代谢组学在微生物学中的初步应用

1、代谢组学在微生物学中的初步应用中国科学院大连化学物理研究所1808组内 容l菌种识别和鉴定l环境和基因改变对菌株代谢的影响分析l抗生素作用机理分析l细菌胞内代谢物提取方法优化lMTG 乳酸乳球菌代谢分析l其他工作代谢组学技术用于菌种识别和鉴定自制LPA筛分介质A恶臭假单胞菌；B.金黄色葡萄球菌；C.鲍曼不动杆菌；D.产酸克雷伯菌 几种常见细菌的几种常见细菌的CE-SSCPCE-SSCP模式模式 A.伤寒沙门菌；B.福氏志贺菌；C.铜绿假单胞菌；D.洋葱伯克霍尔得菌 Zhao C et al., J Sep Sci. 2005 Apr;28(6):513-21. 几种常见细菌的几种常见细菌的CE

2、-RFLPCE-RFLP模式模式 直接毛细管电泳分析-微米颗粒Electrophoresis. 2006 May;27(9):1784-9. 直接毛细管电泳分析-纳米颗粒代谢组学技术用于环境和基因改变对菌株代谢的影响分析嗜碱菌不同培养条件下胞内代谢物的嗜碱菌不同培养条件下胞内代谢物的GC指纹图指纹图反相高效液相色谱法测定嗜碱微生物发酵液中的有机酸. 食品与发酵工业,2007,33(4):116-118. 发酵时间发酵时间Ferment time 天冬氨酸天冬氨酸Asp谷氨酸谷氨酸Glu天冬酰胺天冬酰胺Asn谷氨酰胺谷氨酰胺Gln丝氨酸丝氨酸Ser苏氨酸苏氨酸Thr甘氨酸甘氨酸Gly脯氨酸脯氨酸

3、Pro丙氨酸丙氨酸Ala缬氨酸缬氨酸Val甲硫氨酸甲硫氨酸Met苯丙氨酸苯丙氨酸Phe赖氨酸赖氨酸Lys4h2.034.121.640.7131.061.111.471.430.7130.8031.880.7131.086h1.954.571.110.3750.5440.6940.8870.7100.3400.3960.8720.3810.7728h1.9905.811.290.3470.4060.6961.150.5960.2680.3210.7640.3231.0610h1.073.130.790.2600.3720.4460.7440.4360.2240.2600.5430.2650.4

4、6212h2.078.121.600.3670.5210.7661.220.5230.2090.3050.5220.3350.72326h1.486.591.120.4360.4621.562.450.3400.1640.1970.2960.4171.0228h1.406.551.010.3310.3960.9031.930.4150.1750.1750.3360.4981.1830h1.986.461.170.3630.4341.913.640.3900.1640.1840.2550.5151.7732h1.376.881.130.3380.4882.212.400.3930.1600.18

5、20.2660.3771.1034h1.166.090.9230.4320.4411.522.060.4150.1670.1840.2720.3981.4136h1.495.070.3100.4560.3030.3621.360.4200.1630.1640.2800.4382.36嗜碱微生物细胞内氨基酸的高效液相色谱测定及代谢途径分析.分析化学，2007，35（7）：1008-1010 两种培养条件下嗜碱菌胞内代谢物的两种培养条件下嗜碱菌胞内代谢物的GC的的PLS-DA得分图得分图Lp3-3优化1012l l14b be ee ef f1618-2-1-10-21g gl lu uc co

6、os se eS Su ur rf fa ac ce e P Pl lo ot t o of f l l v vs s b be ee ef f, , g gl lu uc co os se e17.29g/l大肠杆菌胞内代谢物大肠杆菌胞内代谢物GC指纹图指纹图E.coli1.1566, DC100sdhAB ,DC101ackA-pta 菌株乳酸Ala磷酸甘油琥珀酸嘧啶AspPhe尸胺尿苷ProGluE.coli0.27-19.10.490.520.111.07-0.20.420.921.35DC1010.160.1516.80.341.610.420.490.0870.20.830.611

7、.11DC1000.30-15.60.382.060.230.160.1130.10.311.724.88菌胞内定性的部分代谢物的相对含量菌胞内定性的部分代谢物的相对含量 大肠杆菌的代谢流分析和中心碳代谢途径分析大肠杆菌的代谢流分析和中心碳代谢途径分析 fructose glucoseackA-ptasdhABWild typeMetabolic flux comparison between wild type and sdhAB grown on glucoseMetabolic flux comparison between wild type and ackA-pta grown on

8、 glucoseClass1, DC101ackA-pta Class2, DC100sdhAB Class3,E.coli1.1566GCXGC-MS,与一维与一维GC-MS相比相比,变量增加约变量增加约2倍倍,达达327个个Class1, DC101ackA-pta Class2,E.coli1.1566Class1, DC100sdhAB Class2,E.coli1.1566Class1, DC101ackA-pta Class2, DC100sdhAB 正在进一步分析三株菌胞内代谢物变化差异，希望通过关键代正在进一步分析三株菌胞内代谢物变化差异，希望通过关键代谢物的变化，找出三株菌各

9、自的代谢规律，并进行比较、分析谢物的变化，找出三株菌各自的代谢规律，并进行比较、分析代谢组学技术用于抗生素作用机理分析抗生素作用差异分析E.coli (ATCC25922)兼性厌氧Grams 阴性杆菌P.aeruginosa (ATCC27853)需氧Grams 阴性杆菌抗生素Table 1 basic information about the 11 antibiotics in the study Class Name Antimicrobial mechanism Applied concentrations interfering synthesis of cell wall Ceft

10、azidime 0.003 g/ml Ceftriaxone 0.0015g/ml Cefepime 0.0015g/ml Piperacillin 0.05g/ml Piperacillin/tazobactam 0.011g/ml Ticarcillin /clavulanic acid 0.0425g/ml Quinolone targeting DNA gyrase & topoisomerase IV Levofloxacin 0.0005g/ml Ciprofloxacin 0.00025g/ml Monobactam blocking the synthesis of c

11、ell wall Aztreonam 0.003g/ml Carbapenem disturbing the synthesis of cell wall Imipenem/Cilastatin 0.001g/ml Meropenem 0.001g/ml Beta-lactam两株菌的代谢物相关分析后的关系J Pharm Biomed Anal. 2007 9;44(1):180-7. 细菌胞内代谢物提取方法优化代谢淬灭优化衍生条件优化MTG 乳酸乳球菌代谢分析Protein-glutamine -glutamyltransferase The gamma-carboxymide groups

12、 of peptide-bound glutamine residues act as acyl donors, and the 6-amino-groups of protein- and peptide-bound lysine residues act as acceptors, to give intra- and inter-molecular 6-N-(5-glutamyl)lysine crosslinks. biocyc脂肪胺类：2-arylethylamine, N-acetyl-2-arylethylamine氨基酸类：cytochrome c-N6-methyl-L-ly

13、sine, L-lysine,cyt c-L-lysine, N6-acyl-lysine,protein-l-lysine亮氨酸类似物：N-alpha-acetylornithine, citrulline,D-ornithine, L-ornithine,N5-(L-1-carboxyethyl)-L-ornithine.UPLC-MSl name fold tstat pvalue mzmed mzmin mzmax rtmed rtmin rtmax npeaks KO WTl1 M118T122 57.741712 -14.061881 0.0004027033 118.0875 1

14、18.0874 118.0878 121.90090 107.80366 131.3408 4 0 4l2 M257T118 181.915483 -13.721746 0.0008313590 257.1467 257.1465 257.1475 117.91472 104.96745 127.1862 4 0 4l3 M258T118 109.403027 -12.589167 0.0009591301 258.1501 258.1499 258.1509 117.71506 104.76487 126.8895 4 0 4l4 M235T126 34.006379 -10.222930

15、0.0016690655 235.1656 235.1193 235.1662 125.90705 108.51273 143.8020 5 1 4l5 M236T124 Inf -10.182104 0.0020187369 236.1687 236.1682 236.1693 123.58813 108.00625 133.9127 4 0 4l6 M418T531 6.621662 -4.929390 0.0026318292 417.5482 417.5480 417.5507 531.10637 520.51009 544.9151 5 1 4l7 M311T104 1.332506

16、 -5.652667 0.0040897591 311.2188 311.2183 311.2198 104.30771 85.52443 139.0771 8 4 4l8 M132T174 8.499006 -4.998239 0.0042219840 132.1030 132.1029 132.1033 174.19586 159.34896 183.7679 5 1 4l9 M167T99 101.776552 7.790087 0.0043549483 167.0136 167.0135 167.0137 99.19683 89.50017 126.5053 4 4 0l10 M321

17、T190 Inf -7.615490 0.0046995378 321.0647 321.0644 321.0655 190.10151 180.82082 200.8785 4 0 4l11 M140T115 50.533902 -7.011364 0.0057711586 140.0691 140.0690 140.0694 114.52012 101.52364 123.5261 4 0 4l12 M227T106 4.884450 -6.130591 0.0064430727 226.9514 226.9511 226.9520 105.55710 89.27044 138.3053

18、8 4 4l13 M263T1030 4.523924 -4.241759 0.0064629491 263.2412 263.2407 263.2413 1030.40386 1029.95168 1030.5799 3 0 3l14 M327T105 2.798364 6.237520 0.0067712714 327.1927 327.1919 327.1936 105.04536 87.75186 139.2701 8 4 4l15 M262T1031 3.097678 -4.057292 0.0074025322 262.2378 262.2373 262.2385 1030.575

19、27 1029.71205 1030.8802 8 4 4l16 M363T102 73.876373 -6.162464 0.0085533443 362.9264 362.9257 362.9266 102.02189 89.27044 102.1660 3 0 3l17 M431T102 46.261435 -6.003476 0.0091287265 430.9157 430.9155 430.9163 101.92157 89.16920 102.0666 3 0 3l18 M306T1064 3.185921 -3.856527 0.0107684578 306.2637 306.

20、2631 306.2648 1063.67863 1063.37003 1064.7373 8 4 4l19 M595T249 Inf -5.124341 0.0143881281 594.6565 594.6561 594.6591 249.46406 236.71399 269.8103 4 0 4l20 M219T989 4.589917 -3.327726 0.0180472918 219.2147 219.2146 219.2149 988.64337 986.56818 989.4739 5 1 4l21 M348T134 5.525715 3.569178 0.018581010

21、7 348.0708 348.0703 348.0716 134.14700 126.44272 161.6339 6 4 2l22 M218T989 3.156974 -3.269740 0.0203347063 218.2114 218.2112 218.2118 988.84680 986.76766 989.7757 7 3 4l23 M166T282 9.045244 -3.758749 0.0233659848 166.0871 166.0870 166.0876 282.48645 273.28102 286.4922 5 1 4l24 M333T131 766.910645 4

22、.226096 0.0242281397 332.5613 332.5611 332.5614 130.74166 120.76575 158.8399 4 4 0l25 M459T267 Inf -4.153616 0.0253662286 458.6816 458.6805 458.6822 266.70016 253.58684 293.7440 4 0 4l26 M299T601 22.016010 -3.746571 0.0322348535 299.1463 299.1457 299.1467 600.65813 545.48755 618.0949 6 0 4l27 M120T2

23、82 12.345868 -3.488852 0.0333437562 120.0819 120.0816 120.0823 282.28362 273.08004 286.3935 5 1 4l28 M230T135 21.714035 3.520655 0.0356771098 230.0957 230.0957 230.0958 135.21679 131.89250 159.8034 3 3 0l29 M284T1030 4.391102 -2.842706 0.0365243884 284.2199 284.2192 284.2202 1030.16604 1029.51085 10

24、30.6969 4 0 4l30 M431T266 Inf -3.577982 0.0373398227 430.7038 430.7035 430.7054 266.35373 253.18496 294.1497 4 0 4l31 M268T168 6.511667 3.197541 0.0384001783 268.1039 268.1037 268.1047 168.40569 158.78840 204.1441 6 4 2l32 M167T279 20.147977 -3.329832 0.0409055758 167.0901 167.0894 167.0911 279.14092 272.67807 286.1962 4 0 4l33 M625T186 Inf -3.449628 0.0409477646