The effect of ß-ionone on bacterial cells: the use of specific lux-biosensors.

The diversity of the biological activity of volatile organic compounds (VOCs), including unsaturated ketone ß-ionone, promising pharmacological, biotechnological, and agricultural agent, has aroused considerable interest. However, the functional role and mechanisms of action of VOCs remain insufficiently studied. In this work, the response of bacterial cells to the action of ß-ionone was studied using specific bioluminescent lux-biosensors containing stress-sensitive promoters. We determined that in Escherichia coli cells, ß-ionone induces oxidative stress (PkatG and Pdps promoters) through a specific response mediated by the OxyR/OxyS regulon, but not SoxR/SoxS (PsoxS promoter). It has been shown that ß-ionone at high concentrations (50 µM and above) causes a weak induction of the expression from the PibpA promoter and slightly induces the PcolD promoter in the E. coli biosensors; the observed effect is enhanced in the ΔoxyR mutants. This indicates the presence of some damage to proteins and DNA. ß-Ionone was found to inhibit the bichaperone-dependent DnaKJE-ClpB refolding of heat-inactivated bacterial luciferase in E. coli wild-type and ΔibpB mutant strains. In the cells of the Gram-positive bacterium Bacillus subtilis 168 pNK-MrgA ß-ionone does not cause oxidative stress. Thus, in this work, the specificity of bacterial cell stress responses to the action of ß-ionone was shown.

A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering.

CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid.

A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering.

CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we generated and characterized a library of 137 promoters and built a de novo Yarrowia lipolytica strain synthetizing 373.8 mg/L homogentisic acid.

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50-100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.

LitR directly upregulates autoinducer synthesis and luminescence in Aliivibrio logei.

LitR is a master-regulator of transcription in the ainS/R and luxS/PQ quorum sensing (QS) systems of bacteria from Vibrio and Aliivibrio genera. Here, we for the first time directly investigated the influence of LitR on gene expression in the luxI/R QS system of psychrophilic bacteria Aliivibrio logei. Investigated promoters were fused with Photorhabdus luminescens luxCDABE reporter genes cassette in a heterological system of Escherichia coli cells, litR A. logei was introduced into the cells under control of P lac promoter. LitR has been shown to upregulate genes of autoinducer synthase (luxI), luciferase and reductase (luxCDABE), and this effect doesn't depend on presence of luxR gene. To a much lesser degree, LitR induces luxR1, but not the luxR2 - the main luxI/R regulator. Enhanced litR expression leads to an increase in a LuxI-autoinducer synthesis and a subsequent LuxR-mediated activation of the luxI/R QS system. Effect of LitR on luxI transcription depends on lux-box sequence in luxI promoter even in absence of luxR (lux-box is binding site of LuxR). The last finding indicates a direct interaction of LitR with the promoter in the lux-box region. Investigation of the effect of LitR A. logei on luxI/R QS systems of mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio salmonicida showed direct luxR-independent upregulation of luxI and luxCDABE genes. To a lesser degree, it induces luxR A. fischeri and luxR1 A. salmonicida. Therefore, we assume that the main role of LitR in cross-interaction of these three QS systems is stimulating the expression of luxI.

Influence of the luxR Regulatory Gene Dosage and Expression Level on the Sensitivity of the Whole-Cell Biosensor to Acyl-Homoserine Lactone.

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescensluxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxRA. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30-100 pM).

The Mode of Action of Cyclic Monoterpenes (-)-Limoneneand (+)-α-Pinene on Bacterial Cells.

A broad spectrum of volatile organic compounds' (VOCs') biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCs-the cyclic monoterpenes (-)-limonene and (+)-α-pinene-on bacteria was studied in this work. We used genetically engineered Escherichia coli bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the luxCDABE genes of Photorhabdus luminescens. We showed that (-)-limonene induces the PkatG and PsoxS promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (-)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-α-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (-)-limonene completely inhibits the DnaKJE-ClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both E. coli wild type and mutant ΔibpB strains. (+)-α-Pinene partially inhibits refolding only in ΔibpB mutant strain.

DNA sequence-specific ligands. XVIII. Synthesis, physico-chemical properties; genetic, virological, and biochemical studies of fluorescent dimeric bisbenzimidazoles DBPA(n).

A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells. Their antiviral activity was tested in model cell lines infected with herpes simplex virus type I. Also, it was found that DBPA(n) could inhibit catalytic activities of HIV-1 integrase at low micromolar concentrations. All of the dimeric bisbenzimidazoles DBPA(n) manifested fluorescent properties, were well soluble in water, nontoxic up to concentrations of 200 µM, and could penetrate into nuclei followed by binding to DNA.

ArdB Protective Activity for Unmodified λ Phage Against EcoKI Restriction Decreases in UV-Treated Escherichia coli.

Anti-restriction proteins ArdB/KlcA specifically inhibit restriction (endonuclease) activity of restriction-modification (RM) type I systems. Molecular mechanisms of ArdB/KlcA-based anti-restriction remain unknown. In this study, we quantitate effects of ArdB on protection of unmodified λ phage DNA from EcoKI restriction. After UV irradiations, which produce significant amounts of unmodified chromosomal DNA in Escherichia coli K12 cells, the protective activity of ArdB decreases. Unlike ArdB, DNA-mimicking protein Ocr retains its ability to protect the unmodified λ phage regardless of UV dose. We hypothesize that the observed decrease in ArdB protective activity in UV-treated cells is due to its binding to unmodified chromosomal DNA, which decreases effective concentrations of free ArdB molecules available for λ phage protection against type I restriction enzymes.

Comparative analysis of Aliivibrio logei luxR1 and luxR2 genes regulation in Escherichia coli cells.

Regulation of Aliivibrio logei luxR1 and luxR2 genes was evaluated in Escherichia coli cells with use of transcriptional fusions of luxR1 and luxR2 promoter/operator regions with the Photorhabdus luminescens luxCDABE reporter gene cassette. Expression of the luxR1 and luxR2 genes was shown to largely depend on the CRP as activator. The hns::kan mutation increases the expression of luxR2 gene by two to three orders of magnitude and luxR1 gene by two to threefold. The LuxR1 and LuxR2 proteins in the presence of autoinducer (N-acyl homoserine lactone, AI) separately as well as together considerably enhanced the transcription of the luxR2 gene. In contrast, the transcription of luxR1 gene decreases depending on AI concentration in the presence of the luxR1 and luxR2 genes combination. It was identified that the promoter region of luxR2 gene consists of two promoters: Pcrp is located downstream of the crp box and Plux-box is located between the crp box and the lux box.

DNA sequence-specific dimeric bisbenzimidazoles DBP(n) and DBPA(n) as inhibitors of H-NS silencing in bacterial cells.

DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used. Escherichia coli lux biosensors were constructed by cloning fragments bearing QS-regulated promoters into the vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE genes. It was shown that the dimeric bisbenzimidazoles DBP(n) and DBPA(n) counteract the H-NS silencing activity. Thus, the presence of DBP(n) or DBPA(n) in the medium leads to an approximately 10-100-fold increase in the level of transcription of QS promoters in E. coli hns+. The largest decrease in the level of H-NS repression was observed using ligands containing a linker with a length of ca. 18Å, such as DBP(2) and DBPA(2). Ligands containing linkers with n=1 and 3 are an order of magnitude less active; ligands with n=4 are inactive. DBPA(2) exhibits activity starting with a concentration of 0.5µM; the minimum concentration of DBP(2) is 5-7 times higher. It is suggested that A-T pairs located at five nucleotide pair intervals, which correspond to the linker length in highly active ligands with n=2, play a key role in the structure of H-NS-binding sites in QS-regulated promoters.

Ketones 2-heptanone, 2-nonanone, and 2-undecanone inhibit DnaK-dependent refolding of heat-inactivated bacterial luciferases in Escherichia coli cells lacking small chaperon IbpB.

Many bacteria, fungi, and plants produce volatile organic compounds (VOCs) emitted to the environment. Bacterial VOCs play an important role in interactions between microorganisms and in bacterial-plant interactions. Here, we show that such VOCs as ketones 2-heptanone, 2-nonanone, and 2-undecanone inhibit the DnaKJE-ClpB bichaperone dependent refolding of heat-inactivated bacterial luciferases. The inhibitory activity of ketones had highest effect in Escherichia coli ibpB::kan cells lacking small chaperone IbpB. Effect of ketones activity increased in the series: 2-pentanone, 2-undecanone, 2-heptanone, and 2-nonanone. These observations can be explained by the interaction of ketones with hydrophobic segments of heat-inactivated substrates and the competition with the chaperones IbpAB. If the small chaperone IbpB is absent in E. coli cells, the ketones block the hydrophobic segments of the polypeptides and inhibit the action of the bichaperone system. These results are consistent with the data on inhibitory effects of VOCs on survival of bacteria. It can be suggested that the inhibitory activity of the ketones indicated is associated with different ability of these substances to interact with hydrophobic segments in proteins.

The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria.

The N-terminal domain of Aliivibrio fischeri LuxR is a target of the GroEL chaperonin.

Here we show that the C-terminal domain of LuxR activates the transcription of Aliivibrio fischeri luxICDABEG in Escherichia coli SKB178 gro(+) and E. coli OFB1111 groEL673 strains to the same level. Using affinity chromatography, we showed that GroEL binds to the N-terminal domain of LuxR, pointing to a GroEL/GroES requirement for the folding of the N-terminal domain of LuxR.