Agnès Rodrigue

Toward inline multiplex biodetection of metals, bacteria, and toxins in water networks: the COMBITOX project

This special issue of Environmental Science and Pollution Research highlights selected papers whose results have been obtained in the course of the COMBITOX project. COMBITIOX is an interdisciplinary research project funded by the French National Research Agency (ANR) aiming at conceiving an inline multiparametric device for the surveillance of water networks using biosensors. This device is not intended to fully replace chemical methods, but when compared to analytical chromatographic methodologies, biological sensors can offer rapid and on-site monitoring of even trace levels of targeted compounds (Sun et al. 2015) and can quickly raise the alarm in the event of an accidental or intentional pollution. Numerous developments have been published to improve the sensitivity, specificity, and time response of various biosensors in laboratory conditions (Xiong et al. 2012) (der Meer et al. 2010), but their actual transfer into technological devices for the surveillance of water networks remains at a conceptual level. Thus, the challenge here is to go a step beyond and validate biosensors under real-life field conditions by incorporating them in a single inline detector. During the course of COMBITOX, we could define the interface between the biosensors and a common light detector as well as the physical conditioning of the bioreagents and usage protocol. Our resulting prototype allow the detection of bioavailable toxic compounds as well as microorganisms, impacting human health through the drinking water network or interfering with the biological process of modern wastewater treatment plants. We also plan to propose this system to meet the emerging threats such as bioterrorism. COMBITOX focuses on three families of Bobjects^ to detect: metals (cadmium, mercury, arsenic, nickel, etc.), environmental and/or food toxins, and pathogenic microorganisms. Whole-cell biosensors based on reporter gene under the control of an inducible promoter are used to detect various metals (Hynninen and Virta 2010), the antibody/antigen interaction for toxins (Makaraviciute and Ramanaviciene 2013), and the specific infection of bacteria by bacteriophages for pathogenic microorganisms (Smartt et al. 2012) (Vinay et al. 2015). In all cases, the signal measured is photochemical (fluorescence, bioluminescence, or chemo-luminescence): such a method to transduce the biological recognition is very sensitive and a single photodetector can be used for all biosensors included in the device. The challenge here rather lies in the design and the optimization of the different biological compounds for their use in the field while maintaining a high sensibility and robustness. As a consequence, the different articles presented in this special issue focus on original strategies for the optimization and the adaptation of the three types of biosensors for their use in a semi-autonomous inline water analyzer. In the case of whole-cell biosensors, improvement of the dose-responses Responsible editor: Philippe Garrigues

RcnB Is a Periplasmic Protein Essential for Maintaining Intracellular Ni and Co Concentrations in Escherichia coli

Nickel and cobalt are both essential trace elements that are toxic when present in excess. The main resistance mechanism that bacteria use to overcome this toxicity is the efflux of these cations out of the cytoplasm. RND (resistance-nodulation-cell division)- and MFS (major facilitator superfamily)-type efflux systems are known to export either nickel or cobalt. The RcnA efflux pump, which belongs to a unique family, is responsible for the detoxification of Ni and Co in Escherichia coli. In this work, the role of the gene yohN, which is located downstream of rcnA, is investigated. yohN is cotranscribed with rcnA, and its expression is induced by Ni and Co. Surprisingly, in contrast to the effect of deleting rcnA, deletion of yohN conferred enhanced resistance to Ni and Co in E. coli, accompanied by decreased metal accumulation. We show that YohN is localized to the periplasm and does not bind Ni or Co ions directly. Physiological and genetic experiments demonstrate that YohN is not involved in Ni import. YohN is conserved among proteobacteria and belongs to a new family of proteins; consequently, yohN has been renamed rcnB. We show that the enhanced resistance of rcnB mutants to Ni and Co and their decreased Ni and Co intracellular accumulation are linked to the greater efflux of these ions in the absence of rcnB. Taken together, these results suggest that RcnB is required to maintain metal ion homeostasis, in conjunction with the efflux pump RcnA, presumably by modulating RcnA-mediated export of Ni and Co to avoid excess efflux of Ni and Co ions via an unknown novel mechanism.

Ni exposure impacts the pool of free Fe and modifies DNA supercoiling via metal-induced oxidative stress in Escherichia coli K-12

The biology of nickel has been widely studied in mammals because of its carcinogenic properties, whereas few studies have been performed in microorganisms. In the present work, changes accompanying stress caused by nickel were evaluated at the cellular level using RNA-Seq in Escherichia coli K-12. Interestingly, a very large number of genes were found to be deregulated by Ni stress. Iron and oxidative stress homeostasis maintenance were among the most highly enriched functional categories, and genes involved in periplasmic copper efflux were among the most highly upregulated. These results suggest that the deregulation of Fe and Cu homeostatic genes is caused by a release of free Cu and Fe ions in the cell which in turn activate the Cu and Fe homeostatic systems. The content of Cu was not significantly affected upon the addition of Ni to the growth medium, nor were the Cus and CopA Cu-efflux systems important for the survival of bacteria under Ni stress In contrast the addition of Ni slightly decreased the amount of cellular Fe and activated the transcription of Fur regulated genes in a Fur-dependent manner. Cu or Fe imbalance together with oxidative stress might affect the structure of DNA. Further experiments revealed that Ni alters the state of DNA folding by causing a relaxed conformation, a phenomenon that is reversible by addition of the antioxidant Tiron or the Fe chelator Dip. The Tiron-reversible DNA relaxation was also observed for Fe and to a lesser extent with Cu but not with Co. DNA supercoiling is well recognized as an integral aspect of gene regulation. Moreover our results show that Ni modifies the expression of several nucleoid-associated proteins (NAPs), important agents of DNA topology and global gene regulation. This is the first report describing the impact of metal-induced oxidative on global regulatory networks.

Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system.

The ZraSR system belongs to the family of TCSs (two-component signal transduction systems). In Escherichia coli, it was proposed to participate in zinc balance and to protect cytoplasmic zinc overload by sequestering this metal ion into the periplasm. This system controls the expression of the accessory protein ZraP that would be a periplasmic zinc scavenger. ZraPSR is functionally homologous with CpxPAR that integrates signals of envelope perturbation, including misfolded periplasmic proteins. The auxiliary periplasmic regulator CpxP inhibits the Cpx pathway by interacting with CpxA. Upon envelope stress sensing, the inhibitory function of CpxP is relieved, resulting in CpxR activation. Similarly to CpxPAR, ZraPSR probably plays a role in envelope stress response as a zinc-dependent chaperone activity was demonstrated for ZraP in Salmonella. We have purified ZraP from E. coli and shown that it is an octamer containing four interfacial metal-binding sites contributing to dimer stability. These sites are located close to the N-terminus, whereas the C-terminus is involved in polymerization of the protein to form a tetramer of dimers. In vitro, ZraP binds copper with a higher affinity than zinc and displays chaperone properties partially dependent on zinc binding. In vivo, zinc-bound ZraP is a repressor of the expression of the zraPSR operon. However, we have demonstrated that none of the Zra proteins are involved in zinc or copper resistance. We propose an integrated mechanism in which zinc is a marker of envelope stress perturbation and ZraPSR TCS is a sentinel sensing and responding to zinc entry into the periplasm.

Data set for transcriptome analysis of Escherichia coli exposed to nickel

Ni is recognized as an element that is toxic to humans, acting as an allergen and a carcinogenic agent, and it is also toxic to plants. The toxicity of Ni has been understudied in microorganisms. The data presented here were obtained by submitting the model bacterium Escherichia coli K-12 to nickel stress. To identify expressed genes, RNA-Seq was performed. Bacteria were exposed to 50 µM NiCl2 during 10 min. Exposure to Ni lead to the deregulation of 57% of the E. coli transcripts. Further analysis using DAVID identified most affected biological pathways. The list of differentially expressed genes and physiological consequences of Ni stress are described in “Ni exposure impacts the pool of free Fe and modifies DNA supercoiling via metal-induced oxidative stress in Escherichia coli K-12” (M. Gault, G. Effantin, A. Rodrigue, 2016) [1].

Semi-autonomous inline water analyzer: design of a common light detector for bacterial, phage, and immunological biosensors

The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.