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

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.

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Lux-biosensors: screening biologically active compounds for genotoxicity

Ecological genetics ◽

10.17816/ecogen14452-62 ◽

2016 ◽

Vol 14 (4) ◽

pp. 52 ◽

Cited By ~ 5

Author(s):

Elena V Igonina ◽

Mariya V Marsova ◽

Serikbai K Abilev

Keyword(s):

Oxidative Stress ◽

Mutagenic Activity ◽

Sos Response ◽

Biologically Active ◽

Published Data ◽

E Coli ◽

Lux Operon ◽

Lux Biosensors ◽

Catalase Peroxidase ◽

Pharmacologically Active

To study the ability of metal salts and pharmacologically active drugs to induce the oxidative stress and SOS response in bacteria, a set of lux-biosensors was used. The sensors were based on three E. coli strains carrying recombinant plasmids with lux-operon fused to the promoters of SoxS (superoxide dismutase), KatG (catalase-peroxidase) and ColD (colicin D) genes. The created biosensors were used to analyze the activity of 47 substances, with 16 of them identified to induce SOS-response and 6 - to induce oxidative stress. The results observed were compared to the previously published data on the mutagenic activity of the same 47 substances evaluated using Ames test. The comparison had shown full coincidence for 42 from 47 substances analyzed. We discuss the possibility to use the lux-based biosensors for the screening of the genetic activity of chemical compounds.

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A (p)ppGpp-Null Mutant of Haemophilus ducreyi Is Partially Attenuated in Humans Due to Multiple Conflicting Phenotypes

Infection and Immunity ◽

10.1128/iai.01994-14 ◽

2014 ◽

Vol 82 (8) ◽

pp. 3492-3502 ◽

Cited By ~ 9

Author(s):

Concerta Holley ◽

Dharanesh Gangaiah ◽

Wei Li ◽

Kate R. Fortney ◽

Diane M. Janowicz ◽

...

Keyword(s):

Oxidative Stress ◽

Parent Strain ◽

Double Mutant ◽

Bacterial Species ◽

Stringent Response ◽

Haemophilus Ducreyi ◽

Content Type ◽

E Coli ◽

Human Volunteers ◽

Increased Sensitivity

ABSTRACT(p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species.Haemophilus ducreyi, the causative agent of chancroid, has homologs of bothrelAandspoT, which primarily synthesize and hydrolyze (p)ppGpp inEscherichia coli. We constructedrelAandrelA spoTdeletion mutants to assess the contribution of (p)ppGpp toH. ducreyipathogenesis. Both therelAsingle mutant and therelA spoTdouble mutant failed to synthesize (p)ppGpp, suggesting thatrelAis the primary synthetase of (p)ppGpp inH. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented intrans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription oflspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. InE. coli, overexpression of DksA compensates for the loss of (p)ppGpp; theH. ducreyidouble mutant expressed higher transcript levels ofdksAthan the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.

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Antagonistic activity of Bacillus subtilis strains isolated from various sources

Ukrainian Journal of Ecology ◽

10.15421/2018_354 ◽

2018 ◽

Vol 8 (2) ◽

pp. 354-364

Author(s):

A. N. Irkitova ◽

A. V. Grebenshchikova ◽

A. V. Matsyura

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Wild Strain ◽

Fish Farming ◽

Antagonistic Activity ◽

Antagonistic Effect ◽

E Coli ◽

Long Period ◽

Test Culture ◽

Agar Media

An important link in solving the problem of healthy food is the intensification of the livestock, poultry and fish farming, which is possible only in the adoption and rigorous implementation of the concept of rational feeding of animals. In the implementation of this concept required is the application of probiotic preparations. Currently, there is an increased interest in spore probiotics. In many ways, this can be explained by the fact that they use no vegetative forms of the bacilli and their spores. This property provides spore probiotics a number of advantages: they are not whimsical, easily could be selected, cultivated, and dried. Moreover, they are resistant to various factors and could remain viable during a long period. One of the most famous spore microorganisms, which are widely used in agriculture, is Bacillus subtilis. Among the requirements imposed to probiotic microorganisms is mandatory – antagonistic activity to pathogenic and conditional-pathogenic microflora. The article presents the results of the analysis of antagonistic activity of collection strains of B. subtilis, and strains isolated from commercial preparations. We studied the antagonistic activity on agar and liquid nutrient medias to trigger different antagonism mechanisms of B. subtilis. On agar media, we applied three diffusion methods: perpendicular bands, agar blocks, agar wells. We also applied the method of co-incubating the test culture (Escherichia coli) and the antagonist (or its supernatant) in the nutrient broth. Our results demonstrated that all our explored strains of B. subtilis have antimicrobial activity against a wild strain of E. coli, but to varying degrees. We identified strains of B. subtilis with the highest antagonistic effect that can be recommended for inclusion in microbial preparations for agriculture.

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Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-67-72 ◽

2019 ◽

Vol 35 (6) ◽

pp. 67-72 ◽

Cited By ~ 1

Author(s):

I.V. Manukhov ◽

L.S. Yaguzhinsky ◽

M.V. Bermeshev ◽

M.A. Zisman ◽

V.G. Pevgov ◽

...

Keyword(s):

Toxic Effect ◽

Atmospheric Oxygen ◽

Inducible Promoter ◽

Oxygen Species ◽

Bacterial Cells ◽

Unique Identifier ◽

E Coli ◽

Lux Biosensors ◽

High Level ◽

Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

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Synthesis of Chalcones and Nucleosides Incorporating [1, 3, 4]Oxadiazolenone Core and Evaluation of their Antifungal and Antibacterial Activities

Current Bioactive Compounds ◽

10.2174/1573407214666180911130110 ◽

2020 ◽

Vol 15 (6) ◽

pp. 665-679

Author(s):

Alok K. Srivastava ◽

Lokesh K. Pandey

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Antibacterial Activity ◽

Bacillus Subtilis ◽

Antifungal Activity ◽

Aspergillus Niger ◽

Gram Negative ◽

E Coli ◽

Gram Positive Bacteria ◽

Good Antibacterial Activity

Background: [1, 3, 4]oxadiazolenone core containing chalcones and nucleosides were synthesized by Claisen-Schmidt condensation of a variety of benzaldehyde derivatives, obtained from oxidation of substituted 5-(3/6 substituted-4-Methylphenyl)-1, 3, 4-oxadiazole-2(3H)-one and various substituted acetophenone. The resultant chalcones were coupled with penta-O-acetylglucopyranose followed by deacetylation to get [1, 3, 4] oxadiazolenone core containing chalcones and nucleosides. Various analytical techniques viz IR, NMR, LC-MS and elemental analysis were used to confirm the structure of the synthesised compounds.The compounds were targeted against Bacillus subtilis, Staphylococcus aureus and Escherichia coli for antibacterial activity and Aspergillus flavus, Aspergillus niger and Fusarium oxysporum for antifungal activity. Methods: A mixture of Acid hydrazides (3.0 mmol) and N, Nʹ- carbonyl diimidazole (3.3 mmol) in 15 mL of dioxane was refluxed to afford substituted [1, 3, 4]-oxadiazole-2(3H)-one. The resulted [1, 3, 4]- oxadiazole-2(3H)-one (1.42 mmol) was oxidized with Chromyl chloride (1.5 mL) in 20 mL of carbon tetra chloride and condensed with acetophenones (1.42 mmol) to get chalcones 4. The equimolar ratio of obtained chalcones 4 and β -D-1,2,3,4,6- penta-O-acetylglucopyranose in presence of iodine was refluxed to get nucleosides 5. The [1, 3, 4] oxadiazolenone core containing chalcones 4 and nucleosides 5 were tested to determined minimum inhibitory concentration (MIC) value with the experimental procedure of Benson using disc-diffusion method. All compounds were tested at concentration of 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.62 mg/mL, 0.31 mg/mL and 0.15 mg/mL for antifungal activity against three strains of pathogenic fungi Aspergillus flavus (A. flavus), Aspergillus niger (A. niger) and Fusarium oxysporum (F. oxysporum) and for antibacterial activity against Gram-negative bacterium: Escherichia coli (E. coli), and two Gram-positive bacteria: Staphylococcus aureus (S. aureus) and Bacillus subtilis(B. subtilis). Result: The chalcones 4 and nucleosides 5 were screened for antibacterial activity against E. coli, S. aureus and B. subtilis whereas antifungal activity against A. flavus, A. niger and F. oxysporum. Compounds 4a-t showed good antibacterial activity whereas compounds 5a-t containing glucose moiety showed better activity against fungi. The glucose moiety of compounds 5 helps to enter into the cell wall of fungi and control the cell growth. Conclusion: Chalcones 4 and nucleosides 5 incorporating [1, 3, 4] oxadiazolenone core were synthesized and characterized by various spectral techniques and elemental analysis. These compounds were evaluated for their antifungal activity against three fungi; viz. A. flavus, A. niger and F. oxysporum. In addition to this, synthesized compounds were evaluated for their antibacterial activity against gram negative bacteria E. Coli and gram positive bacteria S. aureus, B. subtilis. Compounds 4a-t showed good antibacterial activity whereas 5a-t showed better activity against fungi.

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Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

Nature Communications ◽

10.1038/s41467-019-13408-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Naomi Shimokawa-Chiba ◽

Claudia Müller ◽

Keigo Fujiwara ◽

Bertrand Beckert ◽

Koreaki Ito ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stop Codon ◽

Release Factor ◽

Positive Bacterium ◽

Gram Positive ◽

E Coli ◽

Cryo Electron Microscopy ◽

Dead End ◽

Bacterium Bacillus Subtilis ◽

Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

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Metabolome and proteome analyses reveal transcriptional misregulation in glycolysis of engineered E. coli

Nature Communications ◽

10.1038/s41467-021-25142-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chun-Ying Wang ◽

Martin Lempp ◽

Niklas Farke ◽

Stefano Donati ◽

Timo Glatter ◽

...

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Metabolic Pathways ◽

Glycerol Production ◽

Inducible Promoters ◽

E Coli ◽

Inhibition Of Glycolysis ◽

Underlying Mechanisms ◽

Rate Limiting ◽

Engineered Bacteria

AbstractSynthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.

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Comparative Proteomic Analysis of Carbonylated Proteins from the Striatum and Cortex of Pesticide-Treated Mice

Parkinson s Disease ◽

10.1155/2015/812532 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Christina Coughlan ◽

Douglas I. Walker ◽

Kelly M. Lohr ◽

Jason R. Richardson ◽

Laura M. Saba ◽

...

Keyword(s):

Oxidative Stress ◽

Pesticide Exposure ◽

Epidemiological Studies ◽

Cytoskeletal Proteins ◽

Synaptic Function ◽

Behavioral Alterations ◽

Mechanisms Of Toxicity ◽

Kegg Pathways ◽

Increased Risk ◽

Proteomic Analyses

Epidemiological studies indicate exposures to the herbicide paraquat (PQ) and fungicide maneb (MB) are associated with increased risk of Parkinson’s disease (PD). Oxidative stress appears to be a premier mechanism that underlies damage to the nigrostriatal dopamine system in PD and pesticide exposure. Enhanced oxidative stress leads to lipid peroxidation and production of reactive aldehydes; therefore, we conducted proteomic analyses to identify carbonylated proteins in the striatum and cortex of pesticide-treated mice in order to elucidate possible mechanisms of toxicity. Male C57BL/6J mice were treated biweekly for 6 weeks with saline, PQ (10 mg/kg), MB (30 mg/kg), or the combination of PQ and MB (PQMB). Treatments resulted in significant behavioral alterations in all treated mice and depleted striatal dopamine in PQMB mice. Distinct differences in 4-hydroxynonenal-modified proteins were observed in the striatum and cortex. Proteomic analyses identified carbonylated proteins and peptides from the cortex and striatum, and pathway analyses revealed significant enrichment in a variety of KEGG pathways. Further analysis showed enrichment in proteins of the actin cytoskeleton in treated samples, but not in saline controls. These data indicate that treatment-related effects on cytoskeletal proteins could alter proper synaptic function, thereby resulting in impaired neuronal function and even neurodegeneration.

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pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

Canadian Journal of Microbiology ◽

10.1139/w98-121 ◽

1998 ◽

Vol 44 (12) ◽

pp. 1186-1192

Author(s):

Guy Daxhelet ◽

Philippe Gilot ◽

Etienne Nyssen ◽

Philippe Hoet

Keyword(s):

Bacillus Subtilis ◽

Binding Site ◽

Transcription Initiation ◽

Promoter Sequence ◽

Initiation Site ◽

Chloramphenicol Acetyltransferase ◽

Ribosome Binding Site ◽

Chloramphenicol Resistance ◽

E Coli ◽

Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

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