Search

To characterize the cellular stress by n-alkanols with different alkyl chain lengths in Escherichia coli, we investigated how n-alkanols damage cell envelope permeability and whether they enhance the promoter activity of the envelope stress response regulator, σE, by using variants of green fluorescent protein (GFP). By using E. coli cells having GFPuv expressing and localizing in the cytoplasm, the inner membrane, and the periplasm, after exposure to n-alkanols, the fluorescent intensity of GFPuv released from cells was examined. Our data showed that at the similar levels of cell death of about 90–97%, ethanol, a short-chain alkanol, at a concentration of 20% damaged the outer membrane more greatly than the inner membrane, whereas a longer-chain alkanol of pentanol at a concentration of 1.125% damaged both of the outer and inner membranes. Then we investigated the envelope stress response to n-alkanols by σE factor by ratiometric analysis of rpoE promoter activity for the downstream GFPuv expression referenced to that of housekeeping sigma 70 (σ70 ) recognizing lacUV5 promoter for red fluorescent protein (RFP) expression. The results indicated that the relative activity of rpoE promoter by pentanol was much greater than that of ethanol. The degree of its sensitization by rpoE deficiency was much more remarkable for cells treated with pentanol than for those with ethanol. The results suggest that the response of the σE plays a significant role in the membrane integrity and survival of E. coli cells treated with n-alkanols depending on the alkyl chain length of the molecule.

Background and objective Particulate matter (PM) is the most dangerous form of air pollution, and causes many diseases. Plants act as bio-filters to help reduce PM in the atmosphere. PM also influences the growth of plants, so selecting suitable plant species for specific environmental conditions is very important. The air pollution tolerance index (APTI) was used to determine the tolerance level of each plant species to air pollution. The purpose of this study was to determine the tolerance to air pollution of various plant species in order to identify plant species that can be grown in polluted environments; this was achieved by evaluating the APTI of plants. This study analyzed the biochemical parameters of 12 plant species at two sites with different air pollution levels (urban forest and roadside) to assess and compare the APTI of plant species. Methods The healthy leaves of 12 plant species (6 broad leaves and 6 needle leaves) that are commonly used in landscapes in Korea were chosen for this study. The same plant species were collected from two sites with different pollution levels and were analyzed immediately; one site was an urban forest (Chungcheongbuk-do Forest Environment Research Institute) with an area of 25 ha and featuring high vegetation coverage, while the other was at a high-traffic roadside next to a crossroads near the Cheongju Express Bus Terminal. We used the leaf samples to analyze four biochemical parameters of each plant: leaf extract pH (pH), relative leaf water content (RWC), total chlorophyll (TChl), and ascorbic acid. Finally, based on these values, APTI values were calculated. Results The APTI values were different between all 12 plant species at both sites with different levels of pollution. APTI had a significant correlation with the biochemical parameters of plants. Plants in the urban forest and at the roadside showed APTI values ranging from 6.89–9.37 and 7.57–9.94, respectively. The APTI of the roadside plant species tended to be higher than that of the plants from the urban forest. Among 12 plant species, Acer palmatum, Acer buergerianum, and Pinus densiflora had high APTI values. These plant species can serve as biofilters in environments with high air pollution. Conclusion The APTI of the 12 plant species in this study can aid in the selection of suitable plant species from environments with different levels of air pollution. The high APTI of some roadside plant species may show their tolerance under environmental pollution-related stress, or demonstrate their adaptability to the polluted environment. In the future, we need to examine more plant species under various environmental conditions to understand their tolerance levels to air pollution and to correlate plants with air pollution. Further, more studies on other air pollutants that can influence plant growth, such as SO2 and NOx, should be conducted.

Background and objective Particulate matter (PM) has a serious impact on health. Recently, studies are conducted to reduce PM in an environmentally friendly way using plants. This study investigated the physiological responses of plants and their ability to remove PM by continuously spraying different PM sources (loam, fly ash, carbon black) to four native plant species, such as Iris sanguinea, Pteris multifida, Vitis coignetiae, and Viburnum odoratissimum var. awabuki. Methods The four plant species were randomly placed in four chambers, and 0.1 g of different PM was injected into each chamber twice a week. We measured chlorophyll, carotenoid, chlorophyll fluorescence (Fv/Fm), total leaf area, amount of leaf wax, PM10 (sPM10) and PM2.5 (sPM2.5) on the leaf surface, and PM10 (wPM10) and PM2.5 (wPM2.5) on the wax layer. Results For I. sanguinea and V. coignetiae, the sources of PM did not affect the growth response. P. multifida showed high chlorophyll a, b, total chlorophyll, and carotenoid content in carbon black as well as high Fv/Fm and total leaf area, thereby proving that carbon black helped plant growth. By PM sources, sPM10 showed a significant difference in three plant species, sPM2.5 in two plant species, and wPM10 in one plant species, indicating that sPM10 was most affected by PM sources. Conclusion Carbon black increased the leaf area by affecting the growth of P. multifida. This plant can be effectively used for PM reduction by increasing the adsorption area. I. sanguinea and V. coignetiae can be used as economical landscaping plants since they can grow regardless of PM sources.

Extended spectrum β-lactamases (ESBL)-producing Escherichia coli have been found in healthy individuals in Indonesia and Vietnam. The ISEcp1-bla CTX-M transposition unit of ESBL-producing bacterial isolates has been considered responsible for the production of CTX-M type ESBL and is important for the dissemination of bla CTX-M . This study aimed to characterize the upstream genetic structure (UGS) of E. coli isolates possessing bla CTX-M-1 group and/or bla CTX-M-9 group genes obtained from healthy individuals in Indonesia and Vietnam. A total of 501 CTX-M type ESBL-producing E. coli isolates possessing bla CTX-M-1 group and/or bla CTX-M-9 group genes were obtained from healthy individuals of the two countries in 2018. UGSs of the ISEcp1-bla CTX-M transposition unit of the 501 ESBL-producing E. coli isolates were amplified by barcode-adaptor-ligation-mediated PCR and analyzed using the Nanopore sequencer. The obtained sequence information was used to classify the UGSs of the ISEcp1-bla CTX-M transposition unit. From the 501 ESBL-producing E. coli isolates, 502 UGSs were obtained, which were classified into 85 UGS types based on the sequence. ISEcp1 of 359 (71.5%) of the 502 UGSs was disrupted by gene insertion, and ISEcp1-bla CTX-M transposition unit of most (87.1%) of the determined UGSs was confirmed as plasmidic. Only 6 (7.1%) of the 85 UGS types were common to both countries. Our results indicated that many different UGSs of ISEcp1-bla CTX-M transposition units were detected in Indonesia and Vietnam; hence, we suggest that structurally different kinds of plasmids harboring bla CTX-M were separately distributed in the two countries. This article is protected by copyright. All rights reserved.

Introduction Because blaCTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of blaCTX-M could be a good indicator for classifying bacterial isolates harboring blaCTX-M in molecular epidemiology. However, determination of blaCTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of blaCTX-M location. Methods Extracted DNA samples obtained from 168 Escherichia coli isolates possessing blaCTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer. Results Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures. Conclusions Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-blaCTX-M transposition unit was useful for classification of bacterial isolates harboring blaCTX-M.

Extended spectrum β-lactamase (ESBL)-producing Escherichia coli have been found in healthy individuals in Indonesia and Vietnam. The ISEcp1-bla

Because blaExtracted DNA samples obtained from 168 Escherichia coli isolates possessing blaNucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures.Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-bla

The effect of cationic liposome prepared from 1,2-dioleoyl-3-trimethyl- ammonium-propane (DOTAP) on the gene expression at the mRNA translation level was investigated using an E. coli cell-free translation system. DOTAP liposome at 3 mM inhibited the mRNA translation of green fluorescent protein (GFP), as indicated both by the fluorescence intensity of GFP and by SDS-PAGE analysis. The role of DOTAP liposome on the inhibition of mRNA translation was revealed that the cationic quaternary amine groups on the liposome surface can interact and neutralize the anionic phosphate groups on mRNA by an electrostatic interaction. mRNA molecules still existed without any degradation in the presence of DOTAP liposome although it could not be translated. These results clearly illustrated that the DOTAP liposome could knock down mRNA and silence its activity of translation in an E. coli cell-free system.

The role of the charged liposome on the in vitro expression of green fluorescent protein (GFP) was investigated, focusing on its elemental steps such as transcription, translation and folding. The total GFP expression was enhanced to 145% when a neutral liposome (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline) was added externally to a cell-free translation system. On the contrary, the addition of the charged liposome composed of POPC with anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) or cationic stearyl amine (SA) inhibited the total GFP expression, depending on the surface charge density of liposome. In transcription, the RNA synthesis was enhanced regardless of the variation of the surface charge, indicating that transcription was enhanced due to the stabilization of RNA structure by its hydrophobic interaction with liposome. Translation was inhibited by cationic liposome although it was enhanced by anionic liposome and neutral liposome. On the other hand, the folding was not inhibited in the presence of neutral liposome, whereas anionic liposome and cationic liposome inhibited the folding in proportion to the their surface charges, suggesting that the total GFP expression was controlled by a charged liposome in the translation step and folding step.

The effective production and secretion of chitosanase from S. griseus cells, in the presence of hydrogen peroxide, were studied by the treatment of the cells with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes under heat condition. The variation of the conformation and activity of the chitosanase caused by the interaction of liposomes with target chitosanase under stress condition was systematically investigated by using circular dichroism (CD) spectra and dielectric dispersion analysis (DDA). The effect of the oxidative stress (hydrogen peroxide) on the lipid and protein peroxide of cell membrane of S. griseus pretreated with and without liposomes under heat stress at 41 degrees C was further carried out. The possible utilization of membrane-membrane interaction between liposomes and cell membrane induced by the heat treatment was further investigated to enhance the production of chitosanase from S. griseus under oxidative stress condition.

A possible role of a model biomembrane, liposome, in gene expression was investigated by using the cell-free translation system. A reporter protein, green fluorescence protein (GFP), was expressed in vitro with and without liposome prepared with 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidyl chorine (POPC) and cholesterol (Ch) (5.7 mM lipid concentration). In the presence of POPC/Ch liposome, the fluorescence intensity of produced GFP was found to be 1.67 times higher than that in the control after 18 h of expression. The results of the SDS-PAGE analysis also show the above promotion effect of the liposome on the net expression of the GFP gene (1.58 times more). The amounts of mRNA were found to be promoted to 1.29 times higher than those in the control. The differences among mRNA, net expression of the GFP gene, and GFP fluorescence indicate that the enhanced GFP expression in the presence of POPC/Ch liposome could primarily affect the transcription and translation of the GFP gene among the possible steps of gene expression. The variation of in vitro gene expression with various liposomes also shows that the biomembrane could act as a modulator to split the genotype and phenotype in a biological cell.

A direct interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes with membrane of Streptomyces griseus cell under the heat stress at 41 degrees C increased the chitosanase production and release to 2.2 times higher than that at 37 degrees C without the POPC liposomes. Amount of chitosanase released across the lipid mimicking cell membrane (LMCM) liposome under a heat at 41 degrees C in the presence of POPC liposomes was 17% of initially-entrapped chitosanase while it was only 1% in the absence of POPC liposomes, even under a heat stress at 41 degrees C, clearly showing the importance of the direct interaction between membrane and membrane.