scholarly journals Loss of topoisomerase I function affects the RpoS-dependent and GAD systems of acid resistance in Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2783-2791 ◽  
Author(s):  
Natalee Stewart ◽  
Jingyang Feng ◽  
Xiaoping Liu ◽  
Devyani Chaudhuri ◽  
John W. Foster ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Blot Analysis ◽  
Topoisomerase I ◽  
Northern Blot Analysis ◽  
Acid Resistance ◽  
Arginine Decarboxylase ◽  
Rnase H ◽  
Loop Formation ◽  
Human Gastrointestinal Tract ◽  
Insertion Mutations

Acid resistance (AR) in Escherichia coli is important for its survival in the human gastrointestinal tract and involves three systems. The first AR system is dependent on the sigma factor RpoS. The second system (the GAD system) requires the glutamate decarboxylase isoforms encoded by the gadA and gadB genes. The third system (the ARG system) requires the arginine decarboxylase encoded by adiA. Loss of topoisomerase I function from topA deletion or Tn10 insertion mutations lowered the resistance to killing by pH 2 or 2·5 treatment by 10-fold to >100-fold. The RpoS and GAD systems were both affected by the topA mutation, but the ARG system of AR was not affected. Northern blot analysis showed that induction of gadA and gadB transcription in stationary phase and at pH 5·5 was decreased in the topA mutant. Western blot analysis showed that the topA mutation did not affect accumulation of RpoS, GadX or GadW proteins. Topoisomerase I might have a direct influence on the transcription of AR genes. This influence does not involve R-loop formation as the overexpression of RNase H did not alleviate the decrease of AR caused by the topA mutation. The effect of the topA mutation could be suppressed by an hns mutation, so topoisomerase I might be required to counteract the effect of H-NS protein on gene expression, in addition to its influence on RpoS-dependent transcription.

scholarly journals Availability of Glutamate and Arginine during Acid Challenge Determines Cell Density-Dependent Survival Phenotype ofEscherichia coli Strains

2001 ◽  
Vol 67 (10) ◽  
pp. 4914-4918 ◽  
Author(s):  
Shenghui Cui ◽  
Jianghong Meng ◽  
Arvind A. Bhagwat
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Acid Resistance ◽  
Arginine Decarboxylase ◽  
Ofescherichia Coli ◽  
Acid Sensitivity ◽  
Density Dependent ◽  
Survival Pathways ◽  
K 12 ◽  
Acid Challenge

ABSTRACT The cell density-dependent acid sensitivity phenotypes ofEscherichia coli strains K-12 and O157:H7 were examined with reference to three possible mechanisms of acid resistance. There was no evidence of any diffusible substance released from dead cells which could influence the cell density-dependent acid survival phenotype. Instead, cell density-dependent acid survival phenotype was associated with induction of glutamate- and arginine-decarboxylase acid survival pathways and concomitant availability of glutamate and arginine during acid challenge.

Poly(A) tail length of oxytocin- and lysine vasopressin-encoding mRNAs increases during development in the porcine hypothalamus

1990 ◽  
Vol 4 (2) ◽  
pp. 151-158 ◽  
Author(s):  
M. Rehbein ◽  
D. Richter
Keyword(s):  
Length Polymorphism ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Developmental Stages ◽  
Tail Length ◽  
Rnase H ◽  
Northern Blots ◽  
Lysine Vasopressin

ABSTRACT Lysine vasopressin- and oxytocin-encoding mRNAs have been analysed in the developing hypothalamus of the pig. The two hormone-encoding mRNAs were first detectable on fetal day 49 by Northern blot analysis. Whereas RNase mapping revealed identical transcripts throughout the developmental stages studied, Northern blots showed that the early transcripts appeared to be shorter (by 100–200 nucleotides) and more heterogeneous in size than those of later stages. This developmentally related length polymorphism was shown to be due to different poly(A) lengths and was abolished by removal of the poly(A) tails with RNase H. These results indicate that maturation of neurones in the developing porcine hypothalamus is accompanied by an increase in length of the poly(A) tail of vasopressin and oxytocin mRNAs.

scholarly journals Arginine-Agmatine Antiporter in Extreme Acid Resistance in Escherichia coli

2003 ◽  
Vol 185 (22) ◽  
pp. 6556-6561 ◽  
Author(s):  
Ram Iyer ◽  
Carole Williams ◽  
Christopher Miller
Keyword(s):  
Escherichia Coli ◽  
Acid Resistance ◽  
Strong Acid ◽  
Enteric Bacteria ◽  
Arginine Decarboxylase ◽  
Transport Protein ◽  
Gene Arrangement ◽  
Coupled Transport ◽  
E Coli ◽  
Acid Environment

ABSTRACT The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach. Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine α-decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm. The arginine system afforded significant protection to wild-type E. coli cells in our acid shock experiments. The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC. Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine. Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli. Evidence for a lysine-dependent XAR system in E. coli is also presented. The protection by lysine, however, is milder than that by arginine.

scholarly journals Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

2021 ◽  
Author(s):  
J Krishna Leela ◽  
Nalini Raghunathan ◽  
J Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Topoisomerase I ◽  
Synthetic Lethality ◽  
Life Forms ◽  
Dna Supercoiling ◽  
Loop Formation ◽  
Chromosomal Dna ◽  
E Coli ◽  
Rnase Hi

Topoisomerase I (Topo I) of Escherichia coli , encoded by topA , acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC -independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including Δ topA , confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued Δ dnaA lethality; (ii) in dnaA + derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by Δ dnaA . We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR. Importance In all life forms, double helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli . That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability, and suggests that in its absence aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

scholarly journals Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor

2020 ◽  
Vol 21 (16) ◽  
pp. 5843
Author(s):  
Pawel Wojtkiewicz ◽  
Daria Biernacka ◽  
Patrycja Gorzelak ◽  
Anna Stupak ◽  
Gracjana Klein ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Folding ◽  
Acid Resistance ◽  
Transcriptional Factors ◽  
Rnase H ◽  
Amino Acid Residues ◽  
Ppiase Activity ◽  
Multicopy Suppressor ◽  
Rate Limiting Step ◽  
Genes Encoding

Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl cis/trans isomerases (PPIs) in Escherichia coli confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of Δ6ppi strains. Interestingly, viability of Δ6ppi bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda. DksA, MetL and Cmk are for the first time shown to exhibit PPIase activity in chymotrypsin-coupled and RNase T1 refolding assays and their overexpression also restores growth of a Δ(dnaK/J/tig) strain, revealing their mechanism of suppression. Mutagenesis of DksA identified that D74, F82 and L84 amino acid residues are critical for its PPIase activity and their replacement abrogated multicopy suppression ability. Mutational studies revealed that DksA-mediated suppression of either Δ6ppi or ΔdnaK/J is abolished if GroL/S and RpoE are limiting, or in the absence of either major porin regulatory sensory kinase EnvZ or RNase H, transporter TatC or LepA GTPase or Pi-signaling regulator PhoU.

scholarly journals YjdE (AdiC) Is the Arginine:Agmatine Antiporter Essential for Arginine-Dependent Acid Resistance in Escherichia coli

2003 ◽  
Vol 185 (15) ◽  
pp. 4402-4409 ◽  
Author(s):  
Shimei Gong ◽  
Hope Richard ◽  
John W. Foster
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Random Mutagenesis ◽  
Acid Resistance ◽  
Arginine Decarboxylase ◽  
Integral Membrane Protein ◽  
Amino Acid Identity ◽  
Homology Search ◽  
System 2 ◽  
System 1

ABSTRACT To survive in extremely acidic conditions, Escherichia coli has evolved three adaptive acid resistance strategies thought to maintain internal pH. While the mechanism behind acid resistance system 1 remains enigmatic, systems 2 and 3 are known to require external glutamate (system 2) and arginine (system 3) to function. These latter systems employ specific amino acid decarboxylases and putative antiporters that exchange the extracellular amino acid substrate for the intracellular by-product of decarboxylation. Although GadC is the predicted antiporter for system 2, the antiporter specific for arginine/agmatine exchange has not been identified. A computer-based homology search revealed that the yjdE (now called adiC) gene product shared an overall amino acid identity of 22% with GadC. A series of adiC mutants isolated by random mutagenesis and by targeted deletion were shown to be defective in arginine-dependent acid resistance. This defect was restored upon introduction of an adiC+ -containing plasmid. An adiC mutant proved incapable of exchanging extracellular arginine for intracellular agmatine but maintained wild-type levels of arginine decarboxylase protein and activity. Western blot analysis indicated AdiC is an integral membrane protein. These data indicate that the arginine-to-agmatine conversion defect of adiC mutants was at the level of transport. The adi gene region was shown to be organized into two transcriptional units, adiAY and adiC, which are coordinately regulated but independently transcribed. The data also illustrate that the AdiA decarboxylase:AdiC antiporter system is designed to function only at acid levels sufficient to harm the cell.

scholarly journals Profiling Early Osmostress-Dependent Gene Expression in Escherichia coli Using DNA Macroarrays

2002 ◽  
Vol 184 (19) ◽  
pp. 5502-5507 ◽  
Author(s):  
Arnim Weber ◽  
Kirsten Jung
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mrna Levels ◽  
Expression In Escherichia Coli ◽  
Array Technology ◽  
Transcriptional Alterations ◽  
Dna Macroarrays

ABSTRACT DNA macroarray technology was used to monitor early transcriptional alterations of Escherichia coli in response to an osmotic upshift imposed by the addition of 0.4 M NaCl. Altered mRNA levels of 152 genes were detected; 45 genes showed increased expression while the expression of the remaining 107 genes was reduced. Northern blot analysis of several selected genes differing in their relative expression values confirmed the results obtained by the array technology.

scholarly journals Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

2021 ◽  
Author(s):  
J Gowrishankar ◽  
J Krishna Leela ◽  
Nalini Raghunathan
Keyword(s):  
Escherichia Coli ◽  
Topoisomerase I ◽  
Synthetic Lethality ◽  
Dna Supercoiling ◽  
Loop Formation ◽  
Chromosomal Replication ◽  
E Coli ◽  
Rnase Hi ◽  
Synthetically Lethal ◽  
Chromosome Iii

Topoisomerase I (Topo I) of <Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including ΔtopA>, confer lethality in derivatives of wild-type E. coli strain  MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. colitopA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR.

scholarly journals Emergence of Fosfomycin-Resistant Isolates of Shiga-Like Toxin-Producing Escherichia coli O26

1999 ◽  
Vol 43 (4) ◽  
pp. 789-793 ◽  
Author(s):  
Toshinobu Horii ◽  
Taku Kimura ◽  
Kumiko Sato ◽  
Keigo Shibayama ◽  
Michio Ohta
Keyword(s):  
Escherichia Coli ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Transport Systems ◽  
Transcriptional Level ◽  
Gene Encoding ◽  
E Coli ◽  
Fosfomycin Resistance

ABSTRACT We evaluated the susceptibilities of 129 Shiga-like toxin-producingEscherichia coli (STEC) isolates to various antibiotics. The numbers of isolates for which MICs were high (≧128 μg/ml) were as follows: 5 for fosfomycin, 14 for ampicillin, 1 for cefaclor, 6 for kanamycin, 22 for tetracycline, and 2 for doxycycline. For two isolates of STEC O26 MICs of fosfomycin were high (1,024 and 512 μg/ml, respectively). Conjugation experiments and glutathioneS-transferase assays suggested that the fosfomycin resistance in these isolates was determined not by a plasmid but chromosomally. The amount of active intracellular fosfomycin in these STEC isolates was 100- to 200-fold less than that in E. coli C600 harboring pREFTT47B408 in the presence of eitherl-α-glycerophosphate or glucose-6-phosphate. Cloning, sequencing, and Northern blot analysis demonstrated that the transcriptional level of the murA gene encoding UDP-N-acetylglucosamine enolpyruvoyl transferase in these isolates was greater than that in E. coli C600. Our results suggest that the fosfomycin resistance in these STEC isolates is due to concurrent effects of alteration of the glpT and/oruhp transport systems and of the enhanced transcription of the murA gene.

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