Biochemical Bases for the Antimetabolite Action of l -Serine Hydroxamate

SummaryIn Escherichia coli transcription of ribosomal RNA (rRNA) is regulated by the H-NS and Fis proteins, as well as by the small signal molecule ppGpp and the initiating nucleotides. During amino acid starvation, the concentration of ppGpp increases, and binding of this alarmone to RNA polymerase (RNAP) leads to inhibition of rRNA transcription, a regulatory event called stringent response. Here we show that in Pseudomonas aeruginosa DksA, a protein with pleiotropic effects, is a negative regulator of rRNA transcription both during exponential growth and stringent conditions. A dksA mutant overexpresses rRNA, without being affected in the production of ppGpp. Cell-fractionation and chromosome immunoprecipitation experiments demonstrate that DksA is associated with DNA, in particular with promoters of ribosomal genes in vivo . The binding to rRNA promoters specifically increases during stringent response, and correlates with the binding of RNAP to these regions. Moreover DksA can be copurified with RNAP subunits in vivo . DNA band shift experiments show that DksA, in synergy with ppGpp, increases the binding of RNAP to ribosomal promoters. Therefore DksA might be a new regulator of rRNA transcription in P. aeruginosa.

Section: Resultsmentioning

confidence: 99%

DksA represses ribosomal gene transcription in Pseudomonas aeruginosa by interacting with RNA polymerase on ribosomal promoters

Perron

Comte

Delden

2005

“…Induction of the stringent response in S. coelicolor A3(2) by serine hydroxamate Serine hydroxamate (SHX), a structural analogue ol L-serine, provokes the stringent response in E. coli by acting as a competitive inhibitor of seryl-tRNA synthetase (Tosa and Pizer, 1971), thus leading to the accumulation of uncharged tRNA^^'. SHX triggers the stringent response in Streptomyces hygroscopicus {Riesenberg ef ai, 1984), presumably by the same mechanism.…”

Section: Resultsmentioning

confidence: 99%

The stringent response in Streptomyces coelicolor A3(2)

Strauch

Takano

Baylis

et al. 1991

189

157

The stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of actIII occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis alone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).

“…5B). In this set of experiments, the stringent response was induced by 3-amino-1,2,4-triazole (3-AT), which inhibits the synthesis of histidine, leading to starvation for the amino acid (Tosa and Pizer, 1971). As with serine hydroxamate, 3-AT caused the disappearance of transcription foci in wild type but not in the relA mutant (Fig.…”

Section: Before Inductionmentioning

confidence: 95%

“…We induced the stringent response in wild-type cells (DJ2599) grown in LB by the addition of serine hydroxamate to the cultures. Serine hydroxamate is an amino acid analogue that causes starvation for the amino acid serine leading to the stringent response (Tosa and Pizer, 1971;Cashel et al, 1996). Cell samples were taken before and at different times after induction of the stringent response.…”

Section: Rnap Redistribution During the Stringent Responsementioning

confidence: 99%

The distribution of RNA polymerase in Escherichia coli is dynamic and sensitive to environmental cues

Cabrera

Jin

2003

169

243

SummaryDespite extensive genetic, biochemical and structural studies on Escherichia coli RNA polymerase (RNAP), little is known about its location and distribution in response to environmental changes. To visualize the RNAP by fluorescence microscopy in E. coli under different physiological conditions, we constructed a functional rpoC-gfp gene fusion on the chromosome. We show that, although RNAP is located in the nucleoid and at its periphery, the distribution of RNAP is dynamic and dramatically influenced by cell growth conditions, nutrient starvation and overall transcription activity inside the cell. Moreover, mutational analysis suggests that the stable RNA synthesis plays an important role in nucleoid condensation.