Reporter Genes lucFF, luxCDABE, gfp, and dsred Have Different Characteristics in Whole-Cell Bacterial Sensors

Hakkila, Kaisa; Maksimow, Mikael; Karp, Matti; Virta, Marko

doi:10.1006/abio.2001.5517

Cited by 181 publications

(100 citation statements)

References 24 publications

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“…Luciferase is comprised of LuxAB, which oxidizes cellular substrates to generate visible light of approximately 490 nm. LuxCDE comprises a reductase complex that generates a reduced substrate for LuxAB (71,72). Expression of the five Lux subunits required for luciferase activity does not negatively affect C. burnetii growth (P. A. Beare and R. A. Heinzen, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

“…This luciferase system has advantages over other reporter systems in being highly sensitive and responsive, detectable in living cells without addition of exogenous substrate, and capable of generating a signal that does not stably accumulate (71,72). Luciferase is comprised of LuxAB, which oxidizes cellular substrates to generate visible light of approximately 490 nm.…”

Section: Discussionmentioning

confidence: 99%

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Essential Role for the Response Regulator PmrA in Coxiella burnetii Type 4B Secretion and Colonization of Mammalian Host Cells

Beare

Sandoz

Larson

et al. 2014

Journal of Bacteriology

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b Successful host cell colonization by the Q fever pathogen, Coxiella burnetii, requires translocation of effector proteins into the host cytosol by a Dot/Icm type 4B secretion system (T4BSS). In Legionella pneumophila, the two-component system (TCS) PmrAB regulates the Dot/Icm T4BSS and several additional physiological processes associated with pathogenesis. Because PmrA consensus regulatory elements are associated with some dot/icm and substrate genes, a similar role for PmrA in regulation of the C. burnetii T4BSS has been proposed. Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-mediated gene regulation. Compared to wild-type bacteria, C. burnetii ⌬pmrA exhibited severe intracellular growth defects that coincided with failed secretion of effector proteins. Luciferase gene reporter assays demonstrated PmrA-dependent expression of 5 of 7 dot/icm operons and 9 of 11 effector-encoding genes with a predicted upstream PmrA regulatory element. Mutational analysis verified consensus sequence nucleotides required for PmrA-directed transcription. RNA sequencing and whole bacterial cell mass spectrometry of wild-type C. burnetii and the ⌬pmrA mutant uncovered new components of the PmrA regulon, including several genes lacking PmrA motifs that encoded Dot/Icm substrates. Collectively, our results indicate that the PmrAB TCS is a critical virulence factor that regulates C. burnetii Dot/Icm secretion. The presence of PmrA-responsive genes lacking PmrA regulatory elements also suggests that the PmrAB TCS controls expression of regulatory systems associated with the production of additional C. burnetii proteins involved in host cell parasitism.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Essential Role for the Response Regulator PmrA in Coxiella burnetii Type 4B Secretion and Colonization of Mammalian Host Cells

Beare

Sandoz

Larson

et al. 2014

Journal of Bacteriology

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“…In this research, luc was employed as the reporter gene because of its high sensitivity and rapid response characteristics (Billard and DuBow 1998;Hakkila et al 2002). Experiments showed that after a 2-h induction, the biosensor produced an obvious increase of RLU at 0.05 μM As (LOD), which was below the WHO permissible level of 10 μg/L (equivalent to 0.13 μM) in potable water (Nordstrom 2002).…”

Section: Application Of Biosensor To Assess the Bioavailable As In DImentioning

confidence: 99%

The impacts of different long-term fertilization regimes on the bioavailability of arsenic in soil: integrating chemical approach with Escherichia coli arsRp::luc-based biosensor

Hou

et al. 2014

Appl Microbiol Biotechnol

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An Escherichia coli arsRp::luc-based biosensor was constructed to measure the bioavailability of arsenic (As) in soil. In previous induction experiments, it produced a linear response (R 2 =0.96, P<0.01) to As from 0.05 to 5 μmol/L after a 2-h incubation. Then, both chemical sequential extraction, Community Bureau of Reference recommended sequential extraction procedures (BCR-SEPs) and E. coli biosensor, were employed to assess the impact of different long-term fertilization regimes containing N, NP, NPK, M (manure), and NPK+ M treatments on the bioavailability of arsenic (As) in soil. Per the BCR-SEPs analysis, the application of M and M+NPK led to a significant (P<0.01) increase of exchangeable As (2-7 times and 2-5 times, respectively) and reducible As (1.5-2.5 times and 1.5-2.3 times, respectively) compared with the no fertilization treated soil (CK). In addition, direct contact assay of E. coli biosensor with soil particles also supported that bioavailable As in manure-fertilized (M and M+NPK) soil was significantly higher (P<0.01) than that in CK soil (7 and 9 times, respectively). Organic carbon may be the major factor governing the increase of bioavailable As. More significantly, E. coli biosensor-determined As was only 18.46-85.17 % of exchangeable As and 20.68-90.1 % of reducible As based on BCR-SEPs. In conclusion, NKP fertilization was recommended as a more suitable regime in As-polluted soil especially with high As concentration, and this E. coli arsRp::luc-based biosensor was a more realistic approach in assessing the bioavailability of As in soil since it would not overrate the risk of As to the environment.

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“…The bioluminescent bioreporter E. coli strains MC1061 (pmerRlux) (9), which responds to inorganic mercury, and MC1061 (pmerRBlux) (11), which responds to both inorganic and organic mercury, were used. The E. coli MC1061 (pDNlux) strain was used as a non-inducible control (11).…”

Section: Bioavailability Of Mercurymentioning

confidence: 99%

Fish Farming Affects the Abundance and Diversity of the Mercury Resistance Gene merA in Marine Sediments

et al. 2011

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Microbes in sediments below fish farming cages are exposed to a variety of compounds used in aquaculture, such as antifoulants, antimicrobials and mercury. Here, we assessed the abundance of the mercury resistance gene merA in the sediment of four fish farms and four pristine sites using quantitative PCR, and at three fish farms and two pristine sites by constructing clone libraries and sequencing merA amplicons. The abundance of merA was consistently greater at fish farm sites than at pristine sites, and the merA phylotypes were different between fish farm and pristine sites. Mercury concentrations were too low to cause selection pressure and did not differ significantly among samples. The primers designed specifically for this study were capable of detecting two new potential merA clades.

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Reporter Genes lucFF, luxCDABE, gfp, and dsred Have Different Characteristics in Whole-Cell Bacterial Sensors

Cited by 181 publications

References 24 publications

Essential Role for the Response Regulator PmrA in Coxiella burnetii Type 4B Secretion and Colonization of Mammalian Host Cells

Essential Role for the Response Regulator PmrA in Coxiella burnetii Type 4B Secretion and Colonization of Mammalian Host Cells

The impacts of different long-term fertilization regimes on the bioavailability of arsenic in soil: integrating chemical approach with Escherichia coli arsRp::luc-based biosensor

Fish Farming Affects the Abundance and Diversity of the Mercury Resistance Gene merA in Marine Sediments

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