Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2

The bacterial bioluminescence system enables light production in living cells without an external luciferin. Due to its relatively low levels of light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with 7-fold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

Development of Cellular and Enzymatic Bioluminescent Assay Systems to Study Low-Dose Effects of Thorium

Thorium is one of the most widespread radioactive elements in natural ecosystems, along with uranium, it is the most important source of nuclear energy. However, the effects of thorium on living organisms have not been thoroughly studied. Marine luminescent bacteria and their enzymes are optimal bioassays for studying low-dose thorium exposures. Luminescent bioassays provide a quantitative measure of toxicity and are characterized by high rates, sensitivity, and simplicity. It is known that the metabolic activity of bacteria is associated with the production of reactive oxygen species (ROS). We studied the effects of thorium-232 (10−11–10−3 M) on Photobacterium phosphoreum and bacterial enzymatic reactions; kinetics of bacterial bioluminescence and ROS content were investigated in both systems. Bioluminescence activation was revealed under low-dose exposures (<0.1 Gy) and discussed in terms of “radiation hormesis”. The activation was accompanied by an intensification of the oxidation of a low-molecular reducer, NADH, during the enzymatic processes. Negative correlations were found between the intensity of bioluminescence and the content of ROS in bacteria and enzyme systems; an active role of ROS in the low-dose activation by thorium was discussed. The results contribute to radioecological potential of bioluminescence techniques adapted to study low-intensity radioactive exposures.

Modeling the underlying environmental factors of milky sea case and luminous bacteria presence in Java Southern Sea in 2019

The milky sea is one of the unique natural phenomena caused by the presence of luminous Vibrio bacteria in marine ecosystems. Recently a milky sea has been reported frequently included in the Java Southern Sea. Simultaneously, numerous remote sensing based approaches have been developed to detect the presence of luminous bacteria and the milky sea. Despite this state of the art, the information of detrimental factors of the marine bioluminescence was still limited. Then this research aims to model the underlying environmental factors causing the milky sea and luminous bacteria presence in the Java Southern Sea in 2019. The remote sensing assessment for the period of July 29-August 6, 2019 shows that the magnitude of bioluminescence measured in radiance was having a maximum value of 255 nanoW/cm2/sr and an average of 107 nanoW/cm2/sr/day (95%CI: 71.9 to 142 nanoW/cm2/sr/day). The milky sea size increased and reached its peak with a size of 44,124 km2 and then declined. The average milky sea size was 37,942 km2 (95% CI: 33,400 to 42,500 km2) and increased with average rate of 16.01% (95%CI: 5.41% to 26.66%). While Akaike Information Criterion (AIC) indicates that the best model to infer the relationship of bacterial bioluminescence with its environmental factors contained Chlorophyll a followed by sea surface temperature factors with AICc values of 101.16 (AICweight: 0.50) and 101.95 (AICweight: 0.34). This indicates that low temperature and high plankton cells is the limiting factors of the bacterial bioluminescence.

Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

Protein Model and Function Analysis in Quorum-Sensing Pathway of Vibrio qinghaiensis sp.-Q67

Bioluminescent bacteria are mainly found in marine habitats. Vibrio qinghaiensis sp.-Q67 (Q67), a nonpathogenic freshwater bacterium, has been a focus due to its wide use in the monitoring of environmental pollution and the assessment of toxicity. However, the lack of available crystal structures limits the elucidation of the structures of the functional proteins of the quorum-sensing (QS) system that regulates bacterial luminescence in Q67. In this study, 19 functional proteins were built through monomer and oligomer modeling based on their coding proteins in the QS system of Q67 using MODELLER. Except for the failure to construct LuxM due to the lack of a suitable template, 18 functional proteins were successfully constructed. Furthermore, the relationships between the function and predicted structures of 19 functional proteins were explored one by one according to the three functional classifications: autoinducer synthases and receptors, signal transmission proteins (phosphotransferases, an RNA chaperone, and a transcriptional regulator), and enzymes involved in bacterial bioluminescence reactions. This is the first analysis of the whole process of bioluminescence regulation from the perspective of nonpathogenic freshwater bacteria at the molecular level. It provides a theoretical basis for the explanation of applications of Q67 in which luminescent inhibition is used as the endpoint.

Evaluation of the Effects of Particle Sizes of Silver Nanoparticles on Various Biological Systems

Seven biological methods were adopted (three bacterial activities of bioluminescence, enzyme, enzyme biosynthetic, algal growth, seed germination, and root and shoot growth) to compare the toxic effects of two different sizes of silver nanoparticles (AgNPs). AgNPs showed a different sensitivity in each bioassay. Overall, the order of inhibitory effects was roughly observed as follows; bacterial bioluminescence activity ≈ root growth > biosynthetic activity of enzymes ≈ algal growth > seed germination ≈ enzymatic activity > shoot growth. For all bacterial activities (bioluminescence, enzyme, and enzyme biosynthesis), the small AgNPs showed statistically significantly higher toxicity than the large ones (p < 0.0036), while no significant differences were observed among other biological activities. The overall effects on the biological activities (except shoot growth) of the small AgNPs were shown to have about 4.3 times lower EC50 (high toxicity) value than the large AgNPs. These results also indicated that the bacterial bioluminescence activity appeared to be an appropriate method among the tested ones in terms of both sensitivity and the discernment of particle sizes of AgNPs.

Comparative Effects of Particle Sizes of Cobalt Nanoparticles to Nine Biological Activities

The differences in the toxicity of cobalt oxide nanoparticles (Co-NPs) of two different sizes were evaluated in the contexts of the activities of bacterial bioluminescence, xyl-lux gene, enzyme function and biosynthesis of β-galactosidase, bacterial gene mutation, algal growth, and plant seed germination and root/shoot growth. Each size of Co-NP exhibited a different level of toxicity (sensitivity) in each biological activity. No revertant mutagenic ratio (greater than 2.0) of Salmonella typhimurium TA 98 was observed under the test conditions in the case of gene-mutation experiments. Overall, the inhibitory effects on all five bacterial bioassays were greater than those on algal growth, seed germination, and root growth. However, in all cases, the small Co-NPs showed statistically greater (total average about two times) toxicity than the large Co-NPs, except in shoot growth, which showed no observable inhibition. These findings demonstrate that particle size may be an important physical factor determining the fate of Co-NPs in the environment. Moreover, combinations of results based on various biological activities and physicochemical properties, rather than only a single activity and property, would better facilitate accurate assessment of NPs’ toxicity in ecosystems.