Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli

The viability of three isogenic relA+/relA strain pairs of Escherichia coli (CP78/CP79; NF 161/ NF162; CP 107/CP 143) was studied during prolonged starvation for amino acids, glucose or phosphate. After amino acid limitation we found a prolonged viability of all relA+ strains which synthesized ppGpp. We suggest that some ppGpp-mediated pleiotropic effects of the stringent response (e.g. glykogen accumulation, enhanced protein turnover) might be involved in this prolongation of survival. After glucose or phosphate starvation there was no difference in the relA+/relA strains either in the ppGpp content or in the survival.

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Inhibition of transcription starting from bacteriophage lambda pR promoter during the stringent response in Escherichia coli: implications for lambda DNA replication.

Acta Biochimica Polonica ◽

10.18388/abp.1995_4615 ◽

1995 ◽

Vol 42 (2) ◽

pp. 233-239 ◽

Cited By ~ 6

Author(s):

A Szalewska-Pałasz ◽

G Wegrzyn

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Dna Replication ◽

Transcriptional Activation ◽

Plasmid Dna ◽

Bacteriophage Lambda ◽

Stringent Response ◽

Lacz Gene ◽

Relative Level ◽

Lambda Dna

Replication of lambda plasmid DNA is halted in amino acid-starved wild type (stringent) strains whereas it proceeds in relA (relaxed) mutants. The only transcription which could be important in lambda plasmid DNA replication in amino acid-starved Escherichia coli cells is that starting from the pR promoter. Using a fusion which consists of the lacZ gene under the control of bacteriophage lambda pR promoter we found that transcription starting from this promoter was inhibited during the stringent, but not the relaxed, response in E. coli. We confirmed our conclusion by estimating the relative level of the pR transcript by RNA-DNA hybridization. We propose that decreased transcription from the pR promoter which serves as transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid replication during the stringent response. The results presented in this paper, combined with our recent findings (published elsewhere), indicate that the transcriptional activation of ori lambda may be a main regulatory process controlling lambda DNA replication not only during the relaxed response but also in normal growth conditions.

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Basal-Level Effects of (p)ppGpp in the Absence of Branched-Chain Amino Acids in Actinobacillus pleuropneumoniae

Journal of Bacteriology ◽

10.1128/jb.00640-19 ◽

2020 ◽

Vol 202 (8) ◽

Author(s):

Gang Li ◽

Qian Zhao ◽

Tian Luan ◽

Yangbo Hu ◽

Yueling Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

De Novo ◽

Model Organism ◽

Stringent Response ◽

Actinobacillus Pleuropneumoniae ◽

Branched Chain Amino Acids ◽

Content Type ◽

Phenotypic Differences ◽

Branched Chain

ABSTRACT The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments, and is linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, its only known natural host. Pig lungs are known to have a low concentration of free branched-chain amino acids (BCAAs) compared to the level in plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae. Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of the wild type (WT) and an relA spoT double mutant [which does not produce (p)ppGpp], we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology, and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively upregulated, regardless of the existence of BCAAs, without accumulation of (p)ppGpp beyond a basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae. (p)ppGpp-mediated regulation in A. pleuropneumoniae is different from that described for the model organism Escherichia coli. Further work will establish whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family. IMPORTANCE (p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branched-chain amino acids (BCAAs) does not elicit a change in the basal-level (p)ppGpp, but this level is sufficient to regulate the expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is different from that of the model organism Escherichia coli but similar to that found in some Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp but also further validates the complexity and multiplicity of (p)ppGpp regulation in microorganisms that occupy different biological niches.

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A Stringent Analysis of Polyphosphate Dynamics inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00070-19 ◽

2019 ◽

Vol 201 (9) ◽

Author(s):

Michael Downey

Keyword(s):

Escherichia Coli ◽

Infection Control ◽

Bacterial Species ◽

Stringent Response ◽

Industrial Applications ◽

Bacterial Cells ◽

Inescherichia Coli ◽

Content Type ◽

Polyphosphate Accumulation ◽

Inorganic Phosphates

ABSTRACTDuring stress, bacterial cells activate a conserved pathway called the stringent response that promotes survival. Polyphosphates are long chains of inorganic phosphates that modulate this response in diverse bacterial species. In this issue, Michael J. Gray provides an important correction to the model of how polyphosphate accumulation is regulated during the stringent response inEscherichia coli(M. J. Gray, J. Bacteriol, 201:e00664-18, 2019,https://doi.org/10.1128/JB.00664-18). With other recent publications, this study provides a revised framework for understanding how bacterial polyphosphate dynamics might be exploited in infection control and industrial applications.

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Increased Mutation Frequency in Redox-Impaired Escherichia coli Due to RelA- and RpoS-Mediated Repression of DNA Repair

Applied and Environmental Microbiology ◽

10.1128/aem.00583-10 ◽

2010 ◽

Vol 76 (16) ◽

pp. 5463-5470 ◽

Cited By ~ 4

Author(s):

Amarjeet Singh ◽

Anis Karimpour-Fard ◽

Ryan T. Gill

Keyword(s):

Escherichia Coli ◽

Dna Repair ◽

Mutation Frequency ◽

Spontaneous Mutation ◽

Regulatory Gene ◽

Stringent Response ◽

Anaerobic Growth ◽

E Coli ◽

Direct Measurements ◽

Order Of Magnitude

ABSTRACT Balancing of reducing equivalents is a fundamental issue in bacterial metabolism and metabolic engineering. Mutations in the key metabolic genes ldhA and pflB of Escherichia coli are known to stall anaerobic growth and fermentation due to a buildup of intracellular NADH. We observed that the rate of spontaneous mutation in E. coli BW25113 (ΔldhA ΔpflB) was an order of magnitude higher than that in wild-type (WT) E. coli BW25113. We hypothesized that the increased mutation frequency was due to an increased NADH/NAD+ ratio in this strain. Using several redox-impaired strains of E. coli and different redox conditions, we confirmed a significant correlation (P < 0.01) between intracellular-NADH/NAD+ ratio and mutation frequency. To identify the genetic basis for this relationship, whole-genome transcriptional profiles were compared between BW25113 WT and BW25113 (ΔldhA ΔpflB). This analysis revealed that the genes involved in DNA repair were expressed at significantly lower levels in BW25113 (ΔldhA ΔpflB). Direct measurements of the extent of DNA repair in BW25113 (ΔldhA ΔpflB) subjected to UV exposure confirmed that DNA repair was inhibited. To identify a direct link between DNA repair and intracellular-redox ratio, the stringent-response-regulatory gene relA and the global-stress-response-regulatory gene rpoS were deleted. In both cases, the mutation frequencies were restored to BW25113 WT levels.

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The Stringent Response and Cell Cycle Arrest in Escherichia coli

PLoS Genetics ◽

10.1371/journal.pgen.1000300 ◽

2008 ◽

Vol 4 (12) ◽

pp. e1000300 ◽

Cited By ~ 94

Author(s):

Daniel J. Ferullo ◽

Susan T. Lovett

Keyword(s):

Escherichia Coli ◽

Cell Cycle ◽

Cell Cycle Arrest ◽

Stringent Response ◽

Cycle Arrest

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Inorganic Polyphosphate Accumulation inEscherichia coliIs Regulated by DksA but Not by (p)ppGpp

Journal of Bacteriology ◽

10.1128/jb.00664-18 ◽

2019 ◽

Vol 201 (9) ◽

Cited By ~ 13

Author(s):

Michael J. Gray

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Rna Polymerase ◽

Elongation Factor ◽

Stringent Response ◽

Inorganic Polyphosphate ◽

Content Type ◽

Polyphosphate Accumulation ◽

Pathogenic Microbes ◽

Polyp Accumulation

ABSTRACTProduction of inorganic polyphosphate (polyP) by bacteria is triggered by a variety of different stress conditions. polyP is required for stress survival and virulence in diverse pathogenic microbes. Previous studies have hypothesized a model for regulation of polyP synthesis in which production of the stringent-response second messenger (p)ppGpp directly stimulates polyP accumulation. In this work, I have now shown that this model is incorrect, and (p)ppGpp is not required for polyP synthesis inEscherichia coli. However, stringent mutations of RNA polymerase that frequently arise spontaneously in strains defective in (p)ppGpp synthesis and null mutations of the stringent-response-associated transcription factor DksA both strongly inhibit polyP accumulation. The loss of polyP synthesis in a mutant lacking DksA was reversed by deletion of the transcription elongation factor GreA, suggesting that competition between these proteins for binding to the secondary channel of RNA polymerase plays an important role in controlling polyP activation. These results provide new insights into the poorly understood regulation of polyP synthesis in bacteria and indicate that the relationship between polyP and the stringent response is more complex than previously suspected.IMPORTANCEProduction of polyP in bacteria is required for virulence and stress response, but little is known about how bacteria regulate polyP levels in response to changes in their environments. Understanding this regulation is important for understanding how pathogenic microbes resist killing by disinfectants, antibiotics, and the immune system. In this work, I have clarified the connections between polyP regulation and the stringent response to starvation stress inEscherichia coliand demonstrated an important and previously unknown role for the transcription factor DksA in controlling polyP levels.

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Replication of plasmids derived from Shiga toxin-converting bacteriophages in starved Escherichia coli

Microbiology ◽

10.1099/mic.0.042820-0 ◽

2011 ◽

Vol 157 (1) ◽

pp. 220-233 ◽

Cited By ~ 10

Author(s):

Bożena Nejman ◽

Beata Nadratowska-Wesołowska ◽

Agnieszka Szalewska-Pałasz ◽

Alicja Węgrzyn ◽

Grzegorz Węgrzyn

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Transcriptional Activation ◽

Stringent Response ◽

Toxin Production ◽

Replication Initiation ◽

Host Cells ◽

Regulation Of Transcription ◽

E Coli ◽

Dna Replication Initiation

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage λ, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA + and relA − cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the λ P R promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for λ P R, were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to λ) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA − hosts). Possible clinical implications of these results are discussed.

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Dimerization of the RelA protein of Escherichia coli

Biochemistry and Cell Biology ◽

10.1139/o01-144 ◽

2001 ◽

Vol 79 (6) ◽

pp. 729-736 ◽

Cited By ~ 14

Author(s):

Xiaoming Yang ◽

Edward E Ishiguro

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Cellular Localization ◽

Stringent Response ◽

Site Directed Mutagenesis ◽

Ribosome Binding ◽

Terminal Domain ◽

Proposed Model ◽

Mutagenesis Study

The RelA protein of Escherichia coli is a ribosome-associated (p)ppGpp synthetase that is activated by amino acid deprivation. It was recently reported that the activity of RelA is regulated by oligomerization mediated by the C-terminal domain of RelA. The oligomerization of RelA is further characterized in this study. The C-terminal domain consisting of amino acids 455744, designated 'RelA, formed homooligomers as well as heterooligomers with RelA as demonstrated by copurification of RelA and 'RelA and by an affinity blotting assay. Glutaraldehyde-induced cross-linking indicated that the oligomer was a dimer. The functional analysis of 'RelA was based on a combination of yeast two-hybrid analysis, the determination of the effects of overexpression of 'RelA derivatives on the stringent response, and the cellular localization of the overexpressed 'RelA derivatives. These studies indicated that two regions, designated 'RelA-1 (amino acids 455538) and 'RelA-2 (amino acids 550682), were involved in dimerization. The involvement of one of these two regions, RelA-2, is consistent with a previous site-directed mutagenesis study. In addition to dimerization, 'RelA-2 apparently contained the main ribosome-binding domain of RelA. The third region, 'RelA-3 (amino acids 682744), was not involved in either dimerization or ribosome binding. The overexpression of 'RelA-1 and 'RelA-2, but not 'RelA-3, inhibited the stringent response. These results support the previously proposed model which suggests a role for oligomerization in the regulation of (p)ppGpp synthetase.Key words: RelA, Escherichia coli, stringent response.

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