Hae Ja Shin

A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida

A new variant type of regulatory activator and relevant promoters (designated capR, Pr and Po) involved in the metabolism of phenolic compounds were cloned from Pseudomonas putida KCTC1452 by using PCR. The deduced amino acid sequence of CapR revealed a difference in nine amino acids from the effector binding domain of DmpR. To measure effector specificity, plasmids were constructed in such a way that the expression of luc gene for firefly luciferase or lacZ for beta-galactosidase as a reporter was under the control of capR. When Escherichia coli transformed with the plasmids was exposed to phenol, dramatic increases in the activity of luciferase or beta-galactosidase were observed in a range of 0.01-1 mM. Among various phenolic compounds tested, other effective compounds included catechol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2-chlorophenol, 4-chlorophenol, 2-nitrophenol, resorcinol, and 2, 5-dimethylphenol. The results indicate that CapR has effector specificity different from other related activators, CatR and DmpR. Waste water and soil potentially containing phenolic compounds were also tested by this system and the results were compared with chemical and GC data. The present results indicate that the biosensor consisting of capR and the promoters may be utilized for the development of a phenolic compounds-specific biosensor in monitoring the environmental pollutant.

Development of highly-sensitive microbial biosensors by mutation of the nahR regulatory gene.

NahR, a transcriptional regulator for naphthalene degradation in response to salicylate, is a central element in the microbial biosensor for detection of naphthalene and salicylate. To maximize the sensitivity of the biosensor, we have chosen a rational design of highly-sensitive microbial biosensors by introducing site directed mutagenesis to nahR gene. Eight single mutants (N169A, N169C, N169K, N169S, R248H, R248M, R248Q, and R248Y) were made at residues 169 and 248 known as the central inducer-recognition and the C-terminal multimerization domain. The effects of these mutations were examined by monitoring expression of a firefly luciferase (luc) reporter gene under the control of NahR. We found that all mutants at residues 248 and N169C show increased sensitivity (maximum ∼50-fold) compared to wild type, respectively. R248M shows response even at toxic concentration, 5mM. The results show the feasibility and potential versatility of mutational approach for the development of the highly-sensitive microbial biosensors.

A RECOMBINANT Escherichia coli BIOSENSOR FOR DETECTING POLYCYCLIC AROMATIC HYDROCARBONS IN GAS AND AQUEOUS PHASES

Recombinant microbial biosensors are known to be simple, cheap, and very efficient monitoring tools for detecting various environmental pollutants in the field. However, although various recombinant microbial biosensors have been developed for aqueous-phase samples, very few are applicable to the gas phase. Here, we report a recombinant Escherichia coli biosensor that can be used to monitor polycyclic aromatic hydrocarbons (PAHs) in both gas and aqueous phases by color development. Among the PAHs, naphthalene and salicylate are often used as model compounds, since they are less toxic than other options and they are widely used in various applications. Here, recombinant E. coli cells carrying nahR (encoding the NahR regulatory protein for naphthalene degradation)::lac Z fusion genes were constructed and suspended (for aqueous measurements) or co-immobilized (for gaseous measurements) with chlorophenol red-ß-D-galactopyranoside (CPRG). Biosensing was then performed by ß-galactosidase, which hydrolyzed CPRG as a substrate, developing detectable red color with the naked eye. The system showed selective responses to salicylate and naphthalene. Importantly, its response to naphthalene was much more sensitive (about 105-fold) in the gas phase compared to the aqueous phase. Thus, this system could potentially be used for the instrument-free, color-change-based monitoring of gaseous pollutants.

Fluorescence and folding properties of Tyr mutant tryptophan synthase α-subunits from Escherichia coli

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.

Comparative evaluation of an electrochemical bioreporter for detecting phenolic compounds

ABSTRACT In this study, we constructed an Escherichia coli-based electrochemical bioreporter (EB) harboring pLZCapR, which encodes the CapR regulatory protein (for phenol degradation) along with β-galactosidase, and examined its ability to detect phenolic compounds as compared with previously reported optical bioreporters (OBs) controlled by CapR and detected using a luminometer (OB-lum) or spectrophotometer (OB-spec). The recombinant E. coli bioreporter cells were immobilized in polyvinyl alcohol (PVA); p-aminophenyl-β-D-galactopyranoside (PAPG) was used as the enzymatic substrate; and electrochemical measurements were taken. The peak current obtained on cyclic voltammetry (CV) was used to measure the redox response of PAPG degradation. Our results revealed that the EB system showed a detection range of 10 nM to 10 mM phenol with a good lower detection limit (30 nM phenol). Furthermore, the detection time was dramatically lower for the EB system (15–20 min) compared to the OBs (∼6 hr). These responses were reliably repeatable with an acceptable standard deviation (±2.7%; n = 6), and the system showed good stability without loss of activity over 7 hr of operation or following 2 weeks of cold storage. Together, these results show that the EB system is faster and has a lower detection limit than the existing optical techniques.

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda

Abstract In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ∼38 kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01–5 μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2 × 104 −2 × 109 CFU/ml, and exhibited a lower detection limit of 2 × 104 CFU/ml.