신규 메탄산화세균들의 분리, 배양학적 특성 및 대사체 분석

Abstract

To isolate methane oxidizing bacteria (MOB) from various types of soils (paddy field, wetland, garbage landfill, etc.), enrichment culture was performed for 2 weeks at 30℃ using nitrate mineral salt (NMS) medium with methane as sole carbon and energy source. Headspace of 300 mL Erlenmeyer flask was replaced with methane/air gas mixture (50/50, v/v). Over thirty methane oxidizing bacteria were isolated from the enrichment culture using a solid NMS medium. During isolation, more than 40 non-methane oxidizing heterotrophic bacteria, which were grown with methane oxidizing bacteria on NMS agar medium at first subculture but not grown on NMS medium at second subculture, were isolated from the LB agar medium.16s rRNA gene sequence analysis of isolated MOB and heterotrophic bacteria were carried out. As a results, Twenty eight MOB strains were identified. They belonged to Methylomonas sp., Methylophilus sp., Methylosarcina sp., Methylosinus sp., Methylocystis sp.. And twenty three heterotroph strains were identigied by 16S rRNA gene analysis. The eight strains that grow fast among the isolated MOBs were belonged to Methylomonas sp., Methylosinus sp., and Methylocystis sp.. In addition, the specific growth rates (μ-1) in NMS medium contained in the 70 mL gas tightly sealed vial supplemented with methane/air gas mixture (50/50, v/v) were 0.031~0.178. Several mixed culture broths showed high OD600 values of 10 within 5 day cultivation. It was found that some of mixed cultures showed higher OD600 values than pure cultures of corresponding MOBs, but the other presented lower OD600 values. This result suggests that a certain MOB and its related heterotrophic bacterium(a) are symbiotic with each other by exchanging carbon sources or growth promoting substances, but are competitive with each other by inhibiting cell growth. In our study, a novel methane-oxidizing bacterium (MOB) isolated from a rice paddy field soil was identified as Methylocystis sp. JR02 by 16s rDNA gene sequencing analysis. Its specific growth rate and doubling time in nitrate mineral salt (NMS) medium contained in the 70 mL gas tightly sealed vial supplemented with methane/air gas mixture (50/50, v/v) were 0.089 hr-1 and 7.79 hr, respectively. The color of the culture broth was milky white throughout the culture period in the NMS medium containing 4.2 mM phosphate. However, when the phosphate concentration in the medium decreased to less than 0.1 mM, the color of the culture broth turned pink, and the colonies of the NMS agar plate showed metallic green sheen. The pink pigment extracted from the bacterial cell showed two absorption peaks (λmax) at 467 nm and 535 nm in methanol at neutral pH. However, λmax was solvents and pH dependent. That was 467 nm at pH 2.0 and 535 nm at pH 12.0. The pigment was purified and analyzed by LC-MS/MS with m/z 338.7, 352.6 and 366.7. These values were the typical characteristics of the prodigiosin analogues consisting of 2-methyl-3-hexyl prodiginine (m/z 338.7), 2-methyl-3-heptyl prodiginine (m/z 352.6) and 2-methyl-3-octyl prodiginine (m/z 366.7), respectively. The maximal concentration of prodigiosin analogues in the medium was obtained in the stationary phase of cell growth, indicating that it was a secondary metabolite. The maximum concentrations produced were 62.9 mg/L for 2-methyl-3-heptyl prodiginine, 8.1 mg/L for 2-methyl-3-octyl prodiginine, and trace amount of 2-methyl-3-hexyl prodiginine, respectively. To our best knowledge, these were the maximum concentrations obtained from MOB. The anticancer efficacy of the extracted pigment against several human cancer cells were demonstrated through cell viability assay. The pigment concentrations at which cell viability fell below 50% were 15.6, 500, 250 and 250 ㎍/㎖ in prostate cancer cell line LNCap and PC3, breast cancer cell line MDA-MB-231, and lung cancer cell line NCI-H460, respectively. Similarly, an immortalized cell line, HaCaT, exhibited ≤50% cell viability with 62.5 ㎍/㎖ prodigiosin analogues produced by JR02. These results showed that the pigment produced by the JR02 strain was effective in suppressing cancer cell lines similar to prodigiosin. The genome of JR02 strain consisted of a total of five contigs of 4,362,918 bp with GC content of 63.39%. Size of all contigs were 3,885,632; 311,335; 158,371; 4,565 and 3,015 bp. Contig1 has 3,624 CDSs, corresponding to 88.5% of the entire genome coding sequence (CDS) and it was analyzed that contig1 has 55 tRNA and 9 rRNA distinct genes than other contigs. Thus, contig1 is bacterial chromosomal DNA. The particulate methane monooxygenase (pMMO) and the soluble methane monooxygenase (sMMO) gene groups were also present in contig1. Sequence analysis of the three pairs of pMMO α and β subunit genes revealed their close resemblance to the genus Methylocystis, similar to the results of the 16S rRNA gene analysis. Methane monooxygnease components A, B, and C, the constituent genes of sMMO, showed highest homology with the genus Methylosinus, while component D of MMO was most homologous with the genus Methylocystis. Among the prodigiosin-related genes, pigC, pigD, pigE, pigF, pigG, pigH, pigI, pigJ, pigM, and pigA were located in contig2 in the form of an operon. PigB, pigK, pigL, pigN, and pigO genes were not found in pig operon of contig2. Instead, two hypothetical protein genes were found between pigJ and pigM genes. Protein blast analysis of each gene of pig operon showed relatively high homology with various types of prodigiosin biosynthetic gene groups such as Hahella chejuensis, Zoshikella ganghwensis, Janthinobacterium lividum, Rugamonas rubra, and Massilia sp. However, the homology was low with the gene group of the well-known prodigiosin-producer Serratia sp. The size of contig2 was about 311 kbp, which is a plasmid size. Considering above facts, it is possible that the JR02 strain has the ability to produce prodigiosin analogues by horizontal gene transfer. In general, phoR and phoB genes related to phosphate uptake are known to involved in prodigiosin biosynthesis depending on the concentration of phosphate and these genes were located in contig1. Homology analysis of these genes showed similarity to the strains of genus Methylocystis and Methylosinus. In addition, genes that regulate prodigiosin biosynthesis, such as tamA, tamB, and rapA, were found in contig1. The culture supernatant of the JR31 strain, a MOB isolate, showed growth promotion effect on MOB strains (JR07, JR09, JR29, JR30) which require cobalamin(vitamin B12) to grow. Culture supernatant of the JR31 strain not only increased the biomass concentration of JR07 strain but also increased the growth rate. In the activity test of the culture supernatant obtained according to the culture period of the JR31 strain, the JR07 strain showed almost the same level of activity in the supernatant after 7 days of culture. The culture supernatant of the JR31 strain was fractionated by HPLC, and demonstrated growth promoting effect on the vitamin-requiring MOB strains with each fraction, and the best effects were observed with fraction No.17 (20.1-21.0 min) and 18 (21.2-22.0 min). Fraction No.11~20 fractions were analyzed for metabolites. In the GC-TOF-MS analysis, among different metabolites, elaidic acid was distinctly present in fractions no.17 and 18 than other fractions. Elaidic acid is an unsaturated trans fatty acid, which is not known to promotes the growth of microorganisms. In UPLC-LTQ-Orbitrap-MS/MS analysis, a total of 14 possible metabolites were found and among them only one metabolite was identified as amino-methyl-oxononanoicacid. As a result of comparing the contents of metabolites, 5 metabolites (NI 1 ~ 5) showed relatively high content in fraction No.17 whereas 9 metabolites (NI 6 ~ 13 & Amino-methyl-oxononanoicacid) showed relatively high content in fraction No.18.