scholarly journals A T7 phage factor required for managing RpoS inEscherichia coli

2018 ◽  
Author(s):  
Aline Tabib-Salazar ◽  
Bing Liu ◽  
Declan Barker ◽  
Lynn Burchell ◽  
Udi Qimron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Stationary Phases ◽  
Specific Inhibitor ◽  
E Coli ◽  
Bacterial Transcription ◽  
Bacterial Rna ◽  
Phage T7 ◽  
T7 Phage

T7 development inEscherichia colirequires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. While the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growingE. colias a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development inE. colicells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.Significance statementVirus that infect bacteria (phages) represent the most abundant living entities on the planet and many aspects of our fundamental knowledge of phage-bacteria relationships have been derived in the context of exponentially growing bacteria. In the case of the prototypicalEscherichia coliphage T7, specific inhibition of the housekeeping form of the RNA polymerase (Eσ70) by a T7 protein, called Gp2, is essential for the development of viral progeny. We now reveal that T7 uses a second specific inhibitor that selectively inhibits the stationary phase RNAP (EσS), which enables T7 to develop well in exponentially growing and stationary phase bacteria. The results have broad implications for our understanding of phage-bacteria relationships and therapeutic application of phages.

scholarly journals T7 phage factor required for managing RpoS inEscherichia coli

2018 ◽  
Vol 115 (23) ◽  
pp. E5353-E5362 ◽  
Author(s):  
Aline Tabib-Salazar ◽  
Bing Liu ◽  
Declan Barker ◽  
Lynn Burchell ◽  
Udi Qimron ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Stationary Phases ◽  
Inescherichia Coli ◽  
E Coli ◽  
Bacterial Transcription ◽  
Bacterial Rna ◽  
Predominant Form ◽  
Phage Development ◽  
T7 Phage

T7 development inEscherichia colirequires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growingE. colias a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development inE. colicells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.

scholarly journals Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Yanyu Zhu ◽  
Mainak Mustafi ◽  
James C. Weisshaar
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscopy ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Mean Square Displacement ◽  
Quiescent State ◽  
Chromosomal Dna ◽  
Content Type ◽  
E Coli

ABSTRACT In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is “compacted” or “supercompacted,” and there are suggestions that the cytoplasm is “glass-like.” Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli. Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

scholarly journals In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM

2007 ◽  
Vol 189 (9) ◽  
pp. 3635-3638 ◽  
Author(s):  
Matthew R. Chenoweth ◽  
Nancy Trun ◽  
Sue Wickner
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Stationary Phase ◽  
Specific Inhibitor ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
E Coli ◽  
Hsp70 Chaperone

ABSTRACT CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

scholarly journals ArgR-Independent Induction and ArgR-Dependent Superinduction of the astCADBE Operon in Escherichia coli

2002 ◽  
Vol 184 (11) ◽  
pp. 2940-2950 ◽  
Author(s):  
Alexandros K. Kiupakis ◽  
Larry Reitzer
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Nitrogen Source ◽  
Nitrogen Limitation ◽  
Nitrogen Sources ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Nitrogen Regulator I

ABSTRACT For Escherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by σ54 and nitrogen regulator I (NRI, also called NtrC). The astCADBE operon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis of E. coli revealed the presence of a σ54-dependent promoter utilized in exponential phase during nitrogen limitation and a σS-dependent promoter active during stationary phase. We used an ast-lacZ fusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NRI, and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the σ54-dependent promoter. Thus, the ast operon exhibits nitrogen source-specific induction that is unique for an NRI-dependent gene. The transcriptional regulation of the ast operon in E. coli differs from that in Salmonella enterica serovar Typhimurium, in which ArgR is required for ast operon expression.

scholarly journals Sensitivity of Escherichia coli O157:H7 to Commercially Available Alkaline Cleaners and Subsequent Resistance to Heat and Sanitizers

2004 ◽  
Vol 70 (3) ◽  
pp. 1795-1803 ◽  
Author(s):  
Manan Sharma ◽  
Larry R. Beuchat
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Stationary Phases ◽  
Cetylpyridinium Chloride ◽  
Sodium Metasilicate ◽  
Cross Protection ◽  
Decimal Reduction Time ◽  
E Coli ◽  
Subsequent Exposure ◽  
Glycol Monobutyl Ether

ABSTRACT The effects of seven commercially available alkaline cleaners used in the food processing industry, 0.025 M NaOH, and 0.025 M KOH on viability of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 in logarithmic and stationary phases of growth were determined. Cells were treated at 4 or 23�C for 2, 10, or 30 min. Cleaners 2, 4, 6, and 7, which contained hypochlorite and <11% NaOH and/or KOH (pH 11.2 to 11.7), killed significantly higher numbers of cells than treatment with cleaner 3, containing sodium metasilicate (pH 11.4) and <10% KOH, and cleaner 5, containing ethylene glycol monobutyl ether (pH 10.4). There were no differences in the sensitivities of logarithmic and stationary-phase cells to the alkaline cleaners. Treatment with KOH or NaOH (pH 12.2) was not as effective as four out of seven commercial cleaners in killing E. coli O157:H7, indicating that chlorine and other cleaner components have bactericidal activity at high pH. Stationary-phase cells of strain EDL 933 that had been exposed to cleaner 7 at 4 or 23�C and strain FRIK 816-3 exposed to cleaner 7 at 23�C had significantly higher D 55�C (decimal reduction time, minutes at 55�C) values than control cells or cells exposed to cleaner 5, indicating that exposure to cleaner 7 confers cross-protection to heat. Cells of EDL 933 treated with cleaner 7 at 12�C showed significantly higher D 55�C values than cells of FRIK 816-3, indicating that rpoS may play a role in cross-protection. Stationary-phase cells treated with cleaner 5 or cleaner 7 at 4 or 12�C were not cross-protected against subsequent exposure to sanitizers containing quaternary ammonium compounds or sodium hypochlorite, or to cetylpyridinium chloride and benzalkonium chloride.

Mannose-Binding Activity of Escherichia coli: a Determinant of Attachment and Ingestion of the Bacteria by Macrophages

1980 ◽  
Vol 29 (2) ◽  
pp. 417-424
Author(s):  
Zvi Bar-Shavit ◽  
Rachel Goldman ◽  
Itzhak Ofek ◽  
Nathan Sharon ◽  
David Mirelman
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Pathogenic Bacteria ◽  
Binding Activity ◽  
Exponential Phase ◽  
Restrictive Temperature ◽  
Filamentous Bacteria ◽  
Phagocytic Cells ◽  
E Coli ◽  
Mannose Binding

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [ 14 C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α- d -mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters ( R = 0.98, P < 0.001). The internalization of the filamentous cells attached to macrophages during 45 min of incubation was much less efficient (20%) compared to that of exponential-phase, stationary-phase, or antibody-coated filamentous bacteria (90%). The results indicate that the mannose-binding activity of E. coli determines the recognition of the organisms by phagocytes. They further suggest that administration of β-lactam antibiotics may impair elimination of certain pathogenic bacteria by inducing the formation of filaments which are inefficiently internalized by the host's phagocytic cells.

scholarly journals The extent of B-form structure in a 6S RNA variant that mimics DNA to bind to the Escherichia coli RNA Polymerase

2018 ◽  
Author(s):  
Nilesh K. Banavali
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Structural Analysis ◽  
Rna Polymerase ◽  
Rna Structure ◽  
Recent Article ◽  
Single Strand ◽  
Bacterial Rna ◽  
6S Rna

AbstractIn a recent article by Darst and coworkers, it was found that a non-coding 6S RNA variant regulates a bacterial RNA polymerase by mimicking B-Form DNA, and a few different nucleic acid duplex parameters were analyzed to understand the extent of B-form RNA structure. In this manuscript, a different structural analysis based on conformational distance from canonical A-form and B-form single-strand structures is presented. This analysis addresses the occurrence and extent of both local and global B-form structure in the published 6S RNA variant model.

scholarly journals Effect of DNA-interacting drugs on phage T7 RNA polymerase.

1998 ◽  
Vol 45 (1) ◽  
pp. 127-132 ◽  
Author(s):  
M Piestrzeniewicz ◽  
K Studzian ◽  
D Wilmańska ◽  
G Płucienniczak ◽  
M Gniazdowski
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
T7 Rna Polymerase ◽  
Distamycin A ◽  
E Coli ◽  
Phage T7 ◽  
Drug Dependent ◽  
Dna Complex ◽  
Kinetics Of

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

scholarly journals The l-Isoaspartyl Protein Repair Methyltransferase Enhances Survival of Aging Escherichia coli Subjected to Secondary Environmental Stresses

1998 ◽  
Vol 180 (10) ◽  
pp. 2623-2629 ◽  
Author(s):  
Jonathan E. Visick ◽  
Hui Cai ◽  
Steven Clarke
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Protein Denaturation ◽  
Environmental Stresses ◽  
Protein Repair ◽  
Repeated Heating ◽  
E Coli ◽  
Competitive Disadvantage ◽  
Biological World

ABSTRACT Like its homologs throughout the biological world, thel-isoaspartyl protein repair methyltransferase ofEscherichia coli, encoded by the pcm gene, can convert abnormal l-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885–9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation inrpoS, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158–4163, 1997). We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new pcmmutant lacks these phenotypes. Interestingly, the newly constructedpcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42°C. The pcmmutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functionalpcm gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E. coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.

scholarly journals Studies on deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Variations of the enzyme activity during growth

1972 ◽  
Vol 129 (2) ◽  
pp. 291-299 ◽  
Author(s):  
K. A. Abraham ◽  
K. J. Andersen ◽  
A. Rognes
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Stationary Phases ◽  
Bacteriophage T4 ◽  
Polymerase Activity ◽  
Exponential Phase ◽  
Enzyme Preparations ◽  
Rna Polymerase Activity ◽  
Enzyme Molecules ◽  
Cellular Dna

1. RNA polymerase activity of Escherichia coli extracts prepared from cells in exponential and stationary phases of growth, when measured in the presence and absence of external template, showed significant qualitative differences. 2. In both extracts, polymerase activity was higher when assayed with external template, suggesting the presence of a pool of enzyme not bound to cellular DNA. 3. In the crude extract, the fraction of enzyme bound to cellular DNA is higher during the exponential phase of growth. 4. A method is described for the purification of enzyme molecules not tightly bound to cellular DNA from exponential- and stationary-phase cultures. 5. Purified enzyme preparations showed differences in template requirement and subunit composition. 6. On phosphocellulose chromatography of stationary-phase enzyme, a major portion of polymerase activity eluted from the column with 0.25m-KCl. In the case of exponential-phase enzyme, polymerase activity eluted from a phosphocellulose column mainly with 0.35m-KCl. 7. Enzyme assays done with excess of bacteriophage T4 DNA showed a strong inhibition of stationary-phase enzyme by this template. The exponential-phase enzyme was only slightly inhibited by excess of bacteriophage T4 DNA.

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