Recombineering: a homologous recombination-based method of genetic engineering

Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

doi:10.1038/nprot.2008.227

Cited by 703 publications

(631 citation statements)

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“…Deletions, single-and multiple-point mutations, and amber codon (UAG) substitutions were introduced at various positions within the dksA, rpoB, and rpoC genes using the QuikChange site-directed mutagenesis kit (Agilent). Chromosomal mutants in rpoB and dksA were generated by oligomediated recombineering using the λ-red system and standard protocols (31). Colony size estimations were made on arrayed colonies on agar plates using the same methodology as previously described for large-scale chemical-genomic screens (32).…”

Section: Methodsmentioning

confidence: 99%

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo-cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.transcription regulation | stringent response | protein cross-linking | molecular modeling | lineage-specific insertions S ensing and responding to nutritional status is one of the major challenges of microbial life. In Escherichia coli, the global regulatory response to amino acid starvation is orchestrated by the second messenger guanosine-3′,5′-bisdiphosphate (ppGpp), which is a widely conserved master regulator (1). Accumulation of ppGpp during amino acid scarcity triggers the stringent response, which down-regulates expression of rRNA and tRNA while increasing expression of amino acid biosynthetic enzymes. In E. coli, ppGpp works synergistically with the transcription factor DksA to initiate the stringent response (2, 3). Both ppGpp and DksA are critical for survival of stress and virulence in many pathogenic proteobacteria (4).DksA is a relatively small protein with a prominent N-terminal coiled-coil domain and a globular C-terminal domain consisting of a Zn 2+ -binding region and a C-terminal α-helix (3). It belongs to a class of regulators that bind directly to RNA polymerase (RNAP) without contacting DNA (5). DksA modulates RNAP activity by preventing formation of or destabilizing the intermediate complex (RPi) on the pathway to the open complex (RPo), which is competent for initiation. For promoters with intrinsically unstable open complexes, such as rRNA promoters, DksA binding leads to decreased transcription (2).DksA is a critical determinant of the stringent response and a model system for an important class of transcription regulators, making it essential to understand how DksA interacts with RNAP at the molecular level. High-resolution structural information of the DksA/RNAP interaction is currently unavailable. Current models agree that the coiled-coil domain of DksA inserts into the secondary channel of RNAP, the channel used by NTPs to access the active site; that the secondary channel rim helices of β' subunit are critical for DksA binding; and that residues at the tip of the coiled-coil of DksA are important for its activity. However, the precise placement of DksA is unknown. With the number of critical features that can be accessed through the secondary channel, even small changes in the model can ...

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Section: Methodsmentioning

confidence: 99%

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…A DNA fragment containing the complete ccd operon from pathogenic E. coli O157:H7 was synthesized and inserted into the 77-bp intergenic region between the folA and apaH genes of E. coli strain BW25113 using lambda red recombination (22). We chose the Escherichia coli BW25113 strain because it is the parent strain of the Keio collection with a known genotype [F Ϫ Δ(araD-araB)567 lacZ4787(Δ)::rrnB3 LAM Ϫ rph1 Δ(rhaD-rhaB)568 hsdR514] and the complete genome is sequenced (23).…”

Section: Resultsmentioning

confidence: 99%

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

et al. 2017

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One of the first identified and best-studied toxin-antitoxin (TA) systems in Escherichia coli is the F-plasmid-based CcdAB system. This system is involved in plasmid maintenance through postsegregational killing. More recently, ccdAB homologs have been found on the chromosome, including in pathogenic strains of E. coli and other bacteria. However, the functional role of chromosomal ccdAB genes, if any, has remained unclear. We show that both the native ccd operon of the E. coli O157 strain (ccd O157 ) and the ccd operon from the F plasmid (ccd F ), when inserted on the E. coli chromosome, lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters, with the O157 operon showing higher protection. While the plasmid-encoded CcdB toxin is a potent gyrase inhibitor and leads to bacterial cell death even under fully repressed conditions, the chromosomally encoded toxin leads to growth inhibition, except at high expression levels, where some cell death is seen. This was further confirmed by transiently activating the chromosomal ccd operon through overexpression of an active-site inactive mutant of F-plasmid-encoded CcdB. Both the ccd F and ccd O157 operons may share common mechanisms for activation under stress conditions, eventually leading to multidrug-tolerant persister cells. This study clearly demonstrates an important role for chromosomal ccd systems in bacterial persistence.IMPORTANCE A large number of free-living and pathogenic bacteria are known to harbor multiple toxin-antitoxin systems, on plasmids as well as on chromosomes. The F-plasmid CcdAB system has been extensively studied and is known to be involved in plasmid maintenance. However, little is known about the function of its chromosomal counterpart, found in several pathogenic E. coli strains. We show that the native chromosomal ccd operon of the E. coli O157 strain is involved in drug tolerance and confers protection from cell death under multiple antibiotic stress conditions. This has implications for generation of potential therapeutics that target these TA systems and has clinical significance because the presence of persisters in an antibiotic-treated population can lead to resuscitation of chronic infection and may contribute to failure of antibiotic treatment.KEYWORDS toxin-antitoxin, persistence I n the early 1940s it was reported that a small fraction of staphylococcal cells, about one in a million, survived prolonged penicillin treatment (1). These cells were termed persisters. Later it was established that persisters are phenotypic variants in a given population that show antibiotic tolerance due to probable differences in their physiology under a given set of conditions. Upon subsequent culturing, they give rise to a population that retains antibiotic sensitivity. They are different from resistant cells in which resistance is genetically encoded and is passed on from one generation to the next. In contrast, persisters are transient and rare, ranging from 10 Ϫ2 to 10 Ϫ6 in frequenc...

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“…All strains used are derivatives of MG1655 or DJ480 (MG1655 ΔlacX74) (33); strains were made either by P1 transduction, selecting for the appropriate antibiotic resistance marker, or by lambda Red recombineering (34) as outlined in Table S2 and SI Materials and Methods. Primers are listed in Table S3.…”

Section: Methodsmentioning

confidence: 99%

Stress sigma factor RpoS degradation and translation are sensitive to the state of central metabolism

Battesti

Majdalani

Gottesman

2015

Proc. Natl. Acad. Sci. U.S.A.

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RpoS, the stationary phase/stress sigma factor of Escherichia coli, regulates a large cohort of genes important for the cell to deal with suboptimal conditions. Its level increases quickly in the cell in response to many stresses and returns to low levels when growth resumes. Increased RpoS results from increased translation and decreased RpoS degradation. Translation is positively regulated by small RNAs (sRNAs). Protein stability is positively regulated by anti-adaptors, which prevent the RssB adaptor-mediated degradation of RpoS by the ClpXP protease. Inactivation of aceE, a subunit of pyruvate dehydrogenase (PDH), was found to increase levels of RpoS by affecting both translation and protein degradation. The stabilization of RpoS in aceE mutants is dependent on increased transcription and translation of IraP and IraD, two known antiadaptors. The aceE mutation also leads to a significant increase in rpoS translation. The sRNAs known to positively regulate RpoS are not responsible for the increased translation; sequences around the start codon are sufficient for the induction of translation. PDH synthesizes acetyl-CoA; acetate supplementation allows the cell to synthesize acetyl-CoA by an alternative, less favored pathway, in part dependent upon RpoS. Acetate addition suppressed the effects of the aceE mutant on induction of the antiadaptors, RpoS stabilization, and rpoS translation. Thus, the bacterial cell responds to lowered levels of acetyl-CoA by inducing RpoS, allowing reprogramming of E. coli metabolism.RssB | ClpXP | acetyl CoA | RpoS | pyruvate dehydrogenase

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Recombineering: a homologous recombination-based method of genetic engineering

Cited by 703 publications

References 50 publications

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

Stress sigma factor RpoS degradation and translation are sensitive to the state of central metabolism

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