scholarly journals Plasticity of the Pjunc Promoter of ISEc11, a New Insertion Sequence of the IS1111 Family

2006 ◽  
Vol 188 (13) ◽  
pp. 4681-4689 ◽  
Author(s):  
Gianni Prosseda ◽  
Maria Carmela Latella ◽  
Mariassunta Casalino ◽  
Mauro Nicoletti ◽  
Stefano Michienzi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Insertion Sequence ◽  
Functional Characterization ◽  
Virulence Plasmid ◽  
E Coli ◽  
Starting Point ◽  
Natural Variant ◽  
The Right

ABSTRACT We describe identification and functional characterization of ISEc11, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEc11 is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEc11 is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the IS1111 family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter (PjuncC) containing a −10 box in the interstitial sequence and a −35 box upstream of the right IR. A natural variant of ISEc11 (ISEc11p), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEc11p forms a junction promoter (PjuncP) which, despite carrying −10 and −35 boxes identical to those of PjuncC, exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of PjuncP is 20-fold higher than the activity of PjuncC. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Substitutions in Bacteriophage T4 AsiA andEscherichia coli ς70 That Suppress T4motA Activation Mutations

2001 ◽  
Vol 183 (7) ◽  
pp. 2289-2297 ◽  
Author(s):  
Marco P. Cicero ◽  
Meghan M. Sharp ◽  
Carol A. Gross ◽  
Kenneth N. Kreuzer
Keyword(s):  
Rna Polymerase ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
In Vitro Transcription ◽  
Positive Control ◽  
Plaque Morphology ◽  
Mutant Proteins ◽  
E Coli

ABSTRACT Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς70 subunit. AmotA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant ς70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. colipromoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

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 Inhibition of Transcription in Staphylococcus aureus by a Primary Sigma Factor-Binding Polypeptide from Phage G1

2009 ◽  
Vol 191 (12) ◽  
pp. 3763-3771 ◽  
Author(s):  
Mohammed Dehbi ◽  
Gregory Moeck ◽  
Francis F. Arhin ◽  
Pascale Bauda ◽  
Dominique Bergeron ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Growth Inhibitory ◽  
Σ Factor ◽  
Transcription In Vitro

ABSTRACT The primary sigma factor of Staphylococcus aureus, σSA, regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with σSA both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of σSA that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-σ factor may occlude the −35 element recognition domain of σSA. As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of σSA to DNA and σSA-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

scholarly journals Transcription of the Rhodobacter sphaeroides cycA P1 Promoter by Alternate RNA Polymerase Holoenzymes

1998 ◽  
Vol 180 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Barbara J. MacGregor ◽  
Russell K. Karls ◽  
Timothy J. Donohue
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rhodobacter Sphaeroides ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Initiation Site ◽  
E Coli ◽  
Rna Polymerase Holoenzyme

ABSTRACT These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroidescytochrome c 2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterizedEscherichia coli heat shock (ς32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Eς32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of −7. A point mutation at position −34 that is towards the E. coliEς32 −35 consensus sequence (G34T) increasedcycA P1 activity ∼20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed −10 or −35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes,cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Eς37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Eς38) (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10–19, 1998).

scholarly journals Rhodobacter capsulatus nifA1 Promoter: High-GC −10 Regions in High-GC Bacteria and the Basis for Their Transcription

2004 ◽  
Vol 186 (3) ◽  
pp. 740-749 ◽  
Author(s):  
Cynthia L. Richard ◽  
Animesh Tandon ◽  
Robert G. Kranz
Keyword(s):  
Rna Polymerase ◽  
Rhodobacter Capsulatus ◽  
Transcription Start Sites ◽  
E Coli ◽  
Protein Protein Interaction ◽  
Enhancer Binding Protein ◽  
Pribnow Box ◽  
Rna Polymerase Subunits

ABSTRACT It was previously shown that the Rhodobacter capsulatus NtrC enhancer-binding protein activates the R. capsulatus housekeeping RNA polymerase but not the Escherichia coli RNA polymerase at the nifA1 promoter. We have tested the hypothesis that this activity is due to the high G+C content of the −10 sequence. A comparative analysis of R. capsulatus and other α-proteobacterial promoters with known transcription start sites suggests that the G+C content of the −10 region is higher than that for E. coli. Both in vivo and in vitro results obtained with nifA1 promoters with −10 and/or −35 variations are reported here. A major conclusion of this study is that α-proteobacteria have evolved a promiscuous sigma factor and core RNA polymerase that can transcribe promoters with high-GC −10 regions in addition to the classic E. coli Pribnow box. To facilitate studies of R. capsulatus transcription, we cloned and overexpressed all of the RNA polymerase subunits in E. coli, and these were reconstituted in vitro to form an active, recombinant R. capsulatus RNA polymerase with properties mimicking those of the natural polymerase. Thus, no additional factors from R. capsulatus are necessary for the recognition of high-GC promoters or for activation by R. capsulatus NtrC. The addition of R. capsulatus σ70 to the E. coli core RNA polymerase or the use of −10 promoter mutants did not facilitate R. capsulatus NtrC activation of the nifA1 promoter by the E. coli RNA polymerase. Thus, an additional barrier to activation by R. capsulatus NtrC exists, probably a lack of the proper R. capsulatus NtrC-E. coli RNA polymerase (protein-protein) interaction(s).

scholarly journals Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms

2021 ◽  
Vol 22 (2) ◽  
pp. 681
Author(s):  
Vaishnavi Amarr Reddy ◽  
Chunhong Li ◽  
Kumar Nadimuthu ◽  
Jessica Gambino Tjhang ◽  
In-Cheol Jang ◽  
...  
Keyword(s):  
Glandular Trichomes ◽  
Functional Characterization ◽  
Rna Seq ◽  
Post Translational Modifications ◽  
E Coli ◽  
Sweet Basil ◽  
Specific Regulation

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

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.

scholarly journals Abstract P-23: Histone N-terminal Tails Reduce Early Nucleosomal Pausing during Transcription

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S21-S21
Author(s):  
Nadezhda Gerasimova ◽  
Fu-Kai Hsieh ◽  
Vasily Studitsky
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Proximal Part ◽  
Histone Tails ◽  
E Coli ◽  
In Vivo Experiments ◽  
Nucleosomal Dna ◽  
Transcript Elongation

Background: Nucleosomes are the barriers to transcript elongation by RNA polymerase 2 (Pol 2) in vitro and in vivo. Formation and overcoming the barrier are important for transcription regulation. N-terminal tails of core histones do not affect the inner structure of nucleosomal core. However, strongly positively charged tails can interact with the DNA, thereby impeding polymerase progression through the template. Removal of histone tails was shown to facilitate transcription through a nucleosome by both yeast and human Pol 2, and the effect was most noticeable at lower ionic strength (40 mM KCl). In vivo experiments established a new mechanism of overcoming of +1 nucleosomal barrier by removal of histone tails by specific regulative proteinase. As +1 nucleosomal barrier is formed mostly by the promoter-proximal part of the nucleosomal DNA, here we address the effects of histone tails on elongation through this part of the nucleosome. Methods: We have studied the effect of histone tails on transcription by yeast Pol 2 and model enzyme E. coli RNA polymerase utilizing very similar mechanisms of elongation through chromatin. 603 nucleosomes were transcribed in vitro using purified proteins and components. To focus on the proximal part of the nucleosome, transcript elongation was conducted for a limited time and at low ionic strength. Results: For the phosphorylated form of yeast Pol 2 and E. coli RNAP, histone tail removal significantly reduces the strong nucleosome-specific pausing that the yeast polymerase encounters ∼15 bp within the 603 nucleosome and further downstream, leading to both increased traversal of the pause and the accumulation of complexes paused at more distal locations. However, tail removal did not lead to a significant increase in full traversal of either nucleosomal template. The effect of histone tails removal was cognate for both enzymes but differs in detailed effect on the barrier. Conclusion: Histone tails provide a significant part of the nucleosomal barrier to transcript elongation by Pol 2-type mechanism. The effect is very pronounced in the promoter-proximal part of the nucleosomal DNA, suggesting that histone tails could play a role during the regulation of the +1 nucleosomal barrier. The role of Pol 2 CTD phosphorylation and formation of the intranucleosomal loops in the regulation of +1 nucleosomal barrier will also be addressed.

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