Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

Katashkina, J. I.; Skorokhodova, A. Yu.; Zimenkov, Danila; Gulevich, A. Yu.; Minaeva, N. I.; Doroshenko, V. G.; Biryukova, I. V.; Mashko, Sergey V.

doi:10.1007/s11008-005-0087-8

Cited by 57 publications

(33 citation statements)

References 23 publications

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“…The plasmid pKD46 [Datsenko and Wanner, 2000], carrying arabinose-inducible Red genes, was used to provide the Red recombination. The pMW118-attL -Cm R -attR [Katashkina et al, 2005], cI ts857 DNA (Fermentas), and chromosomal DNA from E. coli MG1655 were used as templates to construct amplimers with excisable Cm R , P L -promoters and with rrnG-AT sequence, respectively. Plasmid pMW-int-xis encoding the Xis/Int recombinase was used to eliminate all DNA fragments that were flanked by attL/R sites at the final steps of the chromosomal modifications.…”

Section: Methodsmentioning

confidence: 99%

“…2 ). For chromosomal rearrangements, the excisable marker attL-Cm R -attR was used for selective DNA integration followed by curing the marker with Xis/Int recombinase, as described earlier [Katashkina et al, 2005]. Several of the strains possessed the rrnG-AT sequence instead of the native nutL sequence downstream of P L ( fig.…”

Section: Structure Of the Pykf Gene In The Chromosomesmentioning

confidence: 99%

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Conditional Silencing of the <i>Escherichia coli pykF</i> Gene Results from Artificial Convergent Transcription Protected from Rho-Dependent Termination

Krylov¹,

Airich²,

Kiseleva³

et al. 2010

Microb Physiol

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PykF is one of two pyruvate kinases in Escherichia coli K-12. λP_L was convergently integrated into the chromosome of the MG1655 strain, downstream of pykF, face-to-face with its native promoter. In the presence of λcIts857, efficient pykF ts-silencing was achieved when the 5′-terminus of the P_L-originated antisense RNA (asRNA), consisting of the rrnG-AT sequence, converted elongation complexes of RNA polymerase to a form resistant to Rho-dependent transcription termination. pykF silencing was detected by the following features: (a) impaired growth of the strain when pykA was also disrupted and when using ribose as a non-phosphotransferase system-transporting carbon source; (b) a pattern of reduced synthesis of the full-sized pykF mRNA, mediated by reverse transcription PCR, and (c) a significant decrease in PykF activity. The advantages of anti-terminated convergent transcription were clearly manifested in the strains where the rho_a-terminator was inserted specifically to interrupt asRNA synthesis. Most likely, the target gene was silenced by transcriptional interference due to collisions between converging RNA polymerases, although, strictly, the role of cis-asRNA effects could not be excluded. While details of the mechanisms have yet to be determined, anti-terminated convergent transcription is a promising new technique for silencing other target genes.

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Section: Methodsmentioning

confidence: 99%

Section: Structure Of the Pykf Gene In The Chromosomesmentioning

confidence: 99%

Conditional Silencing of the <i>Escherichia coli pykF</i> Gene Results from Artificial Convergent Transcription Protected from Rho-Dependent Termination

Krylov¹,

Airich²,

Kiseleva³

et al. 2010

Microb Physiol

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“…To construct the MG1655Δ aroE strain, an inframe deletion of the aroE gene was created using λRed-mediated integration of a DNA fragment containing the excisable marker λ attL - cat -λ attR (primers P10, P11). The recombinant plasmids pKD46 and pMW118-λ attL - cat -λ attR were used as a helper for the λRed-mediated integration and as a template for the excisable marker λ attL - cat -λ attR , respectively [19, 23]. The marker was excised using the helper plasmid pMW- int - xis [23].…”

Section: Methodsmentioning

confidence: 99%

Characterization of theCorynebacterium glutamicumdehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

Shmonova

Voloshina

Ovsienko

et al. 2020

Preprint

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2 The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum 3 encoded by the qsuB gene is related to the previously described QuiC1 protein 4 (39.9% identity) from Pseudomonas putida. QuiC1 and QsuB are both two-domain 5 bacterial DSDs. The N-terminal domain provides dehydratase activity, while the 6 C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate 7 dioxygenase. Here, the QsuB protein and its DSD domain (N-QsuB) were 8 expressed in the T7 system, purified and characterized. QsuB was present mainly 9 in octameric form (60%), while N-QsuB had a predominantly monomeric structure 10 (80%) in solution. Both proteins possessed DSD activity with one of the following 11 cofactors (listed in order of decreasing activity): Co 2+ , Mg 2+ , Mn 2+ or Ca 2+ . The K m 12 and k cat values for QsuB were two and three times higher, respectively (K m ~ 1 13 mM, k cat ~ 61 s -1 ) than those for N-QsuB. Notably, 3,4-DHBA inhibited both 14 enzymes via an uncompetitive mechanism. QsuB and N-QsuB were tested for 3,4-15 DHBA production from glucose in E. coli. MG1655aroE P lac qsuB produced at 16 least two times more 3,4-DHBA than MG1655aroE P lac n-qsuB in the presence 17 of isopropyl β-D-1-thiogalactopyranoside.18

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“…The DNA fragment containing P j10 -yddG was obtained via PCR amplification and ligation by the overlap extension of two Katashkina et al (2005) fragments containing a promoter region of gene j10 of phage T7 (amplified from DNA of pET23 plasmid) and yddG (amplified from chromosomal DNA of MG1655 strain). The DNA fragment containing P j10 -yddG was obtained via PCR amplification and ligation by the overlap extension of two Katashkina et al (2005) fragments containing a promoter region of gene j10 of phage T7 (amplified from DNA of pET23 plasmid) and yddG (amplified from chromosomal DNA of MG1655 strain).…”

Section: Genetic Techniquesmentioning

confidence: 99%

“…Construction of pMDV3-Cm r containing P u10 -yddG Plasmid construction was made in E. coli TG1. The DNA fragment containing P j10 -yddG was obtained via PCR amplification and ligation by the overlap extension of two Katashkina et al (2005) fragments containing a promoter region of gene j10 of phage T7 (amplified from DNA of pET23 plasmid) and yddG (amplified from chromosomal DNA of MG1655 strain). The obtained fragment digested with BglII and KpnI was cloned between miniMu ends onto the pMDV3-Cm r plasmid.…”

Section: Genetic Techniquesmentioning

confidence: 99%

YddG fromEscherichia colipromotes export of aromatic amino acids

Doroshenko

Airich

Vitushkina

et al. 2007

FEMS Microbiology Letters

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The inner membrane protein YddG of Escherichia coli is a homologue of the known amino acid exporters RhtA and YdeD. It was found that the yddG gene overexpression conferred resistance upon E. coli cells to the inhibiting concentrations of l-phenylalanine and aromatic amino acid analogues, dl-p-fluorophenylalanine, dl-o-fluorophenylalanine and dl-5-fluorotryptophan. In addition, yddG overexpression enhanced the production of l-phenylalanine, l-tyrosine or l-tryptophan by the respective E. coli-producing strains. On the other hand, the inactivation of yddG decreased the aromatic amino acid accumulation by these strains. The cells of the E. colil-phenylalanine-producing strain containing overexpressed yddG accumulated less l-phenylalanine inside and exported the amino acid at a higher rate than the cells of the isogenic strain containing wild-type yddG. Taken together, these results indicate that YddG functions as an aromatic amino acid exporter.

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Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

Cited by 57 publications

References 23 publications

Conditional Silencing of the <i>Escherichia coli pykF</i> Gene Results from Artificial Convergent Transcription Protected from Rho-Dependent Termination

Conditional Silencing of the <i>Escherichia coli pykF</i> Gene Results from Artificial Convergent Transcription Protected from Rho-Dependent Termination

Characterization of theCorynebacterium glutamicumdehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

YddG fromEscherichia colipromotes export of aromatic amino acids

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