scholarly journals The strong efficiency of the Escherichia coli gapA P1 promoter depends on a complex combination of functional determinants

2004 ◽  
Vol 383 (2) ◽  
pp. 371-382 ◽  
Author(s):  
Benoit THOUVENOT ◽  
Bruno CHARPENTIER ◽  
Christiane BRANLANT
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Site Directed Mutagenesis ◽  
Dna Interactions ◽  
Template Strand ◽  
Chemical Probing ◽  
Upstream Promoter ◽  
Promoter Dna ◽  
High Level ◽  
Protein Pocket

The Escherichia coli multi-promoter region of the gapA gene ensures a high level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. In the exponential phase of growth, gapA mRNAs are mainly initiated at the highly efficient gapA P1 promoter. In the present study, by using site-directed mutagenesis and chemical probing of the RPo (open complex) formed by Eσ70 (holoenzyme associated with σ70) RNAP (RNA polymerase) at promoter gapA P1, we show that this promoter is an extended −10 promoter that needs a −35 sequence for activity. The −35 sequence compensates for the presence of a suboptimal −10 hexamer. A tract of thymine residues in the spacer region, which is responsible for a DNA distortion, is also required for efficient activity. We present the first chemical probing of an RPo formed at a promoter needing both a −10 extension and a −35 sequence. It reveals a complex array of RNAP–DNA interactions. In agreement with the fact that residue A-11 in the non-template strand is flipped out in a protein pocket in previously studied RPos, the corresponding A residue in gapA P1 promoter is protected in RPo and is essential for activity. However, in contrast with some of the previous findings on RPos formed at other promoters, the −12 A:T pair is opened. Strong contacts with RNAP occur both with the −35 sequence and the TG extension, so that the σ4 and σ2 domains may simultaneously contact the promoter DNA. RNAP–DNA interactions were also detected immediately downstream of the −35 hexamer and in a more distal upstream segment, reflecting a wrapping of RNAP by the core and upstream promoter DNA. Altogether, the data reveal that promoter gapA P1 is a very efficient promoter sharing common properties with both extended −10 and non-extended −10 promoters.

scholarly journals Resistance and Virulence Mechanisms of Escherichia coli Selected by Enrofloxacin in Chicken

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Jun Li ◽  
Haihong Hao ◽  
Menghong Dai ◽  
Heying Zhang ◽  
Jianan Ning ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pumps ◽  
Genetic Alterations ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
Genetic Characteristics ◽  
Content Type ◽  
E Coli ◽  
Low Level ◽  
High Level

ABSTRACT This study aimed to investigate the genetic characteristics, antibiotic resistance patterns, and novel mechanisms involved in fluoroquinolone (FQ) resistance in commensal Escherichia coli isolates. The E. coli isolates were recovered from a previous clinical study and subjected to antimicrobial susceptibility testing and molecular typing. Known mechanisms of FQ resistance (target site mutations, plasmid-mediated quinolone resistance [PMQR] genes, relative expression levels of efflux pumps and porins) were detected using DNA sequencing of PCR products and real-time quantitative PCR. Whole-genome shotgun sequencing was performed on 11 representative strains to screen for single nucleotide polymorphisms (SNPs). The function of a key SNP (A1541G) was investigated by site-directed mutagenesis and allelic exchange. The results showed that long-term enrofloxacin treatment selected multidrug-resistant (MDR) E. coli isolates in the chicken gut and that these E. coli isolates had diverse genetic backgrounds. Multiple genetic alterations, including double mutations on GyrA (S83L and D87N), a single mutation on ParC (S80I) and ParE (S458E), activation of efflux pumps, and the presence of the QnrS1 protein, contributed to the high-level FQ resistance (enrofloxacin MIC [MICENR] ≥ 128 μg/ml), while the relatively low-level FQ resistance (MICENR = 8 or 16 μg/ml) was commonly mediated by decreased expression of the porin OmpF, besides enhancement of the efflux pumps. No significant relationship was observed between resistance mechanisms and virulence genes. Introduction of the A1541G mutation on aegA was able to increase FQ susceptibility by 2-fold. This study contributes to a better understanding of the development of MDR and the differences underlying the mechanisms of high-level and low-level FQ resistance in E. coli.

Transcriptional takeover by σ appropriation: remodelling of the σ 70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1729-1740 ◽  
Author(s):  
Deborah M. Hinton ◽  
Suchira Pande ◽  
Neelowfar Wais ◽  
Xanthia B. Johnson ◽  
Madhavi Vuthoori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Molecular Level ◽  
Bacteriophage T4 ◽  
Good Match ◽  
Promoter Sequences ◽  
Recognition Element ◽  
Upstream Promoter ◽  
Promoter Specificity ◽  
Promoter Dna

Activation of bacteriophage T4 middle promoters, which occurs about 1 min after infection, uses two phage-encoded factors that change the promoter specificity of the host RNA polymerase. These phage factors, the MotA activator and the AsiA co-activator, interact with the σ 70 specificity subunit of Escherichia coli RNA polymerase, which normally contacts the −10 and −35 regions of host promoter DNA. Like host promoters, T4 middle promoters have a good match to the canonical σ 70 DNA element located in the −10 region. However, instead of the σ 70 DNA recognition element in the promoter's −35 region, they have a 9 bp sequence (a MotA box) centred at −30, which is bound by MotA. Recent work has begun to provide information about the MotA/AsiA system at a detailed molecular level. Accumulated evidence suggests that the presence of MotA and AsiA reconfigures protein–DNA contacts in the upstream promoter sequences, without significantly affecting the contacts of σ 70 with the −10 region. This type of activation, which is called ‘σ appropriation’, is fundamentally different from other well-characterized models of prokaryotic activation in which an activator frequently serves to force σ 70 to contact a less than ideal −35 DNA element. This review summarizes the interactions of AsiA and MotA with σ 70, and discusses how these interactions accomplish the switch to T4 middle promoters by inhibiting the typical contacts of the C-terminal region of σ 70, region 4, with the host −35 DNA element and with other subunits of polymerase.

scholarly journals High-Level Resistance to Ceftazidime Conferred by a Novel Enzyme, CTX-M-32, Derived from CTX-M-1 through a Single Asp240-Gly Substitution

2004 ◽  
Vol 48 (6) ◽  
pp. 2308-2313 ◽  
Author(s):  
Monica Cartelle ◽  
Maria del Mar Tomas ◽  
Francisca Molina ◽  
Rita Moure ◽  
Rosa Villanueva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Site Directed Mutagenesis ◽  
Clinical Strain ◽  
Directed Mutagenesis ◽  
M Gene ◽  
High Level ◽  
Level Resistance

ABSTRACT A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (bla CTX-M-32) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis

scholarly journals Structural origins of Escherichia coli RNA polymerase open promoter complex stability

2021 ◽  
Vol 118 (40) ◽  
pp. e2112877118
Author(s):  
Ruth M. Saecker ◽  
James Chen ◽  
Courtney E. Chiu ◽  
Brandon Malone ◽  
Johanna Sotiris ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
De Novo ◽  
Promoter Strength ◽  
Transcription Bubble ◽  
Template Strand ◽  
Solution Studies ◽  
Active Site Cleft ◽  
Promoter Dna

The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA–RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand “scrunches” inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

scholarly journals Structural origins of Escherichia coli RNA polymerase open promoter complex stability

2021 ◽  
Author(s):  
Ruth M. Saecker ◽  
James Chen ◽  
Courtney E. Chiu ◽  
Brandon Malone ◽  
Johanna Sotiris ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
De Novo ◽  
Promoter Strength ◽  
Transcription Bubble ◽  
Template Strand ◽  
Solution Studies ◽  
Active Site Cleft ◽  
Promoter Dna

AbstractThe first step of gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo but their structural origins remain largely unknown. Here we use cryo-electron microscopy to determine structures of RPo formed de novo at three promoters with widely differing lifetimes at 37°C: λPR (t1/2 ∼ 10 hours), T7A1 (t1/2 ∼ 4 minutes), and a point mutant in λPR (λPR-5C) (t1/2 ∼ 2 hours). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate-strand “scrunches” inside the active site cleft; the template-strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate RPo lifetime and affect the subsequent steps of the transcription cycle.

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