domain: sde sdv

text:
Bruno Maucourt

Stéphane Vuilleumier

Françoise Bringel
email: francoise.bringel@unistra.fr

Transcriptional regulation of organohalide pollutant utilisation in bacteria

Keywords: dehalogenation, gene expression, organohalide pollutants, regulation, transcriptional regulators, ligands

Organohalides are organic molecules formed biotically and abiotically, both naturally and through industrial production. They are usually toxic and represent a health risk for living organisms, including humans. Bacteria capable of degrading organohalides for growth express dehalogenase genes encoding enzymes that cleave carbon-halogen bonds. Such bacteria are of potential high interest for bioremediation of contaminated sites. Dehalogenase genes are often part of gene clusters that may include regulators, accessory genes and genes for transporters and other enzymes of organohalide degradation pathways. Organohalides and their degradation products affect the activity of regulatory factors, and extensive genome-wide modulation of gene expression helps dehalogenating bacteria to cope with stresses associated with dehalogenation, such as intracellular increase of halides, dehalogenase-dependent acid production, organohalide toxicity, and misrouting and bottlenecks in metabolic fluxes. This review focuses on transcriptional regulation of gene clusters for dehalogenation in bacteria, as studied in laboratory experiments and in situ. The diversity in gene content, organization, and regulation of such gene clusters is highlighted for representative organohalidedegrading bacteria. Selected examples illustrate a key, overlooked role of regulatory processes, often strain-specific, for efficient dehalogenation and productive growth in presence of organohalides.

Highlights

. This review addresses a neglected aspect of dehalogenation: the regulation of dehalogenase production, mainly at the transcriptional level, and the impact of different factors and processes on organohalide degradation. To our knowledge, only the regulation of reductive dehalogenases has recently been reviewed in-depth (Maillard and Willemin 2019). Here, we will focus on acquired knowledge of regulated gene clusters for growth with organohalide pollutants released into the environment as the result of agricultural and industrial practices, and emphasize both general and substrate-specific processes involved in the detection, detoxification and degradation of these chemicals. We chose not to include pharmaceutically relevant organohalides, such as antibiotics (e.g. chloramphenicol, vancomycin) and biocides (e.g. triclosan), since reviews on such compounds have also recently become available (e.g. Novotna, Kwun and Hong 2016). Most of the examples discussed in this review will involve organochlorine pollutants, which traditionally have been better studied. However, much of this research is also relevant to homologous (notably brominated) organohalides, which are often transformed by the same systems (Chae and

Introduction

Organic compounds containing one or several carbon-halogen bonds are produced by both biotic and abiotic processes [START_REF] Gribble | Naturally Occurring Organohalogen Compounds -A Comprehensive Update[END_REF]Fig. 1). They are often toxic to animals [START_REF] Jayaraj | Organochlorine pesticides, their toxic effects on living organisms and their fate in the environment[END_REF], fungi [START_REF] Bernat | 2,4-dichlorophenoxyacetic acid-induced oxidative stress: Metabolome and membrane modifications in Umbelopsis isabellina, a herbicide degrader[END_REF], plants [START_REF] Clausen | Toxicity of 56 substances to trees[END_REF], and micro-organisms [START_REF] Tobajas | Assessment of toxicity and biodegradability on activated sludge of priority and emerging pollutants[END_REF]). Many such compounds have been massively produced industrially, and released into the environment. They may thereby threaten human health, especially at the high concentrations found in chemical production and usage sites. Such compounds have been diversely referred to in the literature as 'halogenated organic compounds', 'organohalogens', or 'organohalides'. In this review, we chose to use the latter term throughout, in agreement with the prevailing consensus among scientists in the field [START_REF] Leys | Organohalide respiration: microbes breathing chlorinated molecules[END_REF]. Some bacteria degrade and use organohalides for growth, as carbon or energy source, or as electron acceptors for respiration [START_REF] Atashgahi | Microbial synthesis and transformation of inorganic and organic chlorine compounds[END_REF]. So far, research on microbial dehalogenation has pesticides are still detected in the environment many years after having been banned by the Stockholm Convention on persistent organic pollutants [START_REF] Wöhrnschimmel | Ten years after entry into force of the Stockholm convention: What do air monitoring data tell about its effectiveness?[END_REF]. In this context, the capacity of specific bacteria to degrade organohalides has attracted much interest, starting already over half a century ago (e.g. [START_REF] Kearney | Enzyme from soil bacterium hydrolyzes phenylcarbamate herbicides[END_REF][START_REF] Wedemeyer | Dechlorination of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane by Aerobacter aerogenes. I. Metabolic products[END_REF][START_REF] Goldman | Carbon halogen bond cleavage. III. Studies on bacterial halidohydrolases[END_REF]). Carbonhalogen bond cleavage by environmental bacteria is catalysed by a wide diversity of enzymes (see e.g. [START_REF] Temme | Presence, diversity, and enrichment of respiratory reductive dehalogenase and non-respiratory hydrolytic and oxidative dehalogenase genes in terrestrial environments[END_REF], and may involve oxidative (e.g. 4-chlorophenylacetate 3,4-dioxygenase; EC 1.14.12.9), reductive (e.g. reductive dehalogenase; EC 1.97.1.8), hydrolytic (e.g. haloalkane dehalogenase; EC 3.8.1.5) or thiolytic (e.g. dichloromethane dehalogenase; EC 4.5.1.3) reactions [START_REF] Agarwal | Enzymatic halogenation and dehalogenation reactions: Pervasive and mechanistically diverse[END_REF][START_REF] Fincker | Biochemistry of catabolic reductive dehalogenation[END_REF][START_REF] Ang | Dehalogenases: From improved performance to potential microbial dehalogenation applications[END_REF]. Regulatory processes of dehalogenase gene expression are often an overlooked aspect of bioremediation of organohalides although dehalogenases are generally among the most abundant proteins in bacteria growing with these compounds as the sole carbon and energy source, reaching up to at least 20% of total soluble proteins [START_REF] Muller | Functional genomics of dichloromethane utilisation in Methylobacterium extorquens DM4[END_REF][START_REF] Van Den Wijngaard | Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2[END_REF]. It has been hypothesized that high cellular levels of dehalogenases may be needed for faster growth with organohalides, especially when enzymes have low turnover and low substrate affinity, or when the substrates are cytotoxic (van den Wijngaard, Reuvekamp and Janssen 1991; [START_REF] Gisi | Effects of bacterial host and dichloromethane dehalogenase on the competitiveness of methylotrophic bacteria growing with dichloromethane[END_REF].

Conversely, the energy required to express dehalogenases at such high levels in the absence of pollutants would put an unnecessary burden on cellular metabolism. Thus, it makes sense that bacteria efficiently regulate the expression of dehalogenases and other genes associated with degradation of organohalides.

Organization and regulation of gene clusters involved in metabolism of organohalides

Dehalogenase-encoding genes are often clustered together with regulator genes, other genes involved in the catabolism and transport of organohalides, and accessory genes (Fig. 2). For example, as recently reviewed specifically [START_REF] Kruse | Comparative genomics and transcriptomics of organohalide-respiring bacteria and regulation of rdh gene transcription[END_REF][START_REF] Maillard | Regulation of organohalide respiration[END_REF], reductive dehalogenase (rdh) gene clusters are generally located immediately adjacent to transcriptional regulatory genes. Gene clusters for organohalide degradation are diverse in genetic organisation, regulatory genes, and genomic localization (e.g. chromosomal or plasmidic, Fig. 2) [START_REF] Sentchilo | Unusual integrase gene expression on the clc genomic island in Pseudomonas sp. Strain B13[END_REF][START_REF] Tabata | Comparison of the complete genome sequences of four γhexachlorocyclohexane-degrading bacterial strains: insights into the evolution of bacteria able to degrade a recalcitrant man-made pesticide[END_REF][START_REF] Nielsen | Evolution of Sphingomonad gene clusters related to pesticide catabolism revealed by genome sequence and mobilomics of Sphingobium herbicidovorans MH[END_REF]. Such oftentimes patchwork assembly can be explained in part by horizontal gene transfer events driven by selective pressure for the capacity to degrade organohalides in contaminated environments. This is confirmed by ample co-occurrence of insertion sequences [START_REF] Schmid-Appert | Association of newly discovered IS elements with the dichloromethane utilisation genes of methylotrophic bacteria[END_REF], transposases [START_REF] Rhee | Detection by PCR of reductive dehalogenase motifs in a sulfidogenic 2-bromophenol-degrading consortium enriched from estuarine sediment[END_REF][START_REF] Maillard | Isolation and characterization of Tn-Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1[END_REF], recombinases [START_REF] Muller | Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. strain VS and its environmental distribution[END_REF], or CRISPR-Cas systems [START_REF] Molenda | Extrachromosomal circular elements targeted by CRISPR-Cas in Dehalococcoides mccartyi are linked to mobilization of reductive dehalogenase genes[END_REF] in gene clusters for dehalogenation. Frequent association of gene clusters for degradation of organohalides with mobile genetic elements, genomic islands or integrative and conjugative elements [START_REF] Sentchilo | Unusual integrase gene expression on the clc genomic island in Pseudomonas sp. Strain B13[END_REF][START_REF] Mcmurdie | Localized plasticity in the streamlined genomes of vinyl chloride respiring Dehalococcoides[END_REF][START_REF] Liang | Horizontal transfer of dehalogenase genes involved in the catalysis of chlorinated compounds: Evidence and ecological role[END_REF] has likely been instrumental for selection and distribution of the most appropriate dehalogenation pathways [START_REF] Liang | Horizontal transfer of dehalogenase genes involved in the catalysis of chlorinated compounds: Evidence and ecological role[END_REF]. Gene clusters for dehalogenation occur both in single polycistronic operons as well as multiple co-regulated operons (i.e., a regulon).

The organization of gene clusters involved in transformation of organohalides may be classified in two groups. The first comprise gene clusters with a dehalogenase gene and vary greatly in size (from 2 to 20 genes including often one or several regulatory genes). In the lindane-degrading strain Sphingobium japonicum UT26, as an impressive example, 16 lin genes associated with 6 dehalogenation reactions of the pathway are found in several gene clusters scattered across two chromosomes and one plasmid (Fig. 2A) [START_REF] Tabata | Comparison of the complete genome sequences of four γhexachlorocyclohexane-degrading bacterial strains: insights into the evolution of bacteria able to degrade a recalcitrant man-made pesticide[END_REF]. This represents a challenge for robust and streamlined applications of dehalogenation metabolism for bioremediation. So far, only a few compact synthetic gene clusters for dehalogenation have been reported, e.g. for utilisation of 1,3dichloropropene and dichloromethane [START_REF] Nikel | Engineering an anaerobic metabolic regime in Pseudomonas putida KT2440 for the anoxic biodegradation of 1,3-dichloroprop-1-ene[END_REF]Michener et al. 2014a) (Fig. 2B).

In contrast, organohalide-relevant catabolic gene clusters lacking dehalogenase genes lead to the formation of halogenated metabolites that often accumulate. For example, the bph gene cluster affects both biphenyl and polychlorinated biphenyl degradation, e.g. in Rhodococcus jostii RHA1 [START_REF] Takeda | Dual two-component regulatory systems are Involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1[END_REF]) (Fig. 2C). Transcription factors in such clusters contribute to regulate expression of cluster genes, and also recognize halogenated homologs of biphenyls [START_REF] Takeda | Dual two-component regulatory systems are Involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1[END_REF].

Transcription factors and their ligands in regulation of organohalide degradation

In gene clusters with dehalogenase genes, regulatory genes are commonly found in direct proximity to those of dehalogenases, and often in opposite orientation, a common characteristic of transcriptional regulators in bacteria [START_REF] Beck | Divergent promoters, a common form of gene organization[END_REF]) (Fig. 2). This facilitates concerted expression [START_REF] Beck | Divergent promoters, a common form of gene organization[END_REF] and production of transcription factors nearby targeted genes, and favours efficient recognition of corresponding DNA-binding sites by preventing spatial diffusion [START_REF] Weng | Spatial organization of transcription in bacterial cells[END_REF]. Transcription factors feature at least two domains: a DNA-binding domain recognizing a specific DNA sequence (often an inverted repeat located upstream or around the target promoter), and a sensor domain, often termed companion domain or effector-binding domain [START_REF] Perez-Rueda | Abundance, diversity and domain architecture variability in prokaryotic DNA-binding transcription factors[END_REF]. Additional domains may also be found, such as an RNA polymerase sigma 54 factor domain which favours RNA polymerase interactions for increased gene expression in the presence of pollutant (see Fig. 3A, MopR).

Transcription factors involved in organohalide detection usually carry a Helix-Turn-Helix (HTH) DNA-binding domain (Fig. 3). This HTH domain is found in about half of known bacterial transcription factors (Rivera-Gomez, Segovia and Perez-Rueda 2011), and indeed in the majority of regulators involved in biodegradation [START_REF] De Lorenzo | Sensing xenobiotic compounds: lessons from bacteria that face pollutants in the environment[END_REF].

Sensor domains in organohalide-responsive transcription factors usually recognize the organohalide itself or one of its metabolites. Binding of small ligands induces conformational changes in transcription factors (Fig. 4), thereby affecting their DNA-binding properties [START_REF] Anantharaman | MEDS and PocR are novel domains with a predicted role in sensing simple hydrocarbon derivatives in prokaryotic signal transduction systems[END_REF][START_REF] Möglich | Structure and signaling mechanism of Per-ARNT-Sim domains[END_REF]. Ligand binding and its role in regulation can be tested using electrophoretic mobility shift assays of DNA and DNase footprints in the presence or absence of regulator proteins [START_REF] Whangsuk | ChpR Is a chlorpyrifos-responsive transcription regulator in Sinorhizobium meliloti[END_REF][START_REF] Starr | Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR[END_REF][START_REF] Sallabhan | The hdhA gene encodes a haloacid dehalogenase that is regulated by the LysR-type regulator, HdhR, in Sinorhizobium meliloti[END_REF][START_REF] Torii | The regulatory mechanism of 2,4,6-trichlorophenol catabolic operon expression by HadR in Ralstonia pickettii DTP0602[END_REF][START_REF] Wagner | Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi[END_REF]. Regulatory processes can also be investigated by comparing global gene expression in cultures in the presence or absence of organohalides, or by using gene-specific transcriptional fusion and RT-qPCR assays (Table 1). New powerful techniques continue to be developed for this purpose. For example, binding of trichloroethene by dehalogenase and a two-component system was demonstrated using unbiased, high-throughput thermal proteome profiling (Türkowsky et al. 2018b).

Crystal structures can suggest how ligands bind to transcription factors (Table 1). Several organohalides act as ligands binding their transcriptional regulators [START_REF] Chae | 4-Chlorobenzoate uptake in Comamonas sp. strain DJ-12 Is mediated by a tripartite ATP-independent periplasmic transporter[END_REF][START_REF] Sanchez | Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134[END_REF][START_REF] Hayes | Structures of the inducer-binding domain of pentachlorophenol-degrading gene regulator PcpR from Sphingobium chlorophenolicum[END_REF]. Some transcription factors feature one cavity in the sensor domain and can bind several different organohalides [START_REF] Ray | Structural basis of selective aromatic pollutant sensing by the effector binding domain of MopR, an NtrC family transcriptional regulator[END_REF]. Other factors have distinct cavities that allow binding of different molecules for regulatory fine-tuning (Fig. 4, PcpR) [START_REF] Hayes | Structures of the inducer-binding domain of pentachlorophenol-degrading gene regulator PcpR from Sphingobium chlorophenolicum[END_REF]. Halogen atoms themselves are not always directly involved in ligand binding (Fig. 4). In some cases, metabolites obtained after cleavage of organohalide carbon, bonds are also recognized by their transcription factors [START_REF] Platero | Transcriptional organization and regulatory elements of a Pseudomonas sp. strain ADP operon encoding a LysR-type regulator and a putative solute transport system[END_REF]. Thus, transcription factors relevant to organohalides degradation are not restricted to organohalides themselves. Ligand specificity is determined by properties of the identified binding sites (volume, solvent accessibility, hydrophobicity) [START_REF] Liang | Anatomy of protein pockets and cavities: Measurement of binding site geometry and implications for ligand design[END_REF], so that detailed structures of transcription factors serve to predict the binding of new ligands by molecular docking, similarly to what can be done for enzymes and their substrates, including dehalogenases [START_REF] Daniel | Mechanism-based discovery of novel substrates of haloalkane dehalogenases using in silico screening[END_REF][START_REF] Yang | Computational design of ligand-binding proteins[END_REF]. Some families of transcription factors have been associated with specific organohalides. For example, transcription factors of the LysR family (e.g. CatR, ClcR, LinR, PcpR; Fig. 2; Table 1) are mainly involved in the detection of aromatic organohalides such as chlorobenzoate or chlorophenol. Transcription factors of the NtrC family (DmpR, MopR, XylR) [START_REF] Ray | Structural basis of selective aromatic pollutant sensing by the effector binding domain of MopR, an NtrC family transcriptional regulator[END_REF]) are able to detect both aromatic compounds with or without halogen substituents, albeit with lesser specificity. On the other hand, transcription factors of the MarR-type, such as RdhR in Dehaloccocoides mccartyi, and of the CRP-FNR type, such as CprK in Desulfitobacterium strains, are often involved in the regulation of halogenated aliphatic solvents [START_REF] Pop | Regulation of anaerobic dehalorespiration by the transcriptional activator CprK[END_REF]) (Fig. 5D). For other transcription factor families, in contrast, no clear trends have been found. For example, transcription factor ChpR, associated with chlorpyrifos detection and transformation, is the only member of the CadC family known to respond to organohalides [START_REF] Whangsuk | ChpR Is a chlorpyrifos-responsive transcription regulator in Sinorhizobium meliloti[END_REF].

Regulatory mechanisms in gene clusters for dehalogenation expression

The mechanistic details of regulatory processes can be studied at many levels, but the transcriptional level has been the most investigated so far. For organohalides, most studies have focused on transcription factors activating or repressing gene expression in response to organohalides. In some cases, the interference of other non-halogenated growth substrates on dehalogenase expression has been studied. The diverse ways by which transcription factors are known to affect gene expression in the context of degradation of organohalides are reviewed in the next paragraphs.

Modulation of dehalogenase expression by transcription factors

Expression of dehalogenases usually increases in the presence of organohalides. Activation of dehalogenase expression can involve binding of transcription factors to DNA, or alternatively their release from DNA, as well as their interaction with other proteins. For example, transcription of the reductive dehalogenase gene cprA is increased when CprK binds to chlorophenol [START_REF] Maillard | Regulation of organohalide respiration[END_REF]. Conversely, other transcription factors bind to operator sequences to block access to RNA polymerase in the absence of their ligands, thereby repressing transcription of downstream genes. This is the case for DcmR (La Roche and Leisinger 1991), HdhR [START_REF] Sallabhan | The hdhA gene encodes a haloacid dehalogenase that is regulated by the LysR-type regulator, HdhR, in Sinorhizobium meliloti[END_REF]) and RdhR1625 [START_REF] Krasper | The MarR-type regulator Rdh2R regulates rdh gene transcription in Dehalococcoides mccartyi strain CBDB1[END_REF], identified as repressors of the expression of genes involved in the degradation of dichloromethane, chloroacetic acid and trichlorobenzene, respectively (Fig. 2). No ligand has yet been demonstrated experimentally for these factors. Interestingly, some regulators can switch between repressing and activating gene expression. In chlorobenzoate degradation, for example, the transcriptional regulator ClcR represses transcription of the chlorocatechol-1,2dioxygenase gene ClcA by promoting DNA bending. Upon binding of 2-chloro-cis,cis-muconate, ClcR switches from repressor to activator (Fig. 5), by interacting directly with the alpha subunit of the RNA polymerase to increase transcription activation [START_REF] Mcfall | Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme[END_REF].

Regulation of dehalogenase expression by multiple transcriptional regulators

Several studies have investigated the effects of multiple copies of gene clusters within a genome on regulation of organohalide degradation. For example, duplication of the bphST genes in Rhodococcus jostii RHA1 (Fig. 2C) and subsequence mutational drift and selection resulted in a bphST gene variant more responsive to chlorinated biphenyls, with unchanged DNA-binding sites but different ligandsensing specificities [START_REF] Takeda | Dual two-component regulatory systems are Involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1[END_REF]. Duplicated bphST copies are highly similar (92 and 97% protein identity between BphS1/BphS2 sensor histidine kinases and BphT1/BphT2 response regulators, respectively). BphS1T1 and BphS2T2 both detect chlorobenzene and 1,2-dichlorobenzene, but only BphS1T1 recognizes biphenyl [START_REF] Takeda | Dual two-component regulatory systems are Involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1[END_REF]. Similarly, Pseudomonas putida RB1 carries two orthologous gene clusters involved in benzoate metabolism: catR-catBCA and clcR-clcABD of which only the latter is specific for chlorinated compounds [START_REF] Mcfall | Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme[END_REF]. Transcriptional regulator CatR activates both cat and clc operons in presence of cis,cis-muconate, whereas ClcR only activates expression of the clc operon in presence of 2-chlorocis,cis-muconate despite being able to bind the cat operon promoter. Thus, duplication of transcription factor genes can lead to ligand binding specificities [START_REF] Perez | Evolution of transcriptional regulatory circuits in bacteria[END_REF].

Repression of dehalogenation metabolism by non-halogenated compounds

Catabolic repression is often observed in bacteria able to utilise other carbon sources and energy sources besides organohalides for growth [START_REF] Govantes | Regulation of the atrazine-degradative genes in Pseudomonas sp. strain ADP[END_REF][START_REF] Weir | A single monooxygenase, Ese, is involved in the metabolism of the organochlorides endosulfan and endosulfate in an Arthrobacter sp[END_REF]. For example, the tricarboxylic acid cycle intermediate fumarate competes with 2-chloro-cis,cis-muconate for binding to the transcription factor ClcR, inhibiting clcABC gene activation for chlorobenzoate utilisation by ClcR [START_REF] Mcfall | A tricarboxylic acid cycle intermediate regulating transcription of a chloroaromatic biodegradative pathway: fumarate-mediated repression of the clcABD operon[END_REF]) (Fig. 5B). Intermediates of the tricarboxylic acid cycle and sugars repress bph gene expression and thereby PCB degradation in Acidovorax sp. KKS102 [START_REF] Ohtsubo | Identification of a response regulator gene for catabolite control from a PCB-degrading beta-proteobacteria, Acidovorax sp. KKS102[END_REF]. This system involves a two-component regulator-sensor (bphPQ) located outside the bph gene cluster.

BphQ recognizes and binds to the pE promoter upstream of the bph gene cluster when carbon sources other than PCBs are present [START_REF] Ohtsubo | Identification of a response regulator gene for catabolite control from a PCB-degrading beta-proteobacteria, Acidovorax sp. KKS102[END_REF]. Catabolic repression of organohalide degradation is not limited to carbon when an organohalide can also serve as a source of nitrogen for bacterial growth.

For example, repression of expression of genes for atrazine catabolism and inhibition of dehalogenation is observed in the presence of an alternative preferential nitrogen source [START_REF] Platero | Transcriptional organization and regulatory elements of a Pseudomonas sp. strain ADP operon encoding a LysR-type regulator and a putative solute transport system[END_REF]). In this specific example, the gene cluster for dehalogenation atzRSTUVW includes a fixation site for transcription factor NtrC, in addition to a nucleoid protein binding site (integration host factor; IHF) [START_REF] Platero | Transcriptional organization and regulatory elements of a Pseudomonas sp. strain ADP operon encoding a LysR-type regulator and a putative solute transport system[END_REF]) (Fig. 5), and NtrC represses transcription of atz dehalogenase genes in the presence of ammonium, nitrate or urea. Similarly, for some organohalides used as both carbon and sulphur source for growth, as in the case of degradation of the pesticide endosulfan by Arthrobacter sp. KW [START_REF] Weir | A single monooxygenase, Ese, is involved in the metabolism of the organochlorides endosulfan and endosulfate in an Arthrobacter sp[END_REF], the presence of other sulphur sources inhibits endosulfan degradation by an unknown mechanism.

Uptake of pollutants

In many cases, transport of organohalides into bacterial cells will also affect dehalogenase expression.

In Comamonas sp. DJ-12 for example, the genes fcbT1T2T3 located in the gene cluster for 4chlorobenzoate degradation encode a transporter essential for 4-chlorobenzoate import which is induced by 4-chlorobenzoate, as the enzymes involved in its degradation [START_REF] Chae | 4-Chlorobenzoate uptake in Comamonas sp. strain DJ-12 Is mediated by a tripartite ATP-independent periplasmic transporter[END_REF].

Transporters involved in organohalide uptake are often encoded by genes found nearby dehalogenase genes (Fig. 2), and can be located at the outer membrane (e.g. beta-barrel transporter [START_REF] Belchik | A β-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in Cupriavidus necator[END_REF])) or at the inner membrane (e.g. ABC-type, TRAP-type or major facilitator-type superfamily transporters; [START_REF] Platero | Transcriptional organization and regulatory elements of a Pseudomonas sp. strain ADP operon encoding a LysR-type regulator and a putative solute transport system[END_REF][START_REF] Chae | 4-Chlorobenzoate uptake in Comamonas sp. strain DJ-12 Is mediated by a tripartite ATP-independent periplasmic transporter[END_REF][START_REF] Hoffmann | 2,4-Dichlorophenoxyacetic acid (2,4-D) utilisation by Delftia acidovorans MC1 at alkaline pH and in the presence of dichlorprop is improved by introduction of the tfdK gene[END_REF][START_REF] Su | Existence of a robust haloacid transport system in a Burkholderia species bacterium[END_REF].

Post-transcriptional regulatory mechanisms

Known post-transcriptional regulatory mechanisms for organohalide gene expression include aspects of both protein expression and dehalogenase activity. Translation can be modulated by formation of stem-loop structures at the level of mRNA leader sequences (riboswitches, metabolite-binding mRNA structures) [START_REF] Speed | An archaeal fluoride-responsive riboswitch provides an inducible expression system for hyperthermophiles[END_REF], or by interactions with proteins such as Hfq and small RNAs (Kavita, de Mets and Gottesman 2018). Some riboswitches specifically bind fluoride halide ions, and to trigger the translation of halide transporters [START_REF] Speed | An archaeal fluoride-responsive riboswitch provides an inducible expression system for hyperthermophiles[END_REF], as well as of enzymes known to be inhibited by fluoride and proteins of unknown function [START_REF] Baker | Widespread genetic switches and toxicity resistance proteins for fluoride[END_REF]. Intriguingly, the dichloromethane dehalogenating strain Methylorubrum extorquens DM4 (formerly Methylobacterium extorquens (Green and Ardley 2018)), contains ten homologs of this "fluoride" riboswitch [START_REF] Baker | Widespread genetic switches and toxicity resistance proteins for fluoride[END_REF].

All these riboswitch copies are located in a genomic island involved in dehalogenation (Bringel and Vuilleumier, unpublished data). This suggests a potential link of halide-specific riboswitches with regulatory processes of organohalide degradation.

Dehalogenases and their associated transcription factors also represent key targets for posttranslational protein modifications to modulate both dehalogenase activity and associated regulatory processes. For instance, acetylation of a two-component system regulates dehalogenase gene pceA transcription in Sulfurospirillum halorespirans (Fig. 5C). In the presence of tetrachloroethene (PCE), acetylation of the DNA-binding response regulator (SHALO_1502) induces conformational changes of the protein that mimics phosphorylation by a PCE-responsive histidine kinase (SHALO_1503) (Türkowsky et al. 2018a). Acetylation was shown to induce pceAB transcription (although less strongly than by phosphorylation), so that pceAB remains transcribed and activated even in absence of PCE. Besides acetylation, other protein modifications such as phosphorylation and glycosylation have been found. However, post-transcriptional regulatory mechanisms have not yet been much investigated in the context of degradation of organohalides. This clearly represents an attractive area for future study.

Global responses in gene expression upon growth with organohalides

Bacteria that grow with organohalides are exposed to different stresses that may trigger changes in gene expression extending beyond the genes associated with dehalogenation per se. Also and as already mentioned, dehalogenase genes are often located on mobile genetic elements [START_REF] Liang | Horizontal transfer of dehalogenase genes involved in the catalysis of chlorinated compounds: Evidence and ecological role[END_REF]) which often have wide-ranging effects on the expression of genes not necessarily linked to the functions encoded by the element [START_REF] Lang | Transcriptome modulations due to A/C2 plasmid acquisition[END_REF][START_REF] San Millan | Interactions between horizontally acquired genes create a fitness cost in Pseudomonas aeruginosa[END_REF]. In Pseudomonas aeruginosa PAO1 for example, acquisition of the genomic island ICEclc for chlorobenzoate degradation, changes patterns of gene expression outside ICEclc itself even in the absence of chlorobenzoate [START_REF] Gaillard | Host and invader impact of transfer of the clc genomic island into Pseudomonas aeruginosa PAO1[END_REF].

Large-scale gene expression changes during growth with chlorinated compounds have been observed in other studies (Table 2), notably by cDNA micro-array and RNA-seq approaches. Frequently, hundreds of transcripts change expression levels between organohalide and non-halogenated substrates. Similarly large changes were also observed in proteomic studies [START_REF] Zhang | Proteomic strategy for the analysis of the polychlorobiphenyldegrading cyanobacterium Anabaena PD-1 exposed to aroclor 1254[END_REF][START_REF] Goris | Proteomics of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates[END_REF][START_REF] Bibi-Triki | N-terminome and proteogenomic analysis of the Methylobacterium extorquens DM4 reference strain for dichloromethane utilization[END_REF]Türkowsky et al. 2018aTürkowsky et al. , 2018b)). Such investigations at the genomescale will clearly continue to provide much-needed valuable detailed information on the global effects of dehalogenation metabolism on bacterial physiology in the future.

Indeed, the global bacterial response to organohalides and their toxicity usually involves several still incompletely characterised aspects of the general stress response, including transcription factors that respond to halogenated pollutants and their degradation products [START_REF] Bringel | Methylotrophs and methylotroph populations for chloromethane degradation[END_REF][START_REF] Heipieper | Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems[END_REF]). For instance, some organohalides act as solvents and disrupt membrane fluidity and integrity, potentially affecting the proton-motive force [START_REF] Murínová | Response Mechanisms of Bacterial Degraders to Environmental Contaminants on the Level of Cell Walls and Cytoplasmic Membrane[END_REF]. In addition, besides the inherent toxicity of organohalides, the process of degradation itself may be a major source of organohalide toxicity, with degradation products being more of a problem than organohalides themselves. For example, glutathione S-transferase driven degradation of dichloromethane yields Schloromethylglutathione, a reactive metabolite which leads to the formation of alkylated DNA adducts [START_REF] Kayser | Dehalogenation of dichloromethane by dichloromethane dehalogenase/glutathione S-transferase leads to formation of DNA adducts[END_REF], with associated mutagenic effects [START_REF] Gisi | Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione S-transferases[END_REF]. Similarly, pentachlorophenol degradation leads to the formation of highly toxic hydroxyl radicals by reaction of the degradation pathway intermediate tetrachlorobenzoquinone with hydrogen peroxide [START_REF] Zhu | Molecular mechanism for metal-independent production of hydroxyl radicals by hydrogen peroxide and halogenated quinones[END_REF]). Higher expression of enzymes of oxidative stress such as catalases or peroxidases is indeed often observed during degradation of organohalides by oxygenases [START_REF] Jennings | Proteomic and transcriptomic analyses reveal genes upregulated by cis-dichloroethene in Polaromonas sp. strain JS666[END_REF][START_REF] Puglisi | Transcriptional response of Rhodococcus aetherivorans I24 to polychlorinated biphenyl-contaminated sediments[END_REF]. Finally, halide production from dehalogenation is another source of stress associated with degradation of organohalides. In dichloromethane dehalogenation, for example, high transcription of the ClcA chloride/proton antiporter gene was shown to be of advantage in laboratory experimental evolution studies of bacterial dichloromethane utilisation (Michener et al. 2014a(Michener et al. , 2014b)). This highlighted the importance of gene expression re-tuning in the host for efficient growth with an organohalide following acquisition of the corresponding gene cluster.

Taken together, the toxic effects associated with organohalides and their degradation contribute to explaining the differential expression of a large variety of stress response genes in the context of organohalide degradation (e.g. [START_REF] Gvakharia | Global transcriptional response of Nitrosomonas europaea to chloroform and chloromethane[END_REF][START_REF] Jennings | Proteomic and transcriptomic analyses reveal genes upregulated by cis-dichloroethene in Polaromonas sp. strain JS666[END_REF][START_REF] Puglisi | Transcriptional response of Rhodococcus aetherivorans I24 to polychlorinated biphenyl-contaminated sediments[END_REF][START_REF] Islam | New insights into Dehalococcoides mccartyi metabolism from a reconstructed metabolic network-based systems-level analysis of D. mccartyi transcriptomes[END_REF]. This was shown to include common chaperones DnaK, GroES and GroEL, known to target a wide range of proteins [START_REF] Bhandari | Substrate interaction networks of the Escherichia coli chaperones: Trigger factor, DnaK and GroEL[END_REF], further emphasizing the major effects of dehalogenation on global gene expression. Moreover, some chaperones encoded within dehalogenase operons are only expressed in the context of dehalogenation metabolism, suggesting a specific role of some chaperones in the folding of structurally complex dehalogenases such as reductive dehalogenases [START_REF] Morita | Functional characterization of the trigger factor protein PceT of tetrachloroethene-dechlorinating Desulfitobacterium hafniense Y51[END_REF][START_REF] Maillard | Redundancy and specificity of multiple trigger factor chaperones in Desulfitobacteria[END_REF][START_REF] Nelly | Functional heterologous production of reductive dehalogenases from Desulfitobacterium hafniense Strains[END_REF].

Aspects of metabolic fluxes and energy balance clearly also impact global gene expression in organohalide metabolism. For example, the energy retrieved from organohalide degradation may sometimes be insufficient to match the energy needs of dehalogenase synthesis and adaptation to toxic organohalides (Cases and de Lorenzo 2005a). This provides a rationale for the often-observed adjustments in central metabolism and corresponding expression levels of genes for enzymes and also transporters of growth-supporting organohalides involved in energy metabolism [START_REF] Jennings | Proteomic and transcriptomic analyses reveal genes upregulated by cis-dichloroethene in Polaromonas sp. strain JS666[END_REF][START_REF] Zhang | Proteomic strategy for the analysis of the polychlorobiphenyldegrading cyanobacterium Anabaena PD-1 exposed to aroclor 1254[END_REF]. Similarly, modulation of the level of expression of genes involved in cofactor and redox balance has been observed such as the glutathione reductase gene found downstream of glutathione S-transferase dehalogenase gene dsmH2 involved in the degradation of the broad-spectrum herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) [START_REF] Li | Identification of two glutathione-dependent 3,6-dichlorogentisate dehalogenases and their roles in the catabolism of the herbicide dicamba in Rhizorhabdus dicambivorans Ndbn-20[END_REF], or the FAD reductase gene tcpX in 2,4,6-trichlorophenol degradation [START_REF] Sanchez | Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134[END_REF]. Moreover, requirements for increased cellular pools of essential dehalogenase cofactors can also trigger higher expression of gene sets involved in cofactor biosynthesis. Reductive dehalogenation, for example, usually involves corrinoid-dependent dehalogenases, with concomitant increased expression of genes for cobalamin biosynthesis upon growth with organohalides [START_REF] Kruse | Comparative genomics and transcriptomics of organohalide-respiring bacteria and regulation of rdh gene transcription[END_REF], such as in Desulfitobacterium hafniense Y51 growing with tetrachloroethene [START_REF] Peng | Global transcriptome analysis of the tetrachloroethenedechlorinating bacterium Desulfitobacterium hafniense Y51 in the presence of various electron donors and terminal electron acceptors[END_REF], or of genes regulated by riboswitches that detect cobalamin [START_REF] Choudhary | Diversity of cobalamin riboswitches in the corrinoid-producing organohalide respirer Desulfitobacterium hafniense[END_REF][START_REF] Rupakula | Functional genomics of corrinoid starvation in the organohaliderespiring bacterium Dehalobacter restrictus strain PER-K23[END_REF].

Another example of dehalogenation-dependent regulation of cobalamin-associated genes was demonstrated in M. extorquens CM4 growing with chloromethane: an additional set of such genes is co-localized with chloromethane dehalogenase on a plasmid, and its expression is specifically regulated in a chloromethane-dependent way, unlike that of the homologous chromosomal gene set [START_REF] Roselli | The 380 kb pCMU01 plasmid encodes chloromethane utilisation genes and redundant genes for vitamin B12-and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: A proteomic and bioinformatics study[END_REF][START_REF] Chaignaud | Genomic and transcriptomic analysis of growthsupporting dehalogenation of chlorinated methanes in Methylobacterium[END_REF].

Moving out of the laboratory: Assessing regulation of dehalogenation expression in the environment

Most transcriptomics studies have been conducted under reproducible, low complexity conditions, such as with pure cultures of reference dehalogenating strains growing with different substrates under controlled nutrients, temperature, pH, and oxygen conditions. In real-world polluted environments, however, physical, chemical and biological parameters are constantly changing, making it likely that complex adaptive processes, prominently also at the transcriptional level, are associated with organohalide degradation and bacterial growth in the environment (Cases and de Lorenzo 2005b).

In this context, it is striking to note that many pollutant-degrading bacteria that show promising results when cultivated in the laboratory have been ineffective in in situ studies aiming at bioremediation [START_REF] Boopathy | Factors limiting bioremediation technologies[END_REF][START_REF] Lovley | Cleaning up with genomics: applying molecular biology to bioremediation[END_REF][START_REF] De Lorenzo | Recombinant bacteria for environmental release: what went wrong and what we have learnt from it[END_REF]. This is also in part because organohalides are usually found together with other contaminants in the environment, resulting in additive or synergistic toxic effects [START_REF] Nirmalakhandan | Additivity in microbial toxicity of nonuniform mixtures of organic chemicals[END_REF], as notably reported for fungicides (White, Potter and Culbreath 2010) and heavy metals [START_REF] Olaniran | Bioavailability of heavy metals in soil: impact on microbial biodegradation of organic compounds and possible improvement strategies[END_REF]. Indeed, the presence of several unrelated pollutants has been shown to affect transcriptional regulation, for example when transcription factors recognize several molecules with antagonistic (e.g. inducing and inhibitory) effects [START_REF] Selifonova | Use of an ipb-lux fusion to study regulation of the isopropylbenzene catabolism operon of Pseudomonas putida RE204 and to detect hydrophobic pollutants in the environment[END_REF].

Other differences between laboratory and in situ experiments include predation of organohalidedegrading strains by other microorganisms [START_REF] Cunningham | Protistan predation affects trichloroethene biodegradation in a bedrock aquifer[END_REF], competition for growth substrates [START_REF] Becker | A modeling study and implications of competition between Dehalococcoides ethenogenes and other tetrachloroethene-respiring bacteria[END_REF], and water stress encountered during the passage of cells grown as liquid cultures under laboratory conditions to in situ generally rather drier environments [START_REF] Moreno-Forero | Comparison of differential gene expression to water stress among bacteria with relevant pollutant-degradation properties[END_REF]. Conversely, organohalide-degrading bacteria may benefit from living with other microorganisms as aggregates [START_REF] Mao | Efficient metabolic exchange and electron transfer within a syntrophic trichloroethene-degrading coculture of Dehalococcoides mccartyi 195 and Syntrophomonas wolfei[END_REF] and as biofilms [START_REF] Yoshida | Enhanced biofilm formation and 3-chlorobenzoate degrading activity by the bacterial consortium of Burkholderia sp. NK8 and Pseudomonas aeruginosa PAO1[END_REF]) in the environment, e.g. also by sharing and exchanging metabolites and cofactors, with underlying regulatory processes differing from those observed in laboratory experiments [START_REF] Mao | Efficient metabolic exchange and electron transfer within a syntrophic trichloroethene-degrading coculture of Dehalococcoides mccartyi 195 and Syntrophomonas wolfei[END_REF][START_REF] Chen | Mutualistic interaction between dichloromethane-and chloromethane-degrading bacteria in an anaerobic mixed culture: Mutualistic degradation of chlorinated methanes[END_REF]) (Table 3). Indeed, many organohalide-respiring bacteria strongly depend on other bacteria for corrinoid production [START_REF] Fincker | Biochemistry of catabolic reductive dehalogenation[END_REF].

The relative scarcity of in situ transcriptomic studies specific of dehalogenation metabolism reflects this complexity, and the difficulty of correlating observed changes in gene expression with dehalogenation metabolism. In this context, laboratory microcosms using environmental samples of water, sediment and soil have increasingly been developed to study bacterial dehalogenation. Such microcosms provide conditions mimicking in situ environmental conditions, while allowing for controlled growth conditions and monitoring [START_REF] Amos | Oxygen effect on Dehalococcoides viability and biomarker quantification[END_REF][START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF][START_REF] Lillis | Quantification of catechol dioxygenase gene expression in soil during degradation of 2,4-dichlorophenol[END_REF][START_REF] Xiu | Effect of bare and coated nanoscale zerovalent iron on tceA and vcrA gene expression in Dehalococcoides spp[END_REF][START_REF] Men | Metagenomic and metatranscriptomic analyses reveal the structure and dynamics of a dechlorinating community containing Dehalococcoides mccartyi and corrinoidproviding microorganisms under cobalamin-limited conditions[END_REF]. For example, pollutant degradation could be quantitatively correlated with transcription of dehalogenase gene cbdbA in water microcosms exposed to hexachlorobenzene at several temperatures [START_REF] Tas | Role of "Dehalococcoides" spp. in the aaerobic transformation of hexachlorobenzene in European rivers[END_REF]. Microcosms also enabled investigations of bacterial dehalogenation in the context of microbial interactions with other types of organisms such as plants [START_REF] Liu | Development of a GFP-based biosensor for detecting the bioavailability and biodegradation of polychlorinated biphenyls (PCBs)[END_REF][START_REF] Scheublin | Transcriptional profiling of Gram-positive Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant phenolic compounds[END_REF][START_REF] Chaignaud | Methanol consumption drives the bacterial chloromethane sink in a forest soil[END_REF][START_REF] Wang | Coupling between nitrogen fixation and tetrachlorobiphenyl dechlorination in a rhizobium-legume symbiosis[END_REF]. Such studies are likely to continue to develop in the future, and to allow improved assessment of environmentally relevant processes, such as the fate of plant-emitted chlorinated C 1 volatile organic compounds and their consumption by dehalogenating methylotrophs (reviewed in Bringel andCouée 2015, Bringel et al., 2019).

Nevertheless, direct future development of in situ studies of polluted sites will remain indispensable to better understand and improve bioremediation processes. In the few available in situ studies on organohalide degradation focussing on gene regulation, differential detection of mRNA of gene biomarkers by RT-qPCR remains by far the preferred approach until now [START_REF] Lee | Quantifying genes and transcripts to assess the in situ physiology of " Dehalococcoides" spp. in a trichloroethene-contaminated groundwater site[END_REF][START_REF] Kranzioch | Chloroethene degradation and expression of Dehalococcoides dehalogenase genes in cultures originating from Yangtze sediments[END_REF][START_REF] Mattes | Abundance and activity of vinyl chloride (VC)-oxidizing bacteria in a dilute groundwater VC plume biostimulated with oxygen and ethene[END_REF][START_REF] Ismaeil | Identification of multiple dehalogenase genes involved in tetrachloroethene-to-ethene dechlorination in a Dehalococcoides-dominated enrichment culture[END_REF][START_REF] Liang Yi | Relationships between the abundance and expression of functional genes from vinyl chloride (VC)-degrading bacteria and geochemical parameters at VC-contaminated sites[END_REF].

The mainly used biomarkers are of course dehalogenases [START_REF] Werner | Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration[END_REF][START_REF] Kranzioch | Chloroethene degradation and expression of Dehalococcoides dehalogenase genes in cultures originating from Yangtze sediments[END_REF][START_REF] Matturro | GeneCARD-FISH: Detection of tceA and vcrA reductive dehalogenase genes in Dehalococcoides mccartyi by fluorescence in situ hybridization[END_REF][START_REF] Ibrahim | A degradome-based polymerase chain reaction to resolve the potential of environmental samples for 2,4-dichlorophenol biodegradation[END_REF][START_REF] Liang Yi | Relationships between the abundance and expression of functional genes from vinyl chloride (VC)-degrading bacteria and geochemical parameters at VC-contaminated sites[END_REF][START_REF] Hermon | Dichloromethane biodegradation in multi-contaminated groundwater: Insights from biomolecular and compound-specific isotope analyses[END_REF], although other genes, for example related to stress or cofactor biosynthesis, may also prove useful for this purpose. To our knowledge, however, regulator genes have not yet been used, likely because of the generally low level of expression of such genes compared to usually highly expressed dehalogenases. The presence and relative abundance of gene transcripts can usually be correlated with biodegradation (for an example see [START_REF] Chow | Identification and transcriptional analysis of trans-DCEproducing reductive dehalogenases in Dehalococcoides species[END_REF], and also uncover specific actively dehalogenating populations during bioremediation [START_REF] Maphosa | Exploiting the ecogenomics toolbox for environmental diagnostics of organohalide-respiring bacteria[END_REF]. In tetrachloroethene and triochloroethene bioremediation for example, key dehalogenase genes bvcA and vcrA were measured at both DNA and RNA levels, and found in 99% and 58% of 95 samples from 6 contaminated environments, respectively [START_REF] Liang Yi | Relationships between the abundance and expression of functional genes from vinyl chloride (VC)-degrading bacteria and geochemical parameters at VC-contaminated sites[END_REF]. The added value of RNA detection compared to DNA is that it may help to distinguish dehalogenase gene copies expressed by living cells from those in extracellular DNA or dead cells. As a caveat, however, RNA transcript abundance is not always strictly correlated with biodegradation [START_REF] Lee | Reductive dehalogenase dene expression as a biomarker for physiological activity of Dehalococcoides spp[END_REF], also because some dehalogenase genes are constitutively expressed [START_REF] Paulin | Abundance and expression of enantioselective rdpA and sdpA dioxygenase genes during degradation of the racemic herbicide (R,S)-2-(2,4dichlorophenoxy)propionate in soil[END_REF][START_REF] Peng | Global transcriptome analysis of the tetrachloroethenedechlorinating bacterium Desulfitobacterium hafniense Y51 in the presence of various electron donors and terminal electron acceptors[END_REF][START_REF] T'syen | Identification of the amidase BbdA that initiates biodegradation of the groundwater micropollutant 2,6-dichlorobenzamide (BAM) in Aminobacter sp. MSH1[END_REF].

At this stage, therefore, interpretation of DNA, mRNA and protein biomarkers in environmental samples still requires some caution, in the context of organohalide degradation studies [START_REF] Heavner | Biomarkers' responses to reductive dechlorination rates and oxygen stress in bioaugmentation culture KB-1TM[END_REF], as well as in the general context of microbial ecology [START_REF] Muller | Determining microbial niche breadth in the environment for better ecosystem fate predictions[END_REF].

Applications of molecular tools based on regulatory processes

In addressing the challenge of worldwide environmental pollution and by organohalides particularly, we still mainly proceed today by tinkering, and trial-and-error. In the future, bioremediation strategies will clearly benefit from bacterial strains with optimised in situ fitness, and we have reviewed here how this may also depend on regulatory processes. Many more studies investigating and improving these processes at the molecular level are still needed to develop the most effective bioremediation processes possible. Clearly, the use of genetically engineered strains continues to be unacceptable to the public in many parts of the world, including for in situ bioremediation. Nevertheless, ongoing progress in our understanding of the molecular processes associated with degradation of organohalides, in the context of the rapid development of synthetic biology approaches that also include the design of efficient and specific regulatory circuits, is fuelling advances in the field. For example, targeted engineering of transcription factors can improve and expand recognition ability for specific organohalides of interest [START_REF] Wise | Generation of novel bacterial regulatory proteins that detect priority pollutant phenols[END_REF][START_REF] Mohn | Surveying biotransformations with à la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes[END_REF][START_REF] Beggah | Mutant HbpR transcription activator isolation for 2chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting[END_REF][START_REF] Lang | Mutational analysis of the inducer recognition sites of the LysR-type transcriptional regulator TfdT of Burkholderia sp. NK8[END_REF], in analogy to what has been done extensively to improve activity of dehalogenases and modify their substrate range [START_REF] Pavlova | Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate[END_REF][START_REF] Dvořák | Bioremediation 3.0 : Engineering pollutant-removing bacteria in the times of systemic biology[END_REF][START_REF] Ang | Dehalogenases: From improved performance to potential microbial dehalogenation applications[END_REF]. Synthetic biology approaches combined with metabolic engineering [START_REF] Dvořák | Bioremediation 3.0 : Engineering pollutant-removing bacteria in the times of systemic biology[END_REF]) may also help design and assemble new and specifically regulated optimised dehalogenation pathways, by applying previously streamlined and optimised synthetic gene clusters (Fig. 2B).

Such synthetic gene clusters for dehalogenation hold the promise of conferring improved fitness to bacteria, for use in bioremediation [START_REF] Zhang | Development of an autofluorescent Pseudomonas nitroreducens with dehydrochlorinase activity for efficient mineralization of γ-hexachlorocyclohexane (γ-HCH)[END_REF] or in phytoremediation [START_REF] Mena-Benitez | Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane[END_REF][START_REF] Abhilash | Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics[END_REF]. They can be driven by either constitutive or regulated heterologous promoters (e.g. lac, tac, nod) placed upstream of dehalogenase genes to optimize dehalogenase gene expression. Some exploratory studies along these lines have already been reported. For example, bacterial bph genes for degradation of polychlorinated biphenyls were expressed from the nod promoter induced by plant-produced flavonoids to enhance biodegradation capacity of Pseudomonas strains in the rhizosphere [START_REF] Villacieros | Polychlorinated biphenyl rhizoremediation by Pseudomonas fluorescens F113 derivatives, using a Sinorhizobium meliloti nod system to drive bph gene expression[END_REF]. Conversely, detrimental growth behaviour may be avoided by deletion of corresponding regulatory genes. For example, growth of P. putida KT2440 with glycerol was prevented, and 1,2,3-trichloropropane utilisation favoured, following deletion of the transcription factor gene glpR [START_REF] Gong | Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane[END_REF].

Better expression of genes involved in the adaptive response to specific organohalides may also improve bacterial growth driven by organohalide degradation, as exemplified above for the chloride/proton antiporter clcA gene (Michener et al. 2014a). In this case, a synthetic construct containing the DCM dehalogenase gene together with clcA under the control of an experimentally evolved clcA-specific promoter was designed to confer improved growth with DCM to appropriate bacterial hosts (Fig. 2B, Michener et al. 2014b). The results of this study indeed suggest that efficient halide extrusion may represent a major, although often overlooked, adaptive advantage for growth with organohalide pollutants.

Finally, regulatory processes can also be exploited not only to improve degradation of organohalides, but also to monitor and predict bioremediation potential, as well as to evaluate their bioavailability and quantify them in natural environments [START_REF] Lopes | Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter[END_REF][START_REF] Hua | Development of a bacterial bioassay for atrazine and cyanuric acid detection[END_REF][START_REF] Whangsuk | Specific detection of the pesticide chlorpyrifos by a sensitive genetic-based whole cell biosensor[END_REF][START_REF] Ul Haque | Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere[END_REF]. Bioreporter strains expressing a fluorescent protein as a function of organohalide identity and concentration have been developed (e.g. [START_REF] Whangsuk | Specific detection of the pesticide chlorpyrifos by a sensitive genetic-based whole cell biosensor[END_REF][START_REF] Zhang | Development of an autofluorescent Pseudomonas nitroreducens with dehydrochlorinase activity for efficient mineralization of γ-hexachlorocyclohexane (γ-HCH)[END_REF]). This approach was successfully applied in several cases, e.g. to demonstrate the expression of polychlorinated biphenyls and chloromethane dehalogenase genes in the rhizosphere and the phyllosphere, respectively [START_REF] Liu | Genetically modified Pseudomonas biosensing biodegraders to detect PCB and chlorobenzoate bioavailability and biodegradation in contaminated soils[END_REF][START_REF] Ul Haque | Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere[END_REF]). In the environment, monitoring of bioreporter activity is often constrained by the limitations of spectrophotometric or fluorimetric approaches, arising e.g. from natural autofluorescence of humic acids. Use of bioreporters coupled with fluorescence-activated cell sorting (FACS) may help to overcome these problems [START_REF] Norman | A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments[END_REF][START_REF] Beggah | Mutant HbpR transcription activator isolation for 2chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting[END_REF].

Bioreporters for organohalide quantification have so far mainly made use of transcription factors that sense cytoplasmic processes. In the future, membrane-based two-component systems may offer the advantage of directly sensing pollutants in the environment without potential biases associated with pollutant uptake [START_REF] Ravikumar | Engineered microbial biosensors based on bacterial twocomponent systems as synthetic biotechnology platforms in bioremediation and biorefinery[END_REF]. Similarly, use of halide-inducible promoters [START_REF] Geldart | Chloride-inducible expression vector for delivery of antimicrobial peptides targeting antibiotic-resistant Enterococcus faecium[END_REF] and halide-specific riboswitches [START_REF] Speed | An archaeal fluoride-responsive riboswitch provides an inducible expression system for hyperthermophiles[END_REF] could also be envisaged, to detect the occurrence of dehalogenation, and to drive dehalogenation-dependent gene expression. Insights along these lines will certainly also emerge from further fundamental explorations on the regulation of organohalide degradation pathways, and will likely provide new, improved tools to monitor bioremediation of organohalides, and help define improved criteria for optimal dehalogenating strain efficiency in bioremediation.

To conclude, many aspects of the regulation of organohalide degradation pathways are still largely unexplored, in contrast to extensive available knowledge on genes and enzymes for degradation of organohalides. It is our contention that regulatory aspects may represent a key, often potentially limiting step for effective biodegradation of organohalides, in particular in natural environments. We hope that this review, together with the opportunities offered by increasingly robust omics approaches available today, will encourage to revisit this specific aspect of bacterial dehalogenation metabolism after some 40 years of intensive research focus on dehalogenases. [START_REF] Esquirol | A novel decarboxylating amidohydrolase involved in avoiding metabolic dead ends during cyanuric acid catabolism in Pseudomonas sp. strain ADP[END_REF], bhb [START_REF] Chen | Comparison of four Comamonas catabolic plasmids reveals the evolution of pBHB to catabolize haloaromatics[END_REF], bph [START_REF] Takeda | Dual two-component regulatory systems are Involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1[END_REF], ccd [START_REF] Bers | A novel hydrolase identified by genomic-proteomic analysis of phenylurea herbicide mineralization by Variovorax sp. strain SRS16[END_REF]), clc (Coco et al. 1993;Miyazaki et al. 2015)), clcA-dcmA (Michener et al. 2014a), cpr [START_REF] Smidt | Transcriptional regulation of the cpr gene cluster in orthochlorophenol-respiring Desulfitobacterium dehalogenans[END_REF], dcm (La Roche and Leisinger 1991), dha [START_REF] Poelarends | Roles of horizontal gene transfer and gene integration in evolution of 1, 3-dichloropropene-and 1, 2-dibromoethane-degradative pathways[END_REF], dsm (Li et al. 2018b), had [START_REF] Torii | The regulatory mechanism of 2,4,6-trichlorophenol catabolic operon expression by HadR in Ralstonia pickettii DTP0602[END_REF], hdh [START_REF] Sallabhan | The hdhA gene encodes a haloacid dehalogenase that is regulated by the LysR-type regulator, HdhR, in Sinorhizobium meliloti[END_REF], lin [START_REF] Tabata | Comparison of the complete genome sequences of four γhexachlorocyclohexane-degrading bacterial strains: insights into the evolution of bacteria able to degrade a recalcitrant man-made pesticide[END_REF], mop [START_REF] Schirmer | Expression, inducer spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250[END_REF], odc (Chen et al. 2018), pce [START_REF] Futagami | Emergence of two types of nondechlorinating variants in the tetrachloroethene-halorespiring Desulfitobacterium sp. strain Y51[END_REF], pcp [START_REF] Cai | Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723[END_REF], rdh [START_REF] Collins | NADPH-driven organohalide reduction by a nonrespiratory reductive dehalogenase[END_REF], tbu [START_REF] Kahng | Characterization and role of tbuX in utilisation of toluene by Ralstonia pickettii PKO1[END_REF], tcp [START_REF] Sanchez | Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134[END_REF] and tfd [START_REF] Trefault | Involvement of several transcriptional regulators in the differential expression of tfd genes in Cupriavidus necator JMP134[END_REF].

A

Figure 1 .

 1 Figure1. Natural and industrial sources of organohalides. Some organohalides are found in the environment as a result of natural abiotic production, biomass combustion and volcano emissions[START_REF] Gribble | Naturally Occurring Organohalogen Compounds -A Comprehensive Update[END_REF]. Biological production involves mainly macro-algae,microorganisms and plants(Diaz-Marrero et al. 2002;[START_REF] Tittlemier | Structure elucidation of four possible biogenic organohalogens using isotope exchange mass spectrometry[END_REF][START_REF] Atashgahi | Microbial synthesis and transformation of inorganic and organic chlorine compounds[END_REF]). Some organohalides have been massively produced synthetically, for agriculture[START_REF] Jayaraj | Organochlorine pesticides, their toxic effects on living organisms and their fate in the environment[END_REF] and for industrial use as refrigerants, solvents[START_REF] Tsai | Fate of chloromethanes in the atmospheric environment: Implications for human health, ozone formation and depletion, and global warming impacts[END_REF], electrical insulators and in plastic production.

Figure 2 .

 2 Figure 2. Gene clusters for organohalide degradation. (A) Gene clusters for dehalogenation in representative bacterial degraders. (B) Synthetic gene clusters. (C) Organohalide-relevant catabolic gene clusters lacking dehalogenase genes. References are for gene clusters atz[START_REF] Esquirol | A novel decarboxylating amidohydrolase involved in avoiding metabolic dead ends during cyanuric acid catabolism in Pseudomonas sp. strain ADP[END_REF], bhb[START_REF] Chen | Comparison of four Comamonas catabolic plasmids reveals the evolution of pBHB to catabolize haloaromatics[END_REF], bph[START_REF] Takeda | Dual two-component regulatory systems are Involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1[END_REF], ccd[START_REF] Bers | A novel hydrolase identified by genomic-proteomic analysis of phenylurea herbicide mineralization by Variovorax sp. strain SRS16[END_REF]), clc (Coco et al. 1993; Miyazaki et al. 2015)), clcA-dcmA(Michener et al. 2014a), cpr[START_REF] Smidt | Transcriptional regulation of the cpr gene cluster in orthochlorophenol-respiring Desulfitobacterium dehalogenans[END_REF], dcm (La Roche and Leisinger 1991), dha[START_REF] Poelarends | Roles of horizontal gene transfer and gene integration in evolution of 1, 3-dichloropropene-and 1, 2-dibromoethane-degradative pathways[END_REF], dsm(Li et al. 2018b), had[START_REF] Torii | The regulatory mechanism of 2,4,6-trichlorophenol catabolic operon expression by HadR in Ralstonia pickettii DTP0602[END_REF], hdh[START_REF] Sallabhan | The hdhA gene encodes a haloacid dehalogenase that is regulated by the LysR-type regulator, HdhR, in Sinorhizobium meliloti[END_REF], lin[START_REF] Tabata | Comparison of the complete genome sequences of four γhexachlorocyclohexane-degrading bacterial strains: insights into the evolution of bacteria able to degrade a recalcitrant man-made pesticide[END_REF], mop[START_REF] Schirmer | Expression, inducer spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250[END_REF], odc(Chen et al. 2018), pce[START_REF] Futagami | Emergence of two types of nondechlorinating variants in the tetrachloroethene-halorespiring Desulfitobacterium sp. strain Y51[END_REF], pcp[START_REF] Cai | Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723[END_REF], rdh[START_REF] Collins | NADPH-driven organohalide reduction by a nonrespiratory reductive dehalogenase[END_REF], tbu[START_REF] Kahng | Characterization and role of tbuX in utilisation of toluene by Ralstonia pickettii PKO1[END_REF], tcp[START_REF] Sanchez | Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134[END_REF] and tfd[START_REF] Trefault | Involvement of several transcriptional regulators in the differential expression of tfd genes in Cupriavidus necator JMP134[END_REF]).

Figure 3 .

 3 Figure 3. Functional domains of selected transcriptional factors associated with dehalogenation. (A) Transcription factors; (B) Proteins of two component systems. The HTH (helix-turn-helix) DNA binding domain (blue) present in almost all transcription factors is fused to organohalide-specific sensor domains (green). Abbreviations: MEDS (MEthanogen/methylotroph DcmR Sensory; (Anantharaman and Aravind 2005)) and PAS (Per sensor domain, Arnt, Sin; (Möglich, Ayers and Moffat 2009)). Accession numbers for BspS1 (WP 011598994.1), BphT1 (WP 011598993.1), CnbR (WP 011255153.1), CbrC (CAI82343.1), CbrD (CAI82344.1), ClcR (WP 012248462), CprK (ACL18797.1), DcmR (AAB68953.1), PcpR (U12290.2), (MopR (WP 004721355), and RdhR (WP 011309983).

Figure 4 .

 4 Figure 4. Interactions between organohalides and transcription factors. Black and green dashed lines represent hydrogen bounds and hydrophobic interactions, respectively. Orange dashed lines indicate interactions between aromatic rings. 2D representations generated by PoseView (Stierand and Rarey 2010).

Figure 5 .

 5 Figure 5. Examples of transcriptional regulation in organohalide degradation in Gram-negative and Gram-positive bacteria. (A) Atrazine utilization in Pseudomonas sp. ADP. Binding of cyanuric acid to AtzR activates expression of atzDEF. Expression of atzR and downstream genes is activated or repressed at low or high nitrogen concentration, respectively (Platero et al. 2012). (B) Chlorobenzoate utilization in Pseudomonas putida PRS2000. Expression is regulated by two homologous transcription regulators CatR ClcR (McFall et al. 1997; McFall, Chugani and Chakrabarty 1998). (C) PCE utilization in Sulfurospirillum halorespirans. A twocomponent system activates expression of pceAB when PCE is detected. It has been proposed that a yet unknown signal subsequently activates an acetyltransferase that post-translationally modifies this twocomponent system (Türkowsky et al. 2018a). (D) 2,4,6-trichlorophenol utilization by Ralstonia pickettii DTP0602. Two regulons hadRXABC and hadSYD, separated by 146 kb in the genome sequence (Torii et al. 2013), are differentially regulated by processes involving the two transcription factors HadR and HadS. HadR binds to 2,4,6-trichlorophenol for induced expression of hadYABC. HadS recognizes an intermediate metabolite produced in 2,4,6-trichlorophenol degradation, probably 2-chloromaleylacetate, to alleviate repression of hadYD (Torii et al. 2013). (E) Dehalogenation of chlorophenols by Desulfitobacterium hafniense DCB-2. The reductive dehalogenase cprA gene is regulated by five cprK paralogs (Gabor et al. 2008). CprK1 and CprK2 recognize ortho-substituted phenols such as 3-chloro-4-hydroxyphenylacetate, while CprK4 recognizes meta-substituted phenols, such as 3,5-dichlorophenol. CprK activates expression of gene cprA when ligands are detected. CprK folding is inhibited by oxygen, which prevents disulfide bond formation.

Table 1 .

 1 Transcription factors involved in detection of organohalides Other ligands are described in Takeda et al. 2010; b ligand co-crystallized with transcription factor

	Transcription	Degradation pathway	Ligand	Target DNA sequence	Crystallographic	Organism	Reference
	factor				structure
	Directly organochlorine pollutants recognized as ligands
	BphS1, BphR1	polychlorinated biphenyls biphenyl; dichlorobenzene a	TTCCGTAGTTTTCCCGGATGTTCG	No	Rhodococcus jostii RHA1	(Takeda et al. 2010)
	HadR	2,4,6-trichlorophenol	2,4,6-trichlorophenol	ATGCCGCTGAGGAAT	No	Cupriavidus pickettii DTP0602	(Torii et al. 2013)
	MopR	3-chlorophenol	3-chlorophenol b	TTCATCAAATAATGGA-8nt-	Yes	Acinetobacter calcoaceticus	(Ray et al. 2016)
				ATGCTGATTCATCAA		NCIB8250
		pentachlorophenol;	pentachlorophenol b ;
	PcpR	2,4,6-trichlorophenol	2,4,6-trichlorophenol b	ATTC-7nt-GAAT	Yes	Sphingobium chlorophenolicum L-1	(Hayes et al. 2014)
	Degradation products recognized as ligands
	CbnR	3-chlorobenzoate	cis,cis-muconate	unknown	Yes	Ralstonia eutropa NH9	(Muraoka et al. 2003)
			orthochlorophenolacetic acid			D. hafniense DCB-2
	CprK1	chlorophenols	b	TTTA-8nt-CTAA-20nt-TATAAT	Yes		(Gábor et al. 2008)
	LinR	lindane	2,5-dichlorohydroquinone;	ATTCACAATCTGAAT	No	Sphingomonas paucimobilis UT26 (Miyauchi et al. 2002)
			chlorohydroquinone
			2,3-dichlorophenol b ;
	Rdh2R	trichlorobenzene	2,4-dichlorophenol b ;	TAGTATACGAT-2nt-TAGTATACGATTA	No	Dehalococcoides mccartyi CBDB1	(Krasper et al. 2016)
			3,4-dichlorophenol b
	Unknown ligands
	CbrC	trichlorobenzene	unknown	unknown	No	Dehalococcoides mccartyi CBDB1	(Adrian et al. 2007)
							(La Roche and
	DcmR	dichloromethane	unknown	unknown	No	Methylorubrum extorquens DM4
							Leisinger 1991)

a

Table 2 .

 2 Differentially expressed genes in pure cultures of bacteria growing with organohalides Number of up-or down-regulated genes / total number of genes in genome. When available, total number of regulated genes / total number of genes in genome are shown in the centre of the columns; b Number of spots on micro-array with differential hybridization (not number of genes); c N.A., Not Applicable, no numbers provided.

					Up-	Down-
	Bacteria	Organohalide substrate	Reference substrate	Culture condition	regulated a	regulated a	Method	Reference
			tryptone soy agar	agar plate	569/4 255	580/4 255
				microcosm with plant
	Arthrobacter chlorophenolicus A6	4-chlorophenol	N.A.	(phyllosphere low humidity)	390/4 255	412/4 255	micro-array (Scheublin et al. 2014)
				microcosm with plant
			N.A.	(phyllosphere high humidity)	430/4 255	474/4 255
	Burkholderia xenovorans LB400	aroclor 1242	biphenyl	liquid culture	28/8 249	19/8 429	RNA-seq	(Parnell et al. 2006)
	Dehalococcoides spp. KB1 TM	vinyl chloride	methanol	microcosm	452/19 200 b	95/19 200 b	micro-array (Waller et al. 2012)
		3-chloro-4-hydroxybenzoate
	Desulfitobacterium hafniense DCB-2	3,5-dichlorophenol	uranyl acetate, sodium selenate, ferric citrate	liquid culture	N.A. c	N.A.	micro-array (Kim et al. 2012)
		ortho-bromophenol
	Desulfitobacterium hafniense Y51	trichloroethene	MMYPF-medium (pyruvate and	liquid culture	24/5 060	N.A./5 060	micro-array (Peng et al. 2012)
			fumarate)
	Methylorubrum extorquens CM4	chloromethane	methanol	liquid culture	137/6 262	13/6 262	RNA-seq	(Chaignaud et al. 2017)
	Methylorubrum extorquens DM4	dichloromethane	methanol	liquid culture	69 /5 701	121/5 701	RNA-seq	(Chaignaud et al. 2017)
	Nitrosomonas europaea	chloromethane	mineral medium	liquid culture	67 /2 460	148/2 460	micro-array (Gvakharia et al. 2007)
		chloroform	mineral medium	liquid culture	175/2 460	501/2 460
	Pigmentiphaga sp. H8	3,5-dibromo-4-hydroxybenzoate non induced culture	liquid culture	98/5 678	31/5 678	RNA-seq	(Chen et al. 2018)
							micro-array,
	Polaromonas sp. JS666	cDCE	glycolate	liquid culture	140/5 569	450/5 569		(Jennings et al. 2009)
							proteomics
				liquid culture	129/3 524	87/3 524
	Rhodococcus aetherivorans I24	aroclor 1254	glucose	microcosm (contaminated	207/3 524	176/3 524	micro-array (Puglisi et al. 2010)
				Rotterdam harbour sediments)	44/3 524	67/3 524
	Salmonella typhimurium TA100	MX chlorinated furanone	dimethyl sulfoxide	liquid culture	169/4 253 b	micro-array (Ward et al. 2007)

a

Table 3 .

 3 Differentially regulated genes in bacterial consortia growing with organohalides

				Partner regulated gene number a	Method	Reference
	Organohalide	Culture condition	Consortia partners
				Up-regulated Down-regulated
			Variovorax sp WDL1 b	1 372
	linuron,	liquid culture	Comamonas testosteroni WDL7	169		RNA-seq	(Albers et al. 2018)
	3,4-dichloroaniline		Hyphomicrobium sulfonivorans WDL	N.A. c
			Dehalococcoides mccartyi 195 b	102		micro-array	(Men et al. 2012)
	trichloroethene	liquid culture	Desulfovibrio vulgaris Hildenborough	N.A.	N.A.
			Methanobacterium congolense	N.A.	N.A.
			Dehalococcoides mccartyi 195 b	28	18
		liquid culture
	trichloroethene		Desulfovibrio vulgaris Hildenborough	N.A.	N.A.	micro-array	(Men et al. 2014)
		with vitamin B12	Pelosinus fermentans R7	N.A.	N.A.
			Dehalococcoides mccartyi 195 b	30	44
		liquid culture
	trichloroethene	without vitamin B12	Desulfovibrio vulgaris Hildenborough	N.A.	N.A.	micro-array	(Men et al. 2014)
			Pelosinus fermentans R7	N.A.	N.A.
		microcosm				RNA-seq,
	trichloroethene	(contaminated groundwater	7 bin genomes	550			(Men et al. 2017)
						micro-array
		with or without cobalamin)
			Dehalococcoides mccartyi 195 b
	trichloroethene	liquid culture		18	196	micro-array	(Mao et al. 2015)
			Syntrophomonas wolfei

a Number of genes regulated; b Dehalogenating partner; c N.A., Not Applicable, no numbers provided.

Funding. BM was funded by a doctoral grant of the French Goverment. FB was awarded with an IdeX (Projet initiative d'excellence) grant of the University of Strasbourg.

Conflicts of Interests. None declared.