Construction and evaluation of nagR-nagAa::lux fusion strains in biosensing for salicylic acid derivatives.
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 coli 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 degrees C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30 degrees 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 microM. 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.
Mitchell
Robert J
RJ
National Research Laboratory on Environmental Biotechnology, Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 1 Oryong-dong, Puk-gu, Gwangju 500-712, South Korea.
Gu
Man Bock
MB
D001554:Benzene / Q000737:chemistry; D019817:Benzoic Acid / Q000737:chemistry; D015374:Biosensing Techniques; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D009281:Naphthalenes / Q000737:chemistry; D009876:Operon; D020637:Photorhabdus / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016958:Pseudomonas putida / Q000737:chemistry; D043367:Ralstonia / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012459:Salicylates / Q000737:chemistry*; D020156:Salicylic Acid / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
C503864:3-methylsalicylic acid; D009281:Naphthalenes; D011993:Recombinant Fusion Proteins; D012459:Salicylates; C031721:naphthalene; D019817:Benzoic Acid; D001554:Benzene; D020156:Salicylic Acid