs into the ribosomal A-site, a circumstance occurring during amino acid starvation [5]. Nutrients limitation causes an intracellular accumulation of (p)ppGpp that dramatically alters bacterial physiology and metabolism [6]. Under this condition, growth rate is slowed, stable RNA (rRNA and tRNA) production is inhibited, and the high levels of modified nucleotide differentially up- or down-regulate the expression of a large number of genes. The first site is at the interface of β’
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