Strains M45 Research Articles

The selective breeding and mutagenesis mechanism of high‐yielding surfactin Bacillus subtilis strains with atmospheric and room temperature plasma

Surfactin, a good biological surfactant, is derived from the metabolites of microorganisms. However, the ability of natural strains to produce surfactin is low, and so the presented study aimed to use a novel mutagenesis technology to increase their yields. Atmospheric and room temperature plasma (ARTP) was used to conduct mutation breeding of Bacillus subtilis CICC 10721, and a mutant strain M45 with a higher surfactin yield of 34.2% and a stable subculture was screened out. From the fermentation kinetics study, it was found that the maximum cell dry weight, maximum growth rate and surfactin synthesis parameters of the mutant strain M45 were all greater than that of the original strain. Scanning electron microscope and laser scanning confocal microscope observations showed that the spore morphology changed after ARTP treating, and the intracellular Ca2+ concentration of the mutant increased. Genome resequencing analysis showed that 66 single nucleotide poymorphism non-synonymous mutation sites occurred in M45, and the identification results of the fermentation broth extract from M45 showed that it is composed of C12 -C16 surfactin. ARTP mutagenesis was found to change the morphology of bacteria, membrane permeability and genes related to the synthesis and secretion of surfactin. The present study provides a basis for industrial production of surfactin and an understanding of the mutagenesis mechanism. © 2021 Society of Chemical Industry.

Isolation, screening antimicrobial activity and identification of fungi from marine sediments of the area Thanh Lan, Co To, Vietnam

Marine environment is rich in natural product resources, including marine microorganisms, especially fungi which are not only seen as a potential source of highly applicable bioactive substances but also can provide for science new chemical structures. The objective of this study is to isolate and screen fungal strains with antibacterial activity from the marine environment. Twenty five strains of fungi were isolated from marine sediments of Thanh Lan, Co To island and assessed on antibiotic activity against 7 tested microbial strains, including three Gram-negative bacteria (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Salmonella enterica ATCC13076), three Gram-positive bacteria (Enterococcus faecalis ATCC29212, Stapphylococus aureus ATCC25923, Bacillus cereus ATCC 13245), and the yeast Candida albicans ATCC10231. The minimum inhibitory concentration (MIC) against the tested microorganisms was determined for the crude extracts obtained from the culture broths after ethyl acetate extraction and vacuum rotary evaporation. Three strains with the highest antimicrobial activity M26, M30 and M45 were capable of inhibiting 4 - 5 of the 7 tested microorganisms with MIC values from 64 to 256 μg/ml, depending on each tested strain. Morphological and phylogenetic investigations based on 18S rRNA gene sequences of the three selected strains showed that strains M26 and M30 belonged to the genus Penicillium, whereas strain M45 belonged to the genus Neurospora. The sequences of 18S rRNA gene of three strains M26, M30 and M45 were registered on GenBank database with accession numbers: MH673730, MH673731, MH673732, respectively. Research results showed that marine environment has a great potential in isolation of fungal strains for the search for antibacterial substances as well as other biologically active compounds.

MinD plays an important role in Aeromonas hydrophila adherence to Anguilla japonica mucus

For a better understanding of the genitic regulation of the adhesion of Aeromonas hydrophila, a mini-Tn10 transposon mutagenesis system was introduced to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOF/Km) and the recipient A. hydrophila strain W. Out of 332 individual colonies, 7 mutants with significantly attenuated adhesion ability were selected. The DNA sequence flanking the mini-Tn10 transposon inserted in the mutant strain M45 was amplified by TAIL-PCR. The results showed that an ORF of approximately 870bp (GenBank accession number KP271930) of the mutant strain M45 was inserted by mini-Tn10. This ORF putatively encodes a deduced 289 amino acid protein that displays the highest identity (100%) with the CobQ/CobB/MinD/ParA family protein of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type W, the mutant strain M45 and the complemented strain C45 were investigated. The results indicated that mutation of MinD gene in A. hydrophila could lead to abnormal cell division and reduce the ability of adhesion, biofilm formation and bacterial motility.

Powder Coating Formulations with Pseudomonas fluorescens M45 Enhance the Emergence Rate and Healthy Stand Establishment of Sesame in Disease-Conducive Soil

Sesame is a major cooking oil crop in Korea. One of the primary problems in sesame cultivation is low healthy stand establishment due to the occurrence of seedling rot and damping-off resulting from a complex of soil-borne pathogens in the field. To address the problem, the bioformulation of Pseudomonas fluorescens M45 was prepared in powder form using clay and vermiculite, and was evaluated for its effect on biological control of soil-borne pathogens in sesame cultivation. In the petri dish trial, the emergence rate was overall good (> 92%) regardless of seeds being pelleted and/or M45-treated. In both pot and field trials containing disease-conducive soils, seed-pelleting substantially reduced emergence rate, whereas seed-pelleting with M45 significantly improved the emergence rate (> 26%). The emergence rate of sesame seeds treated with the strain M45 was greater than 30% regardless of seed pelletization. We also found that M45r colonized in the roots at the density of 1.6×10 5 cfu/g. With aid of the bioformulation, however, root colonization of the strain was significantly increased to 4.0×10 6 cfu/g. The powder formulation with strain M45 enhanced the rate of healthy stand establishment in disease-conducive soil. Therefore, bioformulation with strain M45 is a promising method to overcome problems associated with the successive cultivation of sesame.

Spore rec-assay by dry sheet medium culture plate for bacterial counts

A simple and rapid method for spore rec-assay by utilizing dry sheet medium culture (Compactdry TC, CTC) for determining numbers of bacteria, instead of the spore agar plate, was developed. One mL of spore suspension (2 x 10(6)/mL) of Bacillus subtilis strain M45 Rec- or H17 Rec+ was inoculated in the center of the CTC plate. In the case of metabolic activation, 1 mL of mixed solution (spore suspension of M45 or H17: 9,000 x g supernatant of rat-liver homogenate treated with Aroclor 1254 = 19:1) was used. The spore suspension spreads over the whole sheet in seconds and gels. A paper disk impregnated with 20-40 microL of the sample solution and 20 microL of the cofactor solution was placed on the surface of CTC plate. For the assay of samples that do not require metabolic activation, use of the cofactor solution can be omitted. After 48 hr incubation at 37 degrees C, 0.01% MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] aqueous solution (0.5 mL) was dropped uniformly on the plate. The plate was left for 5 min, and the diameter of the inhibition circle was measured with slide calipers. The samples for which the difference in inhibition zone between M45 and H17 was more than 2 mm were judged positive. Under these conditions, the DNA damaging activities of sodium sulfite, sodium benzoate and citric acid, used as food additives, were investigated by the proposed method. Sodium sulfite and sodium benzoate gave positive results and citric acid gave a negative result with or without metabolic activation, in agreement with the results obtained by the conventional method.

Mutagenic and membranal effect of a phytotoxic molecule isolated from olive leaves parasitized by the fungusCycloconium oleaginum Cast

A phytotoxic substance (C23H44O3) which is named 'Substance A', was purified from olive leaves infected with Cycloconium oleaginum Cast. The mutagenic effect of this substance was detected using TA 100 and TA 102 strains of Salmonella in the Ames test using Bacillus subtilis strains M45 rec-, H17 rec+ in the rec assay. Another substance manifesting the mutagenic effect was found in the extract from the Cycloconium oleaginum culture. This substance was not detected in the extract from contaminated olive leaves. Substance A increased electrolytes leakage from tissue of olive leaves, thus manifesting its phytotoxicity.

Activity of quinolones in the Ames Salmonella TA102 mutagenicity test and other bacterial genotoxicity assays

Eight quinolones were examined for their bacterial mutagenicity in the Ames Salmonella TA102 assay and for their effects in other bacterial genotoxicity assays. In the quantitative Ames plate incorporation assay, all eight quinolones induced His+ deletion reversion in Salmonella tester strain TA102, with maximum reversion observed at about two to eight times the MIC. The quinolones also induced the SOS response. At quinolone concentrations close to the MIC, SOS cell filamentation gene sulA was induced in sulA::lacZ fusion strain Escherichia coli PQ37. RecA-mediated cleavage of lambda repressor in lambda::lacZ fusion strain E. coli BR513 was measurable at about 10 times the MIC, though no induction occurred at 20 micrograms of nalidixic or oxolinic acid per ml. Genotoxicity of quinolones also was observed in the Bacillus subtilis DNA repair assay, in which the mutant strain M45 (recA) was more susceptible to quinolones than its parent strain, H17 (rec+). The results from these analyses indicate that quinolones induce SOS functions and are mutagenic in bacteria; these properties correspond to their antimicrobial activities.

Mutagenicity of extracts from ceylon cinnamon in the rec assay

The extraction of about 1.9 kg of Ceylon cinnamon ( Cinnamomum zeylanicum Nees) with 10 litres each of petroleum ether, chloroform and ethanol in a Soxhlet apparatus produced extracts weighing 76, 28 and 270 g respectively for the three solvents. In the preliminary test the ethanol extract showed no mutagenic activity. However, both the petroleum ether and the chloroform extracts showed mutagenicity when tested in the rec assay using Bacillus subtilis strains H17 ( rec +) and M45 ( rec −). When these extracts were studied quantitatively by the liquid and spore rec-assay methods, the minimum inhibitory concentrations of the extracts against strain H17 were higher than those against strain M45. However, in the presence of the liver S-9 mix, the minimum inhibitory concentrations of the petroleum ether and chloroform extracts against both strains of B. subtilis were equal, indicating that the mutagenicity of the extracts had been inactivated.

Mutagenicity screening of anesthetics for fishes

3 fish anesthetics, eugenol, phenthiazamine and tricaine, were tested for mutagenicity in the rec assay (repair test) with Bacillus subtilis strains M45 (rec −) and H17 (rec +), and in the reverse mutation assay with Salmonella typhimurium strains TA100, TA98, TA1535, TA1537 and TA1538. The rec assay was negative for all the compounds. In the reverse mutation assay, phenthiazamine was mutagenic in strains TA100, TA98 and TA1537 after metabolic activation with rat hepatic S9 mix. Eugenol and tricaine were non-mutagenic in the presence and absence of S9 mix.

Genotoxic and mutagenic assays of halothane metabolites in Bacillus subtilis and Salmonella typhimurium.

Reactions of N-acetylcysteine with the halothane metabolite, 2-chloro-1,1-difluoroethylene (CF2CHCl), and two related probable metabolites, 2-bromo-1,1-difluoroethylene (CF2CHBr) and 2-bromo-2-chloro-1,1difluoroethylene (CF2CBrCl), afforded the saturated conjugates, RSCF2CH2Cl, RSCF2CH2Br, and RSCF2CHBrCl, as well as the unsaturated analogs, RSCFCHCl and RSCFCHBr; R = −CH2CH(COOH)NHCOCH3. The mutagenic and genotoxic potential of these conjugates was evaluated in the Salmonella/microsome system described by Ames and a “rec” DNA repair system developed by Kada employing recombination proficient and deficient strains of Bacillus subtilis. When screened for mutagenicity with Salmonella typhimurium strains TA1535 and TA100, the saturated and the unsaturated conjugates were found to be nonmutagenic. However, in a preliminary test using strain TA100 in logarithmic growth, compounds RSCF2CHBrCl and RSCFCHCl were mutagenic. Furthermore, screening for DNA-damaging ability in the B. subtilis “rec” assay with strains H17 and M45 revealed that the urinary halothane metabolite, RSCF2CHBrCl, and the unsaturated analogs, RSCFCHCl and RSCFCHBr, preferentially inhibited the growth of strain M45, which is deficient in its ability to repair DNA. In view of the reported correlation between known mutagens and their differential lethal action on rec− versus rec+ bacteria, the present findings of the DNA-damaging effects of the nonvolatile halothane metabolites and related probable metabolites suggest a possible relationship between halothane metabolism and reported toxic effects associated with occupational anesthetic exposure.