scholarly journals Inhibition of Escherichia coli chromosome replication by rifampicin treatment or during the stringent response is overcome by de novo DnaA protein synthesis

2020 ◽  
Vol 114 (6) ◽  
pp. 906-919 ◽  
Author(s):  
Leise Riber ◽  
Anders Løbner‐Olesen
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
De Novo ◽  
Stringent Response ◽  
Chromosome Replication ◽  
Dnaa Protein ◽  
Rifampicin Treatment

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

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.

scholarly journals Genetic Analysis of the Invariant Residue G791 in Escherichia coli 16S rRNA Implicates RelA in Ribosome Function

2009 ◽  
Vol 191 (7) ◽  
pp. 2042-2050 ◽  
Author(s):  
Hong-Man Kim ◽  
Sang-Mi Ryou ◽  
Woo-Seok Song ◽  
Se-Hoon Sim ◽  
Chang-Jun Cha ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
16S Rrna ◽  
Stringent Response ◽  
State Level ◽  
Synthetic Activity ◽  
Wild Type ◽  
Multicopy Suppressor ◽  
Invariant Residue ◽  
In Cells

ABSTRACT Previous studies identified G791 in Escherichia coli 16S rRNA as an invariant residue for ribosome function. In order to establish the functional role of this residue in protein synthesis, we searched for multicopy suppressors of the mutant ribosomes that bear a G-to-U substitution at position 791. We identified relA, a gene whose product has been known to interact with ribosomes and trigger a stringent response. Overexpression of RelA resulted in the synthesis of approximately 1.5 times more chloramphenicol acetyltransferase (CAT) protein than could be synthesized by the mutant ribosomes in the absence of RelA overexpression. The ratio of mutant rRNA to the total ribosome pool was not changed, and the steady-state level of CAT mRNA was decreased by RelA overexpression. These data confirmed that the phenotype of RelA as a multicopy suppressor of the mutant ribosome did not result from the enhanced synthesis of mutant rRNA or CAT mRNA from the plasmid. To test whether the phenotype of RelA was related to the stringent response induced by the increased cellular level of (p)ppGpp, we screened for mutant RelA proteins whose overexpression enhances CAT protein synthesis by the mutant ribosomes as effectively as wild-type RelA overexpression and then screened for those whose overexpression does not produce sufficiently high levels of (p)ppGpp to trigger the stringent response under the condition of amino acid starvation. Overexpression of the isolated mutant RelA proteins resulted in the accumulation of (p)ppGpp in cells, which was amounted to approximately 18.2 to 38.9% of the level of (p)ppGpp found in cells that overexpress the wild-type RelA. These findings suggest that the function of RelA as a multicopy suppressor of the mutant ribosome does not result from its (p)ppGpp synthetic activity. We conclude that RelA has a previously unrecognized role in ribosome function.

scholarly journals Arresting chromosome replication upon energy starvation in Escherichia coli

2021 ◽  
Author(s):  
Godefroid Charbon ◽  
Jakob Frimodt-Møller ◽  
Anders Løbner-Olesen
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Protein Synthesis ◽  
Genome Stability ◽  
Normal Growth ◽  
Cell Cycle Checkpoints ◽  
Initiator Protein ◽  
Dnaa Protein ◽  
Chromosomal Duplication ◽  
Periods Of Stress

AbstractMost organisms possess several cell cycle checkpoints to preserve genome stability in periods of stress. Upon starvation, the absence of chromosomal duplication in the bacterium Escherichia coli is ensured by holding off commencement of replication. During normal growth, accumulation of the initiator protein DnaA along with cell cycle changes in its activity, ensure that DNA replication starts only once per cell cycle. Upon nutrient starvation, the prevailing model is that an arrest in DnaA protein synthesis is responsible for the absence of initiation. Recent indications now suggest that DnaA degradation may also play a role. Here we comment on the implications of this potential new layer of regulation.

scholarly journals Effects of inhibitors of protein synthesis on lysis of Escherichia coli induced by beta-lactam antibiotics.

1996 ◽  
Vol 40 (4) ◽  
pp. 899-903 ◽  
Author(s):  
D G Rodionov ◽  
E E Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Binding Proteins ◽  
Rapid Development ◽  
Stringent Response ◽  
Beta Lactam ◽  
Penicillin Binding Proteins ◽  
Beta Lactam Antibiotics ◽  
Inhibition Of Protein Synthesis

The role of protein synthesis in ampicillin-induced lysis of Escherichia coli was investigated. The inhibition of protein synthesis through amino acid deprivation resulted in the rapid development of ampicillin tolerance as a consequence of the stringent response, as previously reported. In contrast, inhibition of protein synthesis by use of ribosome inhibitors such as chloramphenicol did not readily confer ampicillin tolerance and, in fact, promoted the development of both stages of the ampicillin-induced lysis process, i.e., (i) an ampicillin-dependent stage which apparently involves the interaction of penicillin-binding proteins with ampicillin and (ii) an ampicillin-independent stage which may represent the events leading to the deregulation of peptidoglycan hydrolase activity. We propose that lysis was facilitated when protein synthesis was inhibited because the production of new penicillin-binding proteins to replace those which were ampicillin inhibited was prevented under these conditions.

scholarly journals The Caulobacter crescentus Homolog of DnaA (HdaA) Also Regulates the Proteolysis of the Replication Initiator Protein DnaA

2015 ◽  
Vol 197 (22) ◽  
pp. 3521-3532 ◽  
Author(s):  
Richard Wargachuk ◽  
Gregory T. Marczynski
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Caulobacter Crescentus ◽  
Extra Chromosome ◽  
Chromosome Replication ◽  
Wild Type ◽  
Content Type ◽  
Antibiotic Development ◽  
E Coli ◽  
Dnaa Protein

ABSTRACTIt is not known how diverse bacteria regulate chromosome replication. Based onEscherichia colistudies, DnaA initiates replication and the homolog of DnaA (Hda) inactivates DnaA using the RIDA (regulatoryinactivation ofDnaA) mechanism that thereby prevents extra chromosome replication cycles. RIDA may be widespread, because the distantly relatedCaulobacter crescentushomolog HdaA also prevents extra chromosome replication (J. Collier and L. Shapiro, J Bacteriol 191:5706–5715, 2009,http://dx.doi.org/10.1128/JB.00525-09). To further study the HdaA/RIDA mechanism, we created aC. crescentusstrain that shuts offhdaAtranscription and rapidly clears HdaA protein. We confirm that HdaA prevents extra replication, since cells lacking HdaA accumulate extra chromosome DNA. DnaA binds nucleotides ATP and ADP, and our results are consistent with the establishedE. colimechanism whereby Hda converts active DnaA-ATP to inactive DnaA-ADP. However, unlikeE. coliDnaA,C. crescentusDnaA is also regulated by selective proteolysis.C. crescentuscells lacking HdaA reduce DnaA proteolysis in logarithmically growing cells, thereby implicating HdaA in this selective DnaA turnover mechanism. Also, wild-typeC. crescentuscells remove all DnaA protein when they enter stationary phase. However, cells lacking HdaA retain stable DnaA protein even when they stop growing in nutrient-depleted medium that induces complete DnaA proteolysis in wild-type cells. Additional experiments argue for a distinct HdaA-dependent mechanism that selectively removes DnaA prior to stationary phase. Related freshwaterCaulobacterspecies also remove DnaA during entry to stationary phase, implying a wider role for HdaA as a novel component of programed proteolysis.IMPORTANCEBacteria must regulate chromosome replication, and yet the mechanisms are not completely understood and not fully exploited for antibiotic development. Based onEscherichia colistudies, DnaA initiates replication, and the homolog of DnaA (Hda) inactivates DnaA to prevent extra replication. The distantly relatedCaulobacter crescentushomolog HdaA also regulates chromosome replication. Here we unexpectedly discovered that unlike theE. coliHda, theC. crescentusHdaA also regulates DnaA proteolysis. Furthermore, this HdaA proteolysis acts in logarithmically growing and in stationary-phase cells and therefore in two very different physiological states. We argue that HdaA acts to help time chromosome replications in logarithmically growing cells and that it is an unexpected component of the programed entry into stationary phase.

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