Abstract
The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia call strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40°C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30°C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 μM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.
| Original language | English |
|---|---|
| Pages (from-to) | 183-197 |
| Number of pages | 15 |
| Journal | Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology |
| Volume | 120 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2005 Mar 1 |
| Externally published | Yes |
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Keywords
- Bacterial biosensor
- Bioluminescence
- NahR
- Naphthalene
- Salicylic acid
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biotechnology
- Bioengineering
Cite this
Construction and evaluation of nagR-nagAa : :lux fusion strains in biosensing for salicylic acid derivatives. / Mitchell, Robert J.; Gu, Man Bock.
In: Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology, Vol. 120, No. 3, 01.03.2005, p. 183-197.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Construction and evaluation of nagR-nagAa
T2 - :lux fusion strains in biosensing for salicylic acid derivatives
AU - Mitchell, Robert J.
AU - Gu, Man Bock
PY - 2005/3/1
Y1 - 2005/3/1
N2 - The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia call strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40°C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30°C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 μM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.
AB - The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia call strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40°C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30°C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 μM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.
KW - Bacterial biosensor
KW - Bioluminescence
KW - NahR
KW - Naphthalene
KW - Salicylic acid
UR - http://www.scopus.com/inward/record.url?scp=19944390242&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=19944390242&partnerID=8YFLogxK
U2 - 10.1385/ABAB:120:3:183
DO - 10.1385/ABAB:120:3:183
M3 - Article
C2 - 15767693
AN - SCOPUS:19944390242
VL - 120
SP - 183
EP - 197
JO - Applied Biochemistry and Biotechnology
JF - Applied Biochemistry and Biotechnology
SN - 0273-2289
IS - 3
ER -