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Constructing of Bacillus subtilis-Based Lux-Biosensors with the Use of Stress-Inducible Promoters.

ABSTRACT: Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H₂O₂. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2'-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.

SUBMITTER: Kessenikh AG

PROVIDER: S-EPMC8431380 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Engineering strong and stress-responsive promoters in Bacillus subtilis by interlocking sigma factor binding motifs.

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Bacillus subtilis WalR (YycF) ChIP-chip Bacillus subtilis; Bacillus subtilis subsp. subtilis str. 168

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Project description:Inorganic trivalent arsenic is a major environmental pollutant and exposure to human results in many pathologies, including keratosis and carcinoma. Here, we analyzed the effects of B. subtilis spores on human normal keratinocytes in the presence of sodium arsenite oxidative stress. Pre-treatment of cells with spores before inducing oxidative stress was able to keep normal levels of intracellular ROS, GSH and lipid peroxidation, as well as to inhibit the activation of the MAPK cascade. Moreover, spores showed a positive effect on cell proliferation, probably due to their binding on the cell surface and the activation of intracellular catalases. We found that spores exert their protective effect by the nuclear translocation of Nrf-2, involved in the activation of stress response genes. This, in turn, resulted in a protective effect against sodium arsenite stress injury, as oxidative stress markers were reported to physiological levels when cells were stressed before incubating them with spores. Therefore, B. subtilis spores can be considered as a new agent to counteract oxidative stress on normal human keratinocytes.

| S-EPMC5788939 | biostudies-literature

Membrane protein overproduction stress in Bacillus subtilis

Project description:Comparison of the transcriptome of Bacillus subtilis under going membrane protein overproduction in the wild type, sigW and cssRS deletion strains. We demonstrate that the dynamics of the stress systems involved in membrane overproduction are far more complicated than was first hypothesised and that many more systems than SigW and CssRS are involved in membrane protein overexpression stress. Interestingly the cssRS genes are repressed in the sigW deletion strain.

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Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis.

Project description:BackgroundEnzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called 'cellulosome'. Hence, we proposed a "biomimic operon" concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis.ResultsAccording to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon.ConclusionsIn this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.

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The transcriptional repressor CcpN from Bacillus subtilis uses different repression mechanisms at different promoters.

Project description:CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under non-repressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP alpha-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.

| S-EPMC2781557 | biostudies-literature

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