Search Results (7)

This review attempts to summarize my three decades-long involvement in, and contribution to, the design, construction and testing of bioluminescent microbial sensor strains (bioreporters). With the understanding that such a document cannot be completely free of bias, the review focuses on studies from my own lab only, with almost no coverage of the parallel progress made by others in similar fields. This admittedly subjective approach by no way detracts from the achievements of countless excellent researchers who are not mentioned here, and whose contributions to the field are at least as important as that of my own. The review covers basic aspects of microbial sensor design, and then progresses to describe approaches to performance improvement of sensor strains aimed at the detection of either specific chemicals, groups of chemicals sharing similar characteristics, or global effects, such as toxicity and genotoxicity. The need for integration of live sensor cells into a compatible hardware platform is highlighted, as is the importance of long-term maintenance of the cells’ viability and activity. The use of multi-member sensors’ panels is presented as a means for enhancing the detection spectrum and sample “fingerprinting”, along with a list of different purposes to which such sensors have been put to use. Full article

The continuous emergence of new illegal compounds, particularly psychoactive chemicals, poses significant challenges for current drug detection methods. Developing new protocols and kits for each new drug requires substantial time, effort, and dedicated manpower. Whole-cell bacterial bioreporters have been proven capable of detecting diverse hazardous compounds in both laboratory and field settings, identifying not only single compounds but also chemical families. We present the development of a microbial bioreporter for the detection of cocaine, the nervous system stimulant that is the second-most widely used illegal drug in the US. Escherichia coli was transformed with a plasmid containing a bacterial luxCDABEG bioluminescence gene cassette, activated by a cocaine-responsive signaling cascade. The engineered bioreporter is demonstrated to be a sensitive and specific first-generation detection system for cocaine, with detection thresholds of 17 ± 8 μg/L and 130 ± 50 μg/L in a buffer solution and in urine, respectively. Further improvement of the sensor’s performance was achieved by altering the nucleotide sequence of the PBen gene promoter, the construct’s sensing element, using accelerated site-directed evolution. The applicability of ready-to-use paper strips with immobilized bioreporter cells was demonstrated for cocaine detection in aqueous solutions. Full article

We present a generic quantitative chemical sensing methodology for assessing the concentration of a target material (TM) in an aqueous solution by using bioluminescent microbial bioreporters as the core sensing elements. Such bioreporters, genetically engineered to respond to the presence of a TM in their microenvironment by emitting bioluminescence, have previously been mostly designed to report the presence or absence of the TM in the sample. We extend this methodology to also assess the TM concentration, by exploiting the dose-dependency of the TM-induced luminescence. To overcome luminescence intensity variations due to bacterial batch differences and the ambient temperature, simultaneous measurements were carried out on sample solutions containing known concentrations of the TM. A “standard ratio” parameter, defined as the ratio between the two measurements, is shown to be independent of the bacterial batch and the temperature, and hence provides the conceptual basis for a generic quantitative chemical sensing methodology. Assessment of 2,4-dinitrotoluene (DNT) concentration in solutions is demonstrated with an accuracy of 2.5% over a wide dynamic range. Full article

Quorum Sensing (QS) is a well-studied intercellular communication mechanism in bacteria, regulating collective behaviors such as biofilm formation, virulence, and antibiotic resistance. However, cell–cell signaling in haloarchaea remains largely unexplored. The coexistence of bacteria and archaea in various environments, coupled with the known cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms and the presence of cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms, suggests a possibility for haloarchaea to possess analogous cell–cell signaling or QS systems. Recently, N-acylhomoserine lactone (AHL)-like compounds were identified in haloarchaea; yet, their precise role—for example, persister cell formation—remains ambiguous. This study investigated the capacity of crude supernatant extract from the haloarchaeon Halorubrum saccharovorum CSM52 to stimulate bacterial AHL-dependent QS phenotypes using bioreporter strains. Our findings reveal that these crude extracts induced several AHL-dependent bioreporters and modulated pyocyanin and pyoverdine production in Pseudomonas aeruginosa. Importantly, our study suggests cross-domain communication between archaea and bacterial pathogens, providing evidence for archaea potentially influencing bacterial virulence. Using Thin Layer Chromatography overlay assays, lactonolysis, and colorimetric quantification, the bioactive compound was inferred to be a chemically modified AHL-like compound or a diketopiperazine-like molecule, potentially involved in biofilm formation in H. saccharovorum CSM52. This study offers new insights into putative QS mechanisms in haloarchaea and their potential role in interspecies communication and coordination, thereby enriching our understanding of microbial interactions in diverse environments. Full article

Landmines and explosive remnants of war pose a global humanitarian problem and cause numerous casualties long after the conflict has ended. The current approaches for locating landmines, such as metal detection, which require one’s physical presence at the minefield, involve high risk to personnel; these methods are also costly, time-consuming, and have a high rate of false-positive results. There is no currently viable technology for the remote detection of buried explosive devices. A possible solution to this may be the use of genetically engineered microorganisms, molecularly “tailored” to emit an optical signal in the presence of trace explosives escaping for the landmine and accumulating in the soil above it. This optical signal, imaged from a remote location, can then be used to generate a physical map of the mine’s location. A few years ago, we have described the remote detection of buried landmines using alginate-encapsulated fluorescent microbial (Escherichia coli) bioreporters spread over the tested minefield. Since then, we have modified the system to one based on bioluminescent (rather than fluorescent) bacteria and have employed several synthetic biology approaches to significantly enhance their major performance parameters: higher signal intensity, faster response time, and lower detection threshold of the target explosives. These molecular approaches and their effect on sensor performance will be described. Full article

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering. Full article

The present study aims to monitor the ability of Salmonella to colonize and compete as a member of the mixed species biofilm within key points at a water bottling plant, in case of a contamination incident with this major foodborne pathogen. To achieve this goal, bacterial communities throughout the production line were collected and their identities were investigated by microbial counts and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). These bacterial communities alone or along with constructed Salmonella enterica serovar Typhimurium (ST) fluorescence-based bioreporters were left to form a biofilm on stainless steel for 6 days at 20 °C. ST bioreporters were constructed by introducing plasmids expressing EYFP (enhanced yellow fluorescent protein) fusions of the genes csgB, csrA, sspH2, and fliD into ST 14028S. The bead vortexing-plate counting method was applied for the enumeration of the biofilm population, while the behavior of the bioreporters was evaluated by fluorescence microscopy. From a set of 16 samples that were collected from the plant, species of Citrobacter, Staphylococcus, Pseudomonas, Bacillus, and Exiguobacterium were identified. The presence of these indigenous bacteria neither inhibited nor enhanced the biofilm formation of ST in mixed bacterial communities (p > 0.05). Furthermore, the csrA-based bioreporter was shown to be induced in multispecies biofilms with Citrobacter. In conclusion, this study enhanced our knowledge of bacterial interactions occurring within a biofilm in a water bottling plant. Full article