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.

Biological activity of volatiles produced by the strains of two Pseudomonas and two Serratia species.

Volatile compounds emitted by bacteria can play a significant role in interacting with microorganisms, plants, and other organisms. In this work, we studied the effect of total gaseous mixtures of organic as well as inorganic volatile compounds (VCs) and individual pure volatile organic compounds (VOCs: ketones 2-nonanone, 2-heptanone, 2-undecanone, a sulfur-containing compound dimethyl disulfide) synthesized by the rhizosphere Pseudomonas chlororaphis 449 and Serratia plymuthica IC1270 strains, the soil-borne strain P. fluorescens B-4117, and the spoiled meat isolate S. proteamaculans 94 strain on Arabidopsis thaliana plants (on growth and germination of seeds). We demonstrated that total mixtures of volatile compounds emitted by these strains grown on Luria-Bertani agar, Tryptone Soya Agar, and Potato Dextrose Agar media inhibited the A. thaliana growth. When studied bacteria grew on Murashige and Skoog (MS) agar medium, volatile mixtures produced by bacteria could stimulate the growth of plants. Volatile compounds of bacteria slowed down the germination of plant seeds; in the presence of volatile mixtures of P. fluorescens B-4117, the seeds did not germinate. Of the individual VOCs, 2-heptanone had the most potent inhibitory effect on seed germination. We also showed that the tested VOCs did not cause oxidative stress in Escherichia coli cells using specific lux-biosensors. VOCs reduced the expression of the lux operon from the promoters of the katG, oxyS, and soxS genes (whose products involved in the protection of cells from oxidative stress) caused by the action of hydrogen peroxide and paraquat, respectively.

Long-Term Antimicrobial Performance of Textiles Coated with ZnO and TiO2 Nanoparticles in a Tropical Climate.

This paper reports the results of the large-scale field testing of composite materials with antibacterial properties in a tropical climate. The composite materials, based on a cotton fabric with a coating of metal oxide nanoparticles (TiO2 and/or ZnO), were produced using high-power ultrasonic treatment. The antibacterial properties of the materials were studied in laboratory tests on solid and liquid nutrient media using bacteria of different taxonomic groups (Escherichia coli, Chromobacterium violaceum, Pseudomonas chlororaphis). On solid media, the coatings were able to achieve a >50% decrease in the number of bacteria. The field tests were carried out in a tropical climate, at the Climate test station "Hoa Lac" (Hanoi city, Vietnam). The composite materials demonstrated long-term antibacterial activity in the tropical climate: the number of microorganisms remained within the range of 1−3% in comparison with the control sample for the duration of the experiment (3 months). Ten of the microorganisms that most frequently occurred on the surface of the coated textiles were identified. The bacteria were harmless, while the fungi were pathogenic and contributed to fabric deterioration. Tensile strength deterioration was also studied, with the fabrics coated with metal oxides demonstrating a better preservation of their mechanical characteristics over time, (there was a 42% tensile strength decrease for the reference non-coated sample and a 21% decrease for the sample with a ZnO + CTAB coating).

Effects of Volatile Organic Compounds on Biofilms and Swimming Motility of Agrobacterium tumefaciens.

Volatile organic compounds (VOCs) emitted by bacteria play an important role in the interaction between microorganisms and other organisms. They can inhibit the growth of phytopathogenic microorganisms, modulate plant growth, and serve as infochemicals. Here, we investigated the effects of ketones, alcohols, and terpenes on the colony biofilms of plant pathogenic Agrobacterium tumefaciens strains and swimming motility, which can play an important role in the formation of biofilms. It was shown that 2-octanone had the greatest inhibitory effect on biofilm formation, acting in a small amount (38.7 g/m3). Ketone 2-butanone and unsaturated ketone ß-ionone reduced the formation of biofilms at higher doses (145.2-580.6 and 387.1-1548.3 g/m3, respectively, up to 2.5-5 times). Isoamyl alcohol and 2-phenylethanol decreased the formation of biofilms at doses of 88.7 and 122.9 g/m3 by 1.7 and 5 times, respectively, with an increased effect at 177.4 and 245.9 g/m3, respectively. The agrobacteria cells in mature biofilms were more resistant to the action of ketones and alcohols. These VOCs also suppressed the swimming motility of agrobacteria; the radius of swimming zones decreased ~from 2 to 5 times. Terpenes (-)-limonene and (+)-α-pinene had no significant influence on the colony biofilms and swimming motility at the doses used. The results obtained represent new information about the effect of VOCs on biofilms and the motility of bacteria.

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.

The Effect of Volatile Organic Compounds on Different Organisms: Agrobacteria, Plants and Insects.

Bacteria and fungi emit a huge variety of volatile organic compounds (VOCs) that can provide a valuable arsenal for practical use. However, the biological activities and functions of the VOCs are poorly understood. This work aimed to study the action of individual VOCs on the bacteria Agrobacterium tumefaciens, Arabidopsis thaliana plants, and fruit flies Drosophila melanogaster. VOCs used in the work included ketones, alcohols, and terpenes. The potent inhibitory effect on the growth of A. tumefaciens was shown for 2-octanone and isoamyl alcohol. Terpenes (-)-limonene and (+)-α-pinene practically did not act on bacteria, even at high doses (up to 400 µmol). 2-Butanone and 2-pentanone increased the biomass of A. thaliana at doses of 200-400 µmol by 1.5-2 times; 2-octanone had the same effect at 10 µmol and decreased plant biomass at higher doses. Isoamyl alcohol and 2-phenylethanol suppressed plant biomass several times at doses of 50-100 µmol. Plant seed germination was most strongly suppressed by isoamyl alcohol and 2-phenylethanol. The substantial killing effect (at low doses) on D. melanogaster was exerted by the terpenes and the ketones 2-octanone and 2-pentanone. The obtained data showed new information about the biological activities of VOCs in relation to organisms belonging to different kingdoms.

Four New Genes of Cyanobacterium Synechococcus elongatus PCC 7942 Are Responsible for Sensitivity to 2-Nonanone.

Microbial volatile organic compounds (VOCs) are cell metabolites that affect many physiological functions of prokaryotic and eukaryotic organisms. Earlier we have demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. Cyanobacteria are very sensitive to ketone action. To investigate the possible molecular mechanisms of the ketone 2-nonanone influence on cyanobacterium Synechococcus elongatus PCC 7942, we applied a genetic approach. After Tn5-692 transposon mutagenesis, several 2-nonanone resistant mutants have been selected. Four different mutant strains were used for identification of the impaired genes (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732, Synpcc7942_0726) that encode correspondingly: 1) a murein-peptide ligase Mpl that is involved in the biogenesis of cyanobacteria cell wall; 2) a putative ABC transport system substrate-binding proteins MlaD, which participates in ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane by aberrantly localized phospholipids transport from outer to inner membranes of bacterial cells; 3) a conserved hypothetical protein that is encoding by gene belonging to phage gene cluster in Synechococcus elongatus PCC 7942 genome; 4) a protein containing the VRR-NUC (virus-type replication-repair nuclease) domain present in restriction-modification enzymes involved in replication and DNA repair. The obtained results demonstrated that 2-nonanone may have different targets in Synechococcus elongatus PCC 7942 cells. Among them are proteins involved in the biogenesis and functioning of the cyanobacteria cell wall (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732) and protein participating in stress response at DNA restriction-modification level (Synpcc7942_0726). This paper is the first report about the genes that encode protein products, which can be affected by 2-nonanone.

Inhibition of cyanobacterial photosynthetic activity by natural ketones.

Microbial volatiles have a significant impact on the physiological functions of prokaryotic and eukaryotic organisms. Various ketones are present in volatile mixtures produced by plants, bacteria, and fungi. Our earlier results demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. In this work, we thoroughly examined the natural ketones, 2-nonanone and 2-undecanone to determine their influence on the photosynthetic activity in Synechococcus sp. PCC 7942. We observed for the first time that the ketones strongly inhibit electron transport through PSII in cyanobacteria cells in vivo. The addition of ketones decreases the quantum yield of primary PSII photoreactions and changes the PSII chlorophyll fluorescence induction curves. There are clear indications that the ketones inhibit electron transfer from QA to QB , electron transport at the donor side of PSII. The ketones can also modify the process of energy transfer from the antenna complex to the PSII reaction center and, by this means, increase both chlorophyll fluorescence quantum yield and the chlorophyll excited state lifetime. At the highest tested concentration (5 mM) 2-nonanone also induced chlorophyll release from Synechococcus cells that strongly indicates the possible role of the ketones as detergents.

SprI/SprR Quorum Sensing System of Serratia proteamaculans 94.

In this study, we investigated the quorum sensing (QS) regulatory system of the psychrotrophic strain Serratia proteamaculans 94 isolated from spoiled refrigerated meat. The strain produced several N-acyl-L-homoserine-lactone (AHL) QS signal molecules, with N-(3-oxo-hexanoyl)-L-homoserine lactone and N-(3-hydroxy-hexanoyl)-L-homoserine lactone as two main types. The sprI and sprR genes encoding an AHL synthase and a receptor regulatory protein, respectively, were cloned and sequenced. Analysis of their nucleotide sequence showed that these genes were transcribed convergently and that their reading frames partly overlapped by 23 bp in the terminal regions. The genes were highly similar to the luxI/luxR-type QS genes of other Gram-negative bacteria. An spr-box (analog of the lux-box) was identified upstream of the sprR gene and found to be overlapped with the sequence of -10 sequence site in the promoter region of this gene. Inactivation of the sprI gene led to the absence of AHL synthesis, chitinolytic activity, and swimming motility; decrease of extracellular proteolytic activity; affected the cellular fatty acid composition; and reduced suppression of the fungal plant pathogen mycelium growth by volatile compounds emitted by strain S. proteamaculans 94. The data obtained demonstrated the important role of the QS system in the regulation of cellular processes in S. proteamaculans 94.

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.

Inhibitory and toxic effects of volatiles emitted by strains of Pseudomonas and Serratia on growth and survival of selected microorganisms, Caenorhabditis elegans, and Drosophila melanogaster.

In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds--ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide--were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.

Microcin C51 plasmid genes: possible source of horizontal gene transfer.

Microcin C51 (MccC51) is an antimicrobial nucleotide-heptapeptide produced by a natural Escherichia coli strain. A 5.7-kb fragment of the pC51 plasmid carrying the genes involved in MccC51 production, secretion, and self-immunity was sequenced, and the genes were characterized. The sequence of the MccC51 gene cluster is highly similar to that of the MccC7 gene. Recombinant plasmids carrying different combinations of the mcc genes involved in the MccC51 production or immunity were constructed to characterize their functional roles. The mccA, mccB, mccD, and mccE genes are involved in MccC51 production, while the mccC and mccE genes are responsible for immunity to MccC51. The mcc gene cluster is flanked by 44-bp direct repeats. Amino acid sequence comparisons allowed us to propose functions for each Mcc polypeptide in MccC51 biosynthesis. Plasmid pUHN containing the cloned mccA, mccB, mccC, and mccE genes, but lacking mccD, directed the synthesis of MccC51p, a substance chemically related to MccC51. MccC51p exhibited weak antibiotic activity against E. coli and was toxic to the producing cells. The immunity to exogenous MccC51 determined by the mccC and mccE genes did not overcome the toxic action of MccC51p on the producing cells. The G+C content of the MccC51 operon, markedly lower than that of the E. coli genome, and the presence of direct repeats suggest the possibility of horizontal transfer of this gene cluster.