3.3. Response of WCBs to Lead
Environmental lead (Pb) is either naturally occurring or resulting from anthropogenic activity given its use in automotive batteries, telecommunications, construction, and its erstwhile use in pipes and paints among others. The toxicity threshold of lead to humans and animals is quite low, with chronic exposure resulting in anemia, neurotoxicity, and severe renal damage
[53][39].
PbrR, a transcriptional regulator and part of the
MerR subfamily, mediates the Pb(II)-induced transcription from its divergent promoter and regulates the
pbr operon as part of some microorganisms’ lead detoxification systems
[54][40]. The
pbr operon was first identified in
Cupriavidus metallidurans CH34 and encodes a particularly comprehensive Pb resistance mechanism which entails transport, efflux, sequestration, precipitation, and biomineralization
[55][41]. As such, components from the
pbr operon can and have been successfully utilized in the conception of Pb-sensing WCBs producing various readouts
[56,57,58][42][43][44].
The indigoidine reporter module described earlier was also utilized in conjunction with a Pb(II) sensory module comprised of the
PbrR transcription regulator and its divergent promoter
[47][45]. The DNA fragments bearing
pbrR the
pbrR-divergent
pbr promoter, borne on the pT-Ppbr plasmid constructed in an earlier study
[59][46], were amplified and cloned into the pT7lac-ind plasmid to yield pPpbr-ind, which was then transformed into
E. coli TOP10 competent cells. The output signal is reliably produced 3 h post-incubation with the inductor present, and it can be spectrophotometrically assessed at λ = 600 nm to derive the concentration of bioavailable Pb(II). The authors noted that WCBs harvested at the lag phase were more apt at detecting Pb within the 0.13–4.17 µM range, whereas those harvested at the exponential phase produced the best results within the 0.26–8.3 µM range. While a color change detectable by the human eye can only be observed at 4.17 µM, the method is still conducive to a rapid and reliable spectrophotometric assessment of Pb content in waters suspected of contamination.
Pb(II)-dependent metalloregulator protein PbrR was also used as a component of a sensory module in an
E. coli TOP10-based WCB leveraging the violacein biosynthetic pathway
[43][38]. The violacein synthetic pathway was first cloned onto a pET21a plasmid to yield pET-vio. Subsequently, the DNA fragment containing gene
pbrR and its divergent promoter
Ppbr, borne on the pT-Ppbr plasmid constructed in an earlier study
[59][46], was PCR-amplified and cloned onto the pET-vio plasmid to yield pPpbr-vio used to transform
E. coli TOP10. Three hours post-incubation in Pb(II)-containing media, the produced violacein could be extracted using butanol and quantified at λ = 490 nm. A linear relationship between Pb(II) concentration and violacein concentration was observed within the 0.1875–1.5 mM range. Beyond the upper limit of the range, the sensor exhibited qualitative properties up to 24 µM, which was the highest tested concentration. An advantage proffered by the use of violacein in this sensor was the ability to visually detect amounts of Pb(II) in the medium at levels below 0.1875 mM.
Building upon this previously mentioned work, the researchers resorted to subcloning techniques to generate plasmids pPpbr-vioABE, pPpbr-vioABDE, and pPpbr-vioABCE from pPpbr-vio
[60][47]. Each of the plasmids including pPpbr-vio was transformed into
E. coli TOP10 to create four WCBs relying on different reporter pigments. The violacein- and deoxyviolacein-producing strains were retained for later assays given that the expected pigment output was produced as opposed to what was observed with the remaining strains. This occurrence can be attributed to the instability of prodeoxyviolacein and proviolacein intermediates as well as the highly branched metabolic pathway culminating in violacein synthesis
[61][48]. The deoxyviolacein-based biosensor displayed considerable efficacy and a narrow dose-dependent response across a broad range of Pb(II) concentrations spanning 2.93–6000 nM, whereas the violacein-based one exhibited a less narrow dose-dependent response between 2.93 nM and 750 nM.
3.4. Response of WCBs to Mercury
The highly toxic nature of bioaccumulative mercury (Hg) coupled with its relative abundance in the environment make it a veritable hazard to public health. Several bacteria have developed resistance mechanisms to the metal. These detoxification mechanisms, enabled by an inducible set of genes arranged in a single
MerR operon under the control of the metal-sensing MerR protein
[66][49], have been exploited in a number of contexts to create crucial biosensors enabling the safeguarding of human and animal health.
The polyvalence of the indigoidine reporter module is further evinced in its robustness at signaling the presence of Hg(II) in an
E. coli-based WCB
[47][45]. This was achieved by coupling the reporter module with a sensor module consisting of the
MerR gene encoding the metalloregulator protein MerR and its divergent
mer promoter region, which had been synthetically produced and introduced into a pET-21a plasmid to generate Ppmer
[67][50]. The DNA fragments contained in Ppmer were PCR amplified and cloned into the previously referenced pT7lac-ind plasmid to generate pPmer-ind, which was then transformed into
E. coli TOP10. WCBs harvested at the exponential phase of bacterial development were found to reliably signal and quantify Hg(II), which induced a dose-responsive indigoidine biosynthesis at a concentration range spanning 0.008–0.52 µM, with the color of the pigment exceeding the human eye detection threshold at Hg(II) concentrations above 0.033 µM.
The violacein reporter developed by Hui et al. and utilized to detect Pb in an earlier work
[43][38] was repurposed in the creation of a Hg(II) biosensor
[68][51]. The Ppmer sensor module which was constructed by Zhang et al.
[67][50] was also utilized in this undertaking. To assemble the sensory and reporter apparatuses, the researchers PCR amplified the DNA fragment containing
merR gene and its divergent
mer promoter from the Ppmer plasmid and cloned them into pET-vio to generate pPmer-vio. The recombinant plasmid was transformed into
E. coli TOP10 to yield a Hg-sensing WCB which would respond to Hg by producing the violet pigment violacein. A dependable readout from WCB cells harvested during the exponential phase was obtained 5 h post-induction, and it exhibited a dose-dependent pigment-based response to Hg(II) in the range of 0.78–12.5 µM. Beyond this upper limit, violacein synthesis reportedly decreased as a result of toxicity. Inductive amounts of Hg(II) equal to and beyond 6.25 µM incurred the production of enough pigment to be detected by the human eye post-extraction using butanol. Sensors harvested at the lag phase exhibited a dose-dependent response to Hg(II) within the 0–0.12 µM range, thus allowing for the quantification of more minute amounts of Hg(II). Violacein was visible with the human eye post-extraction at Hg(II) concentrations withing the 0.006–0.098 µM range and the intensity of the violet color diminished past the Hg(II) concentration of 0.024 µM due to cytotoxic effects.
Transcription regulator MerR has also been used as a sensor module in a
P. aeruginosa WCB employing reporter genes
phzM and
phzS, encoding for the enzymes methyltransferase and flavin-containing monooxygenase, respectively
[69][52]. These enzymes catalyze the synthesis of pyomelanin
[70][53], a red–brown pigment with potent antioxidative properties protecting microorganisms from oxidative stress
[71][54]. A recombinant plasmid carrying
merR under the control of native promoter and the
phzM and
phzS genes under the control of the
mer promoter, was transformed into
P. aeruginosa PAO1. The WCB worked well within a broad pH range, proved to be highly selective by responding poorly to other metal ions and produced a dose-dependent response to Hg(II) between 25 and 1000 nM. Prior to spectrophotometric quantification of pyocyanin to derive Hg(II) concentrations, the hydrophobic pigment must be extracted from the cells using chloroform and hydrochloric acid.
3.5. Response of WCBs to Arsenic
Over one hundred million people are effected by arsenic (As) water contamination across the world and are thus prone to developing skin lesions and gastrointestinal distress in case of chronic exposure to low doses, although high concentrations pose a much greater toxicity risk to human health
[72][55]. A number of WCBs were elaborated utilizing the
ars operon, which consists of two regulatory genes (
arsR and
arsD) and three structural genes (
arsA,
arsB, and
arsC) and contributes to arsenite and arsenate resistance by detoxifying the cell
[73][56]. Highly contaminated areas include Indian and Bangladeshi industrial zones, and remediation must be enabled by access to cheap and dependable technologies.
The bright colors of carotenoid pigments spheroiden and spheroidenone were exploited in the creation of an arsenite biosensor
[74][57]. In its wild form, photosynthetic bacterium
Rhodovulum sulfidophilum produces the red pigment spheroidenone in semi-aerobic conditions via the spheroidone pathway enabled by genes
crtF and
crtA. The former encodes for O-methyltransferase which acts upon the C-1 hydroxy group of demethylspheroidene resulting in the synthesis of yellow spheroiden, and the latter codes for a monooxygenase subjecting the spheroiden produced in the earlier step to a C-2 ketolation thus yielding red spheroidenone. As such, a mutant strain with the
crtA gene deleted, such as
R. sulfidophilum CDM2, would accumulate yellow pigments. To create the arsenite sensor, Fujimoto et al. relied on a strategy predicated on a color shift from yellow to red using
R. sulfidophilum CDM2. To that end, they constructed a reporter module consisting of a promoter-less fragment of the
crtA gene which was cloned onto a broad-host-range plasmid pRK415 together with the
E. coli-derived sensory module comprising the arsenite responsive
E. coli DNA fragment containing the operator/promoter of the
ars operon (
O/pars) as well as the the
arsR repressor. The recombinant plasmid, pSENSE-As, was transformed into
E. coli S17-1 and transferred into
R. sulfidophilum CDM2 through conjugation. Preliminary assessments confirmed that
E. coli O/Pars was recognizable by CDM2 RNA polymerase and that no transcription repression by an endogenous protein occurred in CDM2.
4. Biomonitoring and Control
4.1. High-Level Producer Detection
Lysine is an amino acid with considerable importance in the context of human and animal nutrition and is the second most abundantly produced essential nutrient worldwide
[82,83][58][59].
Corynebacterium glutamicum is an effective production platform of L-lysine and other amino acids, and engineered strains have been turned into industrial workhorses specially created for this purpose
[84][60].
A notable drawback on pSenLys-based sensors is their inability to accurately report the overproduction of a specific amino acid among lysine, histidine, and arginine. The non-specificity of pSenLys prompted the exploration of different avenues. Liu et al. detailed the development of a
C. glutamicum based colorimetric WCB with greater lysine specificity, utilizing lycopene as a reporter pigment
[11]. In response to L-Lys,
LysG activates the expression of
crtI encoding phytoene desaturase which then catalyzes the production of lycopene with the characteristic red color. To that end, a
C. glutamicum mutant strain, deficient of the carotenogenic gene cluster
crtIYe/fEb,
crtB2I21/2, and
LysEG was first generated as a sensor chassis. To construct the plasmid, transcriptional regulator
lysG; its binding site region
lysE promoter; and the phytoene desaturase gene
crtI were amplified using the genome of
C. glutamicum as a template. The expression cassette was fused using overlap extension PCR and cloned into a pTRCmob vector plasmid dubbed pSensorI. The sensor plasmid was transformed into
C. glutamicum WT-
ΔlysEGΔcrtIYEbB22 and this transformed strain bearing pSensorI served as a control as other optimizations were implemented. To remedy the poor specificity of
lysG and diminish false positives induced by docking of L-histidine and L-arginine,
LysG was subjected to site-directed saturation mutagenesis to screen for mutants with reduced affinity to L-histidine and L-arginine. Substitutions at positions 123 and 125—where L-glutamate was substituted with L-L-tyrosine and where L-glutamate was replaced with L-alanine, respectively—were found to confer the modified binding site
lysG* a drastically reduced affinity to L-arginine and L-histidine and an uncompromised colorimetric linear response to L-lysine. To increase the range of dose-responsiveness up from the reported 40 mM limit, the researchers resorted to promoter engineering of
pLysE and 5 promoters were screened. Promoter
pLysE-3 was selected as the most apt candidate and was found to engender an increased range of responsiveness of up to 300 mM. To enhance the performance of the sensor from a color-rendering standpoint and thus facilitate overproducer detection, the
CrtR transcriptional regulator, which is known to repress the
crt operon
[86][61], was deleted by electroporating a suicide plasmid into WT-
ΔlysEGΔcrtIYEbB2 to construct WT-
ΔlysEGΔcrtIYEbB2R.
4.2. Pathogen Detection
N-acyl homoserine lactone (AHL) is a signal molecule utilized by a number of gram-negative bacteria for cell-to-cell communication as part of the quorum sensing mechanism. The utility of WCBs leveraging the quorum sensing apparatus of microorganisms has a number of benefits namely signaling the presence of possibly pathogenic species
[87][62], monitoring bacterial populations in bioreactor settings
[88][63], and modulating the microbial composition of a medium
[89][64].
N-butyryl-L-homoserine lactone (BHL) is an AHL and a small diffusible signaling molecule implicated in quorum sensing, the control of gene expression, and cellular metabolism
[90][65]. To detect minute amounts of BHL within a wide concentration ambit, Yong and Zhong developed a
P. aeruginosa-based biosensor
[91][66]. The researchers used strain
P. aeruginosa CGMCC 1.860, which is naturally capable of producing blue–green pigments upon detecting BHL. This is achieved through the RhlR-RhlI quorum-sensing system, which comprises the transcription activator protein RhlR and the BHL synthase RhlI
[92][67]. To create a biosensor, the researchers deleted the
rhlIR gene cluster, thus creating
P. aeruginosa ΔrhlIR, and overexpressed
rhlR through multi-copy plasmids. As such, the bacteria regained the capacity of sensing BHL while avoiding the production of the analyte by endogenous activity. The recombinant biosensor strain is thus capable of producing the pigment upon sensing of exogenous BHL which can diffuse in the cell and be recognized by the RhlR regulator. Upon BHL binding, this transcription regulator activates the expression of pigment synthases. The resulting WCB whose pigment output can be extracted using chloroform and quantified at λ = 299 nm exhibited dose-dependent pigment production within the 0.11–49.7 µM AHL range.
4.3. Micronutrient Quantification
Micronutrient deficiencies are significant concern of global ambit although gauging the veritable magnitude of the issue remains challenging
[102][68]. In remote settings and in impoverished parts of the world, access to reliable testing is limited due to elevated costs and logistical difficulties. Colorimetric WCBs as part of field-ready kits can be handled by agents with minimal training to provide in situ testing in remote areas, identify micronutrient deficiencies, and help remedy health complications quickly and reliably.
Zinc is an essential micronutrient; deficiencies have incurred public health burdens of significant magnitude, and one billion people across the world are presumed to be at risk of zinc deficiency
[103][69]. Efforts to provide access to impoverished and remote areas of the world have yielded the development of colorimetric biosensors.
In the context of early efforts to develop a colorimetric biosensor compatible with zinc serum levels, Watstein and Styczynski generated an
E. coli-DH10B-based sensor capable of producing three different reporters: violacein, lycopene, and β-carotene
[12]. A violacein operon was cloned onto a plasmid bearing zinc-responsive transcription regulator
ZntR, proprietary ribosomal binding sites, and the gene encoding the Zur metalloregulator protein, which acts as a zinc-responsive repressor. Gene cluster
vioABCDE was placed under the control of a
PznuC repressor actuated by Zur–zinc complexes.
5. Conclusions
The development of whole-cell biosensors keeps up with the pace of broad ranging advancements in genetic engineering and practically puts to use novel approaches stemming from recent advancements. As such, it allows for more nuance to materialize and for a greater understanding of processes and mechanisms to be gleaned. WCBs are a highly versatile platform enabling the development of accessible and inexpensive analytical devices which, in some circumstances, replace their less portable laboratory analogues
[109][70]. They can be adapted to a considerable range of analytes that grows as more regulatory as well as biosynthetic pathways are characterized. Moreover, they benefit from a wide selection of thoroughly understood microbial chassis to transform based on the biosensor’s purpose. Indeed, the caveat of these assessments being conducted in highly controlled settings must be borne in mind. In effect, they were mostly undertaken using solutions of known analyte concentrations and compositions. Their effectiveness in the field may be less pronounced due to a host of causes including the general complexity of natural matrices which may contain compounds of bacterial origin or otherwise which may affect their performance or result in false positives and negatives.