Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains

Sagi, Eran; Hever, Navit; Rosen, Rachel; Bartolome, Amelita; Premkumar, J. Rajan; Ulber, Roland; Lev, Ovadia; Scheper, Thomas; Belkin, Shimshon

doi:10.1016/s0925-4005(03)00014-5

Cited by 96 publications

(51 citation statements)

References 30 publications

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“…These phenotypes were no longer dependent upon osmotic stress and were also observed during growth on regular LB agar plates (0.09 M NaCl). To better visualize this phenomenon, the cells were transformed with a plasmid containing a green fluorescent protein gene (pES2, recAЈ::gfpUV) (45). Furthermore, a ⌬yjbE rpoS::Tn10 double mutant was found to exhibit identical colony (not shown) and cellular (Fig.…”

Section: Resultsmentioning

confidence: 99%

Overproduction of Exopolysaccharides by an Escherichia coli K-12 rpoS Mutant in Response to Osmotic Stress

Ionescu

Belkin

2009

Appl Environ Microbiol

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The yjbEFGH operon is implicated in the production of an exopolysaccharide of an unknown function and is induced by osmotic stress and negatively regulated by the general stress response sigma factor RpoS. Despite the obvious importance of RpoS, negative selection for rpoS has been reported to take place in starved cultures, suggesting an adaptive occurrence allowing the overexpression of RpoD-dependent uptake and nutrientscavenging systems. The trade-off of the RpoS-dependent functions for improved nutrient utilization abilities makes the bacterium more sensitive to environmental stressors, e.g., osmotic stress. In this work, we addressed the hypothesis that overinduction of genes in rpoS-deficient strains indicates their essentiality. Using DNA microarrays, real-time PCR, and transcriptional fusions, we show that genes of the wca operon, implicated in the production of the colanic acid exopolysaccharide, previously shown to be induced by osmotic stress, are also negatively controlled by RpoS. Both exopolysaccharides in the synthesis of which yjb and wca are involved are overproduced in an rpoS mutant during osmotic stress. We also show that both operons are essential in an rpoS-deficient strain but not in the wild type; promoters of both operons are constitutively active in yjb rpoS mutants; this strain produces extremely mucoid colonies, forms long filaments, and exhibits a reduced growth capability. In addition, the wca rpoS mutant's growth is inhibited by osmotic stress. These results indicate that although induced in the wild type, both operons are much more valuable for an rpoS-deficient strain, suggesting that the overproduction of both exopolysaccharides is an adaptive action.

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Section: Resultsmentioning

confidence: 99%

Overproduction of Exopolysaccharides by an Escherichia coli K-12 rpoS Mutant in Response to Osmotic Stress

Ionescu

Belkin

2009

Appl Environ Microbiol

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“…The monitoring of immobilization stress is a complicated task; the best results have been obtained using engineered microorganisms selectively responding to particular stress causes by BLM or fluorescence (Premkumar et al 2001(Premkumar et al , 2002aSagi et al 2003;Perullini et al 2008). Changes in light emission or the production of fluorescent compounds by microorganisms might also be used as signals of unspecified stress (Kuncová et al 2004;Tessema et al 2006).…”

Section: Blmmentioning

confidence: 99%

Bioluminescence of Pseudomonas fluorescens HK44 in the course of encapsulation into silica gel. Effect of methanol

2010

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The bioluminescence (BLM) and colony-forming units (CFU) of Pseudomonas fluorescens HK44 were monitored during encapsulation into pre-polymerized Si(OMe)₄. The non-induced BLM of free cells was increased in the presence of 0.5-2.5 % MeOH. After mixing silica sol with the cell suspension, both BLM and CFU dropped to 1-3 and 8-18 %, respectively; both remained lowered as long as the silica biofilm contained residual MeOH. The kinetics of MeOH being released from silica biofilms (a thickness of 2-6 mm) were first-order. The decrease of bacterial activity due to encapsulation was proportional to the biofilm thickness. MeOH evolving during encapsulation is probably the principal stress factor but not the only one.

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“…Bioluminescence signals exhibit rapid response and lower detection thresholds. This arises from the enzymatic ef?ciency of the reporter in producing detectable signals even at low catalyst concentration (Sagi et al 2003). The ?uorescent marker possess a relatively prolonged life time in comparison to the short bioluminescent reaction making it a desired addition to the marker system (Fernandez-Suarez and Ting 2008).…”

Section: Introductionmentioning

confidence: 99%

Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

et al. 2014

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We report the use of bioluminescence signal enhancement via proximity to deposited silver nanoparticles for bioactive compound discovery. This approach employs a whole-cell bioreporter harboring a plasmid-borne fusion of a speci?c promoter incorporated with a bioluminescence reporter gene. The silver deposition process was ?rst optimized to provide optimal nanoparticle size in the reaction time dependence with ?uorescein. The use of silver deposition of 350 nm particles enabled the doubling of the bioluminescent signal amplitude by the bacterial bioreporter when compared to an untouched nonsilver-deposited microtiter plate surface. This recording is carried out in the less optimal but necessary far-?eld distance. SEM micrographs provided a visualization of the proximity of the bioreporter to the silver nanoparticles. The electromagnetic ?eld distributions around the nanoparticles were simulated using Finite Difference Time Domain, further suggesting a reexcitation of non-chemically excited bioluminescence in addition to metal-enhanced bioluminescence. The possibility of an antiseptic silver effect caused by such a close proximity was eliminated disregarded by the dynamic growth curves of the bioreporter strains as seen using viability staining. As a highly attractive biotechnology tool, this silver deposition technique, coupled with whole-cell sensing, enables increased bioluminescence sensitivity, making it especially Guest Editors: Mustafa Culha, Rawil F. Fakhrullin, Ratnesh Lal This article is part of the topical collection on Nanobiotechnology Electronic supplementary material The online version of this article (useful for cases in which reporter luminescence signals are very weak.

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Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains

Cited by 96 publications

References 30 publications

Overproduction of Exopolysaccharides by an Escherichia coli K-12 rpoS Mutant in Response to Osmotic Stress

Overproduction of Exopolysaccharides by an Escherichia coli K-12 rpoS Mutant in Response to Osmotic Stress

Bioluminescence of Pseudomonas fluorescens HK44 in the course of encapsulation into silica gel. Effect of methanol

Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

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