Improved <i>gfp</i> and <i>inaZ</i> Broad-Host-Range Promoter-Probe Vectors

Miller, William G.; Leveau, Johan H. J.; Lindow, Steven E.

doi:10.1094/mpmi.2000.13.11.1243

Cited by 505 publications

(381 citation statements)

References 29 publications

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“…Expression of C-terminal histidine-tagged B3023 protein under the arabinose-inducible araBAD promoter (P BAD ) from this plasmid was induced by 0.5% L-arabinose (Acros Organics, Morris Plains, NJ, USA). The promoter-probe vector pPROBE-gfp[tagless] (Miller et al, 2000) was used to construct pPyncC-gfp, which carries the yncC promoter-gfp fusion.…”

Section: Methodsmentioning

confidence: 99%

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Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

2008

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Quorum-sensing signal autoinducer 2 (AI-2) stimulates Escherichia coli biofilm formation through the motility regulator MqsR that induces expression of the putative transcription factor encoded by yncC. Here, we show that YncC increases biofilm formation by repressing overproduction of the exopolysaccharide identified as colanic acid (corroborated by decreasing mucoidy and increased sensitivity to bacteriophage P1 infection). Differential gene expression and gel shift assays demonstrated that YncC is a repressor of the predicted periplasmic protein-encoding gene, ybiM, which was corroborated by the isogenic yncC ybiM double mutation that repressed the yncC phenotypes (biofilm formation, colanic acid overproduction, mucoidy and bacteriophage resistance). Through nickel-enrichment DNA microarrays and additional gel shift assays, we found that the putative transcription factor B3023 (directly upstream of mqsR) binds the yncC promoter. Overexpressing MqsR, AI-2 import regulators LsrR/LsrK and AI-2 exporter TqsA induced yncC transcription, whereas the AI-2 synthase LuxS and B3023 repressed yncC. MqsR has a toxic effect on E. coli bacterial growth, which is partially reduced by the b3023 mutation. Therefore, AI-2 quorum-sensing control of biofilm formation is mediated through regulator MqsR that induces expression of the transcription factor YncC. YncC inhibits the expression of periplasmic YbiM, which prevents overproduction of colanic acid (excess colanic acid causes mucoidy) and prevents YbiM from inhibiting biofilm formation.

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Section: Methodsmentioning

confidence: 99%

“…After double digestion with restriction enzymes, BamHI and SacI, the DNA fragment was cloned into the promoter-probe vector plasmid pPROBE-gfp[tagless] (Miller et al, 2000), which led to the plasmid pPyncC-gfp.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

2008

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“…The PCR product was digested with HindIII and EcoRI (restriction sites underlined) and ligated with an equally digested promoter probe vector, pProbeAT (Miller et al, 2000). The resulting construct pRG13 was confirmed by sequencing.…”

Section: Strain Constructionmentioning

confidence: 99%

“…Mobilizing strain, RP4 tra genes integrated in the chromosome, thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu Km R (Simon et al, 1983) Plasmids pminiCTX3a Suicide vector permitting site-specific integration at attB in P. aeruginosa, Tc R (Hoang et al, 2000) pProbeAT Broad-host-range vector with a promoterless gfp, Cb R (Miller et al, 2000) pRG12 1200 bp ampicillin/carbenicillin resistance gene cloned with its native promoter in pminiCTX3a…”

Section: Growth Assaysmentioning

confidence: 99%

Negative regulation of bacterial quorum sensing tunes public goods cooperation

Gupta

Schuster

2013

The ISME Journal

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Bacterial quorum sensing (QS) often coordinates the expression of other, generally more costly public goods involved in virulence and nutrient acquisition. In many Proteobacteria, the basic QS circuitry consists of a synthase that produces a diffusible acyl-homoserine lactone and a cognate receptor that activates public goods expression. In some species, the circuitry also contains negative regulators that have the potential to modulate the timing and magnitude of activation. In this study, we experimentally investigated the contribution of this regulatory function to the evolutionary stability of public goods cooperation in the opportunistic pathogen Pseudomonas aeruginosa. We compared fitness and public goods expression rates of strains lacking either qteE or qscR, each encoding a distinct negative regulator, with those of the wild-type parent and a signalblind receptor mutant under defined growth conditions. We found that (1) qteE and qscR mutations behave virtually identically and have a stronger effect on the magnitude than on the timing of expression, (2) high expression in qteE and qscR mutants imposes a metabolic burden under nutrient conditions that advance induction and (3) high expression in qteE and qscR mutants increases population growth when QS is required, but also permits invasion by both wild-type and receptor mutant strains. Our data indicate that negative regulation of QS balances the costs and benefits of public goods by attenuating expression after transition to the induced state. As the cells cannot accurately assess the amount of cooperation needed, such bet-hedging would be advantageous in changing parasitic and nonparasitic environments.

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“…The fimB and fimE were PCR-amplified and cloned into the KpnI/XbaI-digested pBAD30 vector, creating pTSH30 and pTSH32, respectively. The fim invertible region in its native phase ''off'' (IRL) orientation was PCR-amplified and cloned into the BamHI/ EcoRI-digested pPROBE-NT (Miller et al, 2000), resulting in pTSH14. The invertible region in the non-native (IRR) orientation was made by expressing FimB in the same host, and this version of invertible region was used in the construction of the expression vector.…”

Section: Construction Of the Expression Plasmidmentioning

confidence: 99%

A tightly regulated inducible expression system utilizing the fim inversion recombination switch

Ham

Lee

Keasling

et al. 2006

Biotech & Bioengineering

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Abstract:The fim inversion system of Escherichia coli (E. coli) can behave as a unidirectional switch in an efficient manner. We have developed a new expression system for E. coli, comprising the arabinose-inducible fimE gene and the fim invertible DNA segment containing a constitutively active promoter. In this system, the target gene is cloned with the promoter in the OFF orientation, resulting in no transcribed product. When induced by arabinose, the active promoter is switched to the ON orientation via FimE-catalyzed DNA inversion, and the gene is expressed. Our expression system exhibited very tightly controlled basal expression and high induced expression, with simple induction by inexpensive arabinose. These characteristics make our system suitable for large-scale expression or for production of toxic proteins.

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Improved gfp and inaZ Broad-Host-Range Promoter-Probe Vectors

Cited by 505 publications

References 29 publications

Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

Negative regulation of bacterial quorum sensing tunes public goods cooperation

A tightly regulated inducible expression system utilizing the fim inversion recombination switch

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