Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors

Bereza-Malcolm, Lara; Aracic, Sanja; Kannan, Ruban; Mann, Gülay; Franks, Ashley E.

doi:10.1016/j.bios.2017.03.029

Cited by 35 publications

(24 citation statements)

References 35 publications

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“…Similarly, in silico analysis results also demonstrate that Cd is the powerful inducer for the expression of CadR and P cadR transcription. In concordance with the present study, Brocklehurst et al (2003) and Bereza-Malcolm et al (2017) constructed P cadR harbouring biosensors that were only induced by Cd. These results may be used to know exactly how the P cadR works in P. aeruginosa , in which Cd ions may have different accessibility to the CadR.…”

Section: Resultssupporting

confidence: 89%

Identification of promoter P_cadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter P_cadR from Pseudomonas aeruginosa BC15

Prabhakaran

Rajkumar²,

Ramprasath

et al. 2018

Toxicol Ind Health

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Cadmium (Cd) remediation in Pseudomonas aeruginosa is achieved through the function of two vital genes, cadA and cadR, that code for P-type ATPase (CadA) and transcription regulatory protein (CadR), respectively. Although numerous studies are available on these metal-sensing and regulatory proteins, the promoter of these genes, metal sensing and binding ability, are poorly understood. The present work is aimed at the characterization of the CadR protein, identification of the P cadR promoter and protein-promoter-metal binding affinity using bioinformatics and to validate the results by cloning the P cadR promoter in Escherichia coli DH5. The promoter regions and its curvature were identified and analysed using PePPER software (University of Groningen, The Netherland) and the Bendit program (Version: v.1.0), respectively. Using Phyre, the three-dimensional structure of CadR was modelled, and the structure was validated by Ramachandran plots. The DNA-binding domain was present in the N-terminal region of CadR. A dimeric interface was observed in helix-turn-helix and metal ion-binding sites at the C-terminal. Docking studies showed higher affinity of Cd to both CadR (Atomic contact energy ¼ À15.04 kcal/Mol) and P cadR (Atomic contact energy ¼ À40.18 kcal/Mol) when compared to other metal ions. CadR with P cadR showed the highest binding affinity (Atomic contact energy¼ À250.40 kcal/Mol) when compared with P cadA. In vitro studies using green fluorescent protein tagged with P cadR (gfp-P cadR) cloned in E. coli-expressed gfp protein in a concentrationdependent manner upon Cd exposure. Based on our in silico studies and in vitro molecular cloning analysis, we conclude that P cadR and CadR are active only in the presence of Cd. The CadR protein has the highest binding affinity with P cadR. As it became apparent that the cadR gene regulates the P cadR activity in the presence of Cd with high specificity, and the cadR and P cadR can be used as a biological tool for development of a microbial biosensor.

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Section: Resultssupporting

confidence: 89%

Identification of promoter P_cadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter P_cadR from Pseudomonas aeruginosa BC15

Prabhakaran

Rajkumar²,

Ramprasath

et al. 2018

Toxicol Ind Health

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“…They can survive at room temperature and have relatively low requirements for pH and humidity. Although relatively more robust, bacterial WCBs are still subject to environmental fluctuations since the expression of the reporter protein relies on cell growth (45,46). To solve this issue, the biosensing components have to be decoupled from cell growth.…”

Section: Discussionmentioning

confidence: 99%

Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection

Jia

Zhao

et al. 2019

Appl Environ Microbiol

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Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in Escherichia coli DH5␣. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a halfsaturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 M, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 M. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs. IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.A rsenic (As)-contaminated groundwater, occurring from mining or agriculture or natural contamination due to the abundance of arsenic in the Earth's crust, is a serious global health issue. Long-term exposure to arsenic can result in various diseases including cancers (1, 2). It is estimated that over 100 million people worldwide may be at risk from consuming water contaminated with arsenic (3). The World Health Organization (WHO) has recommended 0.01 mg/liter (0.13 M) as a safe permissible level for arsenic in drinking water (4), and the Food and Agriculture Organization (FAO) has set a maximum contamination level (MCL) for arsenic of 0.01 mg/liter in irrigation water (5).

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“…The metabolic status of the cells substantially affects the performance of bacterial bioreporters and cells that are in the exponential phase have mostly been used in cadmium biosensor assays (Riether, Dollard, and Billard 2001;Hou et al 2015;Bereza-Malcolm et al 2017). However, the optimal growth phase of particular bioreporter strain should be determined to obtain the best and reproducible performance.…”

Section: Fluorescence Response At Different Growth Phasesmentioning

confidence: 99%

Inorganic Cadmium Detection Using a Fluorescent Whole-Cell Bacterial Bioreporter

Elçin

Öktem

2020

Analytical Letters

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Cadmium pollution has become a serious environmental issue due to its toxicity and frequent entrance into environment components such as soil, water, and air via many anthropogenic sources. Over the last two decades, whole-cell bacterial bioreporters have been acknowledged as bio-sentinels for the determination of toxic heavy metals. Herein a sensitive and quite specific bacterial bioreporter was developed to cope with the need for the rapid and simple determination of cadmium. The construction and characterization of a fluorescence-based whole-cell cadmium bioreporter strain, Escherichia coli MG1655 (pBR-PzntA), was described which is based on the expression of green fluorescent protein under the control of the zntA gene promoter of heavy metal resistance determinant. The developed bioreporter was able determine cadmium at 5 mg/L after 3.5 hours of induction in a defined medium while the cadmium detection limit was improved to 2 mg/L after 1.5 hours by the use of an inorganic phosphate-limiting defined medium. Drastic changes in cadmium sensitivity were obtained between bioreporter cells induced at different growth phases. The maximum fluorescence performance was obtained for early exponential growth phase cells. This cadmium bioreporter was found to be more sensitive and specific to cadmium ions than to a wide range of heavy metals and was sensitive to only cadmium at drinking water quality standard concentrations. These findings will lead to future studies including integration of the bioreporter cells into a portable device to assess bioavailable cadmium levels in environmental samples which will provide a rapid and practical field detection technique.

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Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors

Cited by 35 publications

References 35 publications

Identification of promoter P_cadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter P_cadR from Pseudomonas aeruginosa BC15

Identification of promoter P_cadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter P_cadR from Pseudomonas aeruginosa BC15

Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection

Inorganic Cadmium Detection Using a Fluorescent Whole-Cell Bacterial Bioreporter

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Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors

Cited by 35 publications

References 35 publications

Identification of promoter PcadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter PcadR from Pseudomonas aeruginosa BC15

Identification of promoter PcadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter PcadR from Pseudomonas aeruginosa BC15

Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection

Inorganic Cadmium Detection Using a Fluorescent Whole-Cell Bacterial Bioreporter

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Product

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Identification of promoter P_cadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter P_cadR from Pseudomonas aeruginosa BC15

Identification of promoter P_cadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter P_cadR from Pseudomonas aeruginosa BC15