The FecI Extracytoplasmic-Function Sigma Factor of Escherichia coli Interacts with the β′ Subunit of RNA Polymerase

ABSTRACT Transcription of the ferric citrate transport system of Escherichia coli K-12 is mediated by the extracytoplasmic-function (ECF) sigma factor FecI, which is activated by ferric citrate in the growth medium. By using a bacterial two-hybrid system, it was shown in vivo that FecI binds to the β′ subunit of RNA polymerase. The inactive mutant protein FecI(K155E) displayed reduced binding to β′, and small deletions along the entire FecI protein led to total impairment of β′ binding. In vitro, FecI was retained on Ni2+-nitrilotriacetic acid agarose loaded with a His-tagged β′1-313 fragment and coeluted with β′1-313. Binding of FecI to β′ and β′1-313 was enhanced by FecR1-85, which represents the cytoplasmic portion of the FecR protein that transmits the inducing signal across the cytoplasmic membrane. Interaction of FecR with FecI was demonstrated by showing that isolated FecR inhibited degradation of FecI by trypsin. This is the first demonstration of binding of an ECF sigma factor of the FecI type to the β′ subunit of RNA polymerase and of binding being enhanced by the protein that activates the ECF sigma factor.

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Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

Journal of Bacteriology ◽

10.1128/jb.01197-07 ◽

2007 ◽

Vol 189 (23) ◽

pp. 8430-8436 ◽

Cited By ~ 2

Author(s):

Olga V. Kourennaia ◽

Pieter L. deHaseth

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Rna Polymerase ◽

Sigma Factor ◽

Stable Complex ◽

Tryptophan Residue ◽

Specific Sequence ◽

Bacterial Rna

ABSTRACT The heat shock sigma factor (σ32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, σ70. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ32, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

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Interaction of sigma factor σN with Escherichia coli RNA polymerase core enzyme

Biochemical Journal ◽

10.1042/bj3520539 ◽

2000 ◽

Vol 352 (2) ◽

pp. 539-547 ◽

Cited By ~ 6

Author(s):

David J. SCOTT ◽

Anna L. FERGUSON ◽

María-Trinidad GALLEGOS ◽

Melinda PITT ◽

Martin BUCK ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Sigma Factor ◽

Analytical Ultracentrifugation ◽

Spectroscopic Properties ◽

Sedimentation Equilibrium ◽

Equilibrium Binding ◽

Kinetics Of

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) σN-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)σN. The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWσN retained its biological activity in stimulating transcription from σN-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp → Ala single mutants of σN were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the σN-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWσN to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315nm (which reports selectively on the distribution of free and bound 7AWσN) established that a 1:1 complex was formed, with a dissociation constant lower than 2µM. The kinetics of the interaction between 7AWσN and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280nm, whereas only the slower of the two phases was observed at 315nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of σN with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between σN and the major sigma factor, σ70, in Escherichia coli are discussed.

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Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-subunit of RNA polymerase

Journal of Bacteriology ◽

10.1128/jb.00252-21 ◽

2021 ◽

Author(s):

Helen Camakaris ◽

Ji Yang ◽

Tadashi Fujii ◽

James Pittard

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Regulatory Protein ◽

Aromatic Amino Acids ◽

Regulation Of Transcription ◽

Plant Interactions ◽

Α Subunit ◽

Partial Suppression ◽

K 12

A novel selection was developed for RpoA α-CTD mutants altered in activation by the TyrR regulatory protein of E. coli K-12. This allowed the identification of an aspartate to asparagine substitution in residue 250 (DN250) as an Act - mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and substitutions DR250, RE310 and RD310 were all shown to be defective in activation. None of these mutations caused defects in UP regulation. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. TyrR Mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77 and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act - tyrR mutants were isolated in an rpoA + strain, adding positions R77, D97, K101, D118, R119, R121 and E141 to known residues, S95 and D103, and defining the ‘activation patch’ on the NTD of TyrR. These results support a model for activation of TyrR-regulated genes where the ‘activation patch’ on the TyrR NTD interacts with the ‘TyrR-specific patch’ on the αCTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed, and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants, as Activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene TyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence, and important plant interactions.

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Synthesis and maturation of lambda receptor in Escherichia coli K-12: in vivo and in vitro expression of gene lamB under lac promoter control.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.77.3.1491 ◽

1980 ◽

Vol 77 (3) ◽

pp. 1491-1495 ◽

Cited By ~ 16

Author(s):

C. Marchal ◽

D. Perrin ◽

J. Hedgpeth ◽

M. Hofnung

Keyword(s):

Escherichia Coli ◽

In Vitro Expression ◽

K 12 ◽

Lac Promoter

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Identification of Escherichia coli Genes That Are Specifically Expressed in a Murine Model of Septicemic Infection

Infection and Immunity ◽

10.1128/iai.70.7.3404-3412.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3404-3412 ◽

Cited By ~ 26

Author(s):

Muhammad A. Khan ◽

Richard E. Isaacson

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Anaerobic Respiration ◽

Disease Process ◽

Antimicrobial Drugs ◽

Reverse Transcription Pcr ◽

Successful Infection ◽

K 12

ABSTRACT Identification and characterization of bacterial genes that are induced during the disease process are important in understanding the molecular mechanism of disease and can be useful in designing antimicrobial drugs to control the disease. The identification of in vivo induced (ivi) genes of an Escherichia coli septicemia strain by using antibiotic-based in vivo expression technology is described. Bacterial clones resistant to chloramphenicol in vivo were recovered from the livers of infected mice. Most of the ivi clones were sensitive to chloramphenicol when grown in vitro. Using reverse transcription-PCR, it was demonstrated that selected ivi clones expressed cat in the livers of infected mice but not during in vitro growth. A total of 750 colonies were recovered after three successive rounds of in vivo selection, and 168 isolated ivi clones were sequenced. The sequence analysis revealed that 37 clones encoded hypothetical proteins found in E. coli K-12, whereas 10 clones contained genes that had no significant homology to DNA sequences in GenBank. Two clones were found to contain transposon-related functions. Other clones contained genes required for amino acid metabolism, anaerobic respiration, DNA repair, the heat shock response, and the cellular repressor of the SOS response. In addition, one clone contained the aerobactin biosynthesis gene iucA. Mutations were introduced in to seven of the identified ivi genes. An in vivo mouse challenge-competition assay was used to determine if the mutants were attenuated. The results suggested that these ivi genes were important for survival in vivo, and three of the seven mutant ivi clones were required for successful infection of mice.

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Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12

Scientific Reports ◽

10.1038/s41598-019-56886-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Tomohiro Shimada ◽

Yui Yokoyama ◽

Takumi Anzai ◽

Kaneyoshi Yamamoto ◽

Akira Ishihama

Keyword(s):

Escherichia Coli ◽

Genetic System ◽

Regulatory Role ◽

E Coli ◽

Warm Blooded Animal ◽

Plant Utilization ◽

K 12

AbstractOutside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood. Here we describe the set of regulatory targets for uncharacterized IclR-family transcription factor YiaJ on the E. coli genome, using gSELEX screening system. Among a total of 18 high-affinity binding targets of YiaJ, the major regulatory target was identified to be the yiaLMNOPQRS operon for utilization of ascorbate from fruits and galacturonate from plant pectin. The targets of YiaJ also include the genes involved in the utilization for other plant-derived materials as nutrients such as fructose, sorbitol, glycerol and fructoselysine. Detailed in vitro and in vivo analyses suggest that L-ascorbate and α-D-galacturonate are the effector ligands for regulation of YiaJ function. These findings altogether indicate that YiaJ plays a major regulatory role in expression of a set of the genes for the utilization of plant-derived materials as nutrients for survival. PlaR was also suggested to play protecting roles of E. coli under stressful environments in nature, including the formation of biofilm. We then propose renaming YiaJ to PlaR (regulator of plant utilization).

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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Adaptation in a Mouse Colony Monoassociated with Escherichia coli K-12 for More than 1,000 Days

Applied and Environmental Microbiology ◽

10.1128/aem.00358-10 ◽

2010 ◽

Vol 76 (14) ◽

pp. 4655-4663 ◽

Cited By ~ 13

Author(s):

Sean M. Lee ◽

Aaron Wyse ◽

Aaron Lesher ◽

Mary Lou Everett ◽

Linda Lou ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Rates ◽

Bacterial Species ◽

Intestinal Flora ◽

Average Density ◽

Host Bacterium ◽

E Coli ◽

K 12

ABSTRACT Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

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