scholarly journals Growth Phase and Growth Rate Regulation of therapA Gene, Encoding the RNA Polymerase-Associated Protein RapA in Escherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 6126-6134 ◽  
Author(s):  
Julio E. Cabrera ◽  
Ding Jun Jin
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rna Polymerase ◽  
Growth Phase ◽  
Gene Encoding ◽  
Rate Regulation ◽  
E Coli ◽  
Growth Rate Control

ABSTRACT The Escherichia coli rapA gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family. We have studied therapA promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro. We have found that the expression of rapA is growth phase dependent, peaking at the early log phase. The growth phase control ofrapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E. colipromoters. We also found that the rapA promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro. Furthermore, we have shown that a GC-rich or discriminator sequence between the −10 and +1 positions of the rapA promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP.

Effect of Tannins on the In Vitro Growth of Escherichia coli O157:H7 and In Vivo Growth of Generic Escherichia coli Excreted from Steers

2007 ◽  
Vol 70 (3) ◽  
pp. 543-550 ◽  
Author(s):  
BYENG R. MIN ◽  
WILLIAM E. PINCHAK ◽  
ROBIN C. ANDERSON ◽  
TODD R. CALLAWAY
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Escherichia Coli O157 ◽  
Tannin Extract ◽  
Bacterial Growth Rate ◽  
Linear Effect ◽  
E Coli

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37°C, was reduced (P < 0.05) with as little as 400 μg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 μg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 μg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 μg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.

scholarly journals DksA Is Required for Growth Phase-Dependent Regulation, Growth Rate-Dependent Control, and Stringent Control of fis Expression in Escherichia coli

2006 ◽  
Vol 188 (16) ◽  
pp. 5775-5782 ◽  
Author(s):  
Prabhat Mallik ◽  
Brian J. Paul ◽  
Steven T. Rutherford ◽  
Richard L. Gourse ◽  
Robert Osuna
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Growth Phase ◽  
Stationary Growth ◽  
Rate Dependent ◽  
Transcription In Vitro ◽  
State Growth

ABSTRACT DksA is a critical transcription factor in Escherichia coli that binds to RNA polymerase and potentiates control of rRNA promoters and certain amino acid promoters. Given the kinetic similarities between rRNA promoters and the fis promoter (Pfis), we investigated the possibility that DksA might also control transcription from Pfis. We show that the absence of dksA extends transcription from Pfis well into the late logarithmic and stationary growth phases, demonstrating the importance of DksA for growth phase-dependent regulation of fis. We also show that transcription from Pfis increases with steady-state growth rate and that dksA is absolutely required for this regulation. In addition, both DksA and ppGpp are required for inhibition of Pfis promoter activity following amino acid starvation, and these factors act directly and synergistically to negatively control Pfis transcription in vitro. DksA decreases the half-life of the intrinsically short-lived fis promoter-RNA polymerase complex and increases its sensitivity to the concentration of CTP, the predominant initiating nucleotide triphosphate for this promoter. This work extends our understanding of the multiple factors controlling fis expression and demonstrates the generality of the DksA requirement for regulation of kinetically similar promoters.

scholarly journals Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

2008 ◽  
Vol 190 (10) ◽  
pp. 3434-3443 ◽  
Author(s):  
Umender K. Sharma ◽  
Dipankar Chatterji
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Sigma Factors ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

scholarly journals Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

2001 ◽  
Vol 183 (15) ◽  
pp. 4543-4550 ◽  
Author(s):  
Mónica A. Delgado ◽  
Marı́a R. Rintoul ◽  
Ricardo N. Farı́as ◽  
Raúl A. Salomón
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Cyclic Peptide ◽  
Nucleotide Sequencing ◽  
Peptide Antibiotic ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Microcin J25

ABSTRACT Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in therpoC gene, encoding the β′ subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-typerpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoCgene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

scholarly journals DNA sequence around the Escherichia coli unc operon. Completion of the sequence of a 17 kilobase segment containing asnA, oriC, unc, glmS and phoS

1984 ◽  
Vol 224 (3) ◽  
pp. 799-815 ◽  
Author(s):  
J E Walker ◽  
N J Gay ◽  
M Saraste ◽  
A N Eberle
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Promoter Sequence ◽  
Gene Encoding ◽  
E Coli ◽  
Extensive Region ◽  
Transcriptional Promoters ◽  
Phosphoribosylpyrophosphate Amidotransferase ◽  
Unc Operon

The nucleotide sequence is described of a region of the Escherichia coli chromosome extending from oriC to phoS that also includes the loci gid, unc and glmS. Taken with known sequences for asnA and phoS this completes the sequence of a segment of about 17 kilobases or 0.4 min of the E. coli genome. Sequences that are probably transcriptional promoters for unc and phoS can be detected and the identity of the unc promoter has been confirmed by experiments in vitro with RNA polymerase. Upstream of the promoter sequence is an extensive region that appears to be non-coding. Conserved sequences are found that may serve to concentrate RNA polymerase in the vicinity of the unc promoter. Hairpin loop structures resembling known rho-independent transcription termination signals are evident following the unc operon and glmS. The glmS gene encoding the amidotransferase, glucosamine synthetase, has been identified by homology with glutamine 5-phosphoribosylpyrophosphate amidotransferase.

scholarly journals Escherichia coli DNA Topoisomerase I Copurifies with Tn5 Transposase, and Tn5 Transposase Inhibits Topoisomerase I

1999 ◽  
Vol 181 (10) ◽  
pp. 3185-3192 ◽  
Author(s):  
Hesna Yigit ◽  
William S. Reznikoff
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Topoisomerase I ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Dna Relaxation ◽  
Tn5 Transposase ◽  
Derivatives Of

ABSTRACT Tn5 transposase (Tnp) overproduction is lethal toEscherichia coli. Genetic evidence suggested that this killing involves titration of E. coli topoisomerase I (Topo I). Here, we present biochemical evidence that supports this model. Tn5 Tnp copurifies with Topo I while nonkilling derivatives of Tnp, Δ37Tnp and Δ55Tnp (Inhibitor [Inh]), show reduced affinity or no affinity, respectively, for Topo I. In agreement with these results, the presence of Tnp, but not Δ37 or Inh derivatives of Tnp, inhibits the DNA relaxation activity of Topo I in vivo as well as in vitro. Other proteins, including RNA polymerase, are also found to copurify with Tnp. For RNA polymerase, reduced copurification with Tnp is observed in extracts from a topA mutant strain, suggesting that RNA polymerase interacts with Topo I and not Tnp.

scholarly journals The RNA Polymerase α Subunit from Sinorhizobium meliloti Can Assemble with RNA Polymerase Subunits from Escherichia coli and Function in Basal and Activated Transcription both In Vivo and In Vitro

2002 ◽  
Vol 184 (14) ◽  
pp. 3808-3814 ◽  
Author(s):  
Melicent C. Peck ◽  
Tamas Gaal ◽  
Robert F. Fisher ◽  
Richard L. Gourse ◽  
Sharon R. Long
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Sinorhizobium Meliloti ◽  
Core Promoter ◽  
Temperature Sensitive ◽  
Protein Sequence Analysis ◽  
Α Subunit ◽  
E Coli

ABSTRACT Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the α subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (α)-rplQ (L17) found in the α-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti α supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti α and E. coli β, β′, and σ subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti α functions are conserved in heterologous host E. coli even though the two α subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.

scholarly journals Escherichia coli RNA Polymerase Recognition of a σ70-Dependent Promoter Requiring a −35 DNA Element and an Extended −10 TGn Motif

2006 ◽  
Vol 188 (24) ◽  
pp. 8352-8359 ◽  
Author(s):  
India Hook-Barnard ◽  
Xanthia B. Johnson ◽  
Deborah M. Hinton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Consensus Sequence ◽  
Perfect Match ◽  
Start Site ◽  
E Coli ◽  
Functional Differences ◽  
Extension Analysis

ABSTRACT Escherichia coli σ70-dependent promoters have typically been characterized as either −10/−35 promoters, which have good matches to both the canonical −10 and the −35 sequences or as extended −10 promoters (TGn/−10 promoters), which have the TGn motif and an excellent match to the −10 consensus sequence. We report here an investigation of a promoter, Pminor, that has a nearly perfect match to the −35 sequence and has the TGn motif. However, Pminor contains an extremely poor σ70 −10 element. We demonstrate that Pminor is active both in vivo and in vitro and that mutations in either the −35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the −10 element more canonical, thus converting the −35/TGn promoter to a −35/−10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with Pminor, the DNA is single stranded from positions −11 to +4. We also demonstrate that transcription from Pminor incorporates nontemplated ribonucleoside triphosphates at the 5′ end of the Pminor transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. Pminor represents one of the few −35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized −10/−35 and extended −10 promoters used by E. coli RNA polymerase.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

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