Construction and evaluation of nagR-nagAa: :lux fusion strains in biosensing for salicylic acid derivatives

Robert J. Mitchell, Man Bock Gu

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 languageEnglish
Pages (from-to)183-197
Number of pages15
JournalApplied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology
Volume120
Issue number3
DOIs
Publication statusPublished - 2005 Mar 1
Externally publishedYes

Fingerprint

Salicylic acid
Salicylic Acid
Fusion reactions
Derivatives
Operon
Genes
Proteins
Benzoic Acid
Benzoic acid
Escherichia coli
Temperature
Ralstonia
Photorhabdus
Escherichia
Pseudomonas putida
Aptitude
Reaction Time
Limit of Detection
Bacteria
Plasmids

Keywords

  • Bacterial biosensor
  • Bioluminescence
  • NahR
  • Naphthalene
  • Salicylic acid

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Bioengineering

Cite this

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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.",
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KW - Bacterial biosensor

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