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1 微生物学通报 Microbiology China Oct. 20, 2017, 44(10): DOI: /j.microbiol.china 研究报告醋酸胁迫对 Acetobacter pasteurianus 产酸呼吸链活力的影响 * 朱传柳夏小乐史伟王武 ( ) 摘要 : 目的以巴氏醋酸杆菌工业菌株沪酿 1.01 和模式菌株 Acetobacter pasteurianus ATCC 为研究对象, 研究工业菌株与模式菌株在高浓度醋酸胁迫下的产酸发酵时呼吸链酶活相关基因转录水平的变化规律方法检测两种菌株在 0 1% 2% 3% 不同初始醋酸浓度下的生物量产酸以及酶活, 并通过实时荧光定量 PCR 检测呼吸链相关酶合成基因的相对转录水平变化结果两种菌株在 1% 初始醋酸浓度下产酸能力最强, 发酵 48 h 平均产酸速度达到 g/(l h); 两种菌株的 ADH ( 乙醇脱氢酶 ) 和 ALDH ( 乙醛脱氢酶 ) 酶活也达到最高, 平均为和 9.77 U/mg; 相关酶合成基因的相对转录水平较无底酸均提高当初始醋酸浓度上升至 2% 和 3% 时, 菌体酶活产酸能力逐渐下降, 呼吸链上的 adh cyt bd cyt o 和 fapa 基因转录水平上升, 其余基因都降低结论确定了巴氏醋酸杆菌在高初始浓度醋酸条件下, 菌体会自发提高 adh 基因的转录水平, 启动底物磷酸化, 并将醋酸泵出到胞外 ; 同时 aldh 基因的表达受到抑制, 降低产酸, 从而维持体内较低醋酸浓度此外, 呼吸链上其他外排相关酶转录水平也会提升, 如 cyt bd 和 cyt o, 辅助底物磷酸化进程, 加快释放能量关键词 : 巴氏醋酸杆菌, 呼吸链, 代谢变化, 初始醋酸浓度, 实时荧光定量 PCR Effects of acetic acid stress on the activity of acetic acid respiratory chains in Acetobacter pasteurianus ZHU Chuan-Liu XIA Xiao-Le SHI Wei WANG Wu * (Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu , China) Abstract: [Objective] To analyze the regularity of the activity of respiratory chains and the transcriptional levels of related genes in Acetobacter pasteurianus under the stress of high acetic acid concentration, A. pasteurianus CICC and A. pasteurianus ATCC were studied. [Methods] The biomass, acid production and enzyme activity of the two strains have been analyzed in different initial acetic acid concentrations of 0, 1%, 2% and 3%. Real-time quantitative PCR was used to analyzed changes of relative transcriptional levels of respiratory chains related enzyme synthesis genes in different concentrations. [Results] It was found that acid production capacity of Foundation item: National Natural Science Foundation of China (No ) *Corresponding author: Tel/Fax: ; wangwu@jiangnan.edu.cn Received: November 24, 2016; Accepted: March 21, 2017; Published online ( April 05, 2017 基金项目 :(No ) * 通讯作者 Tel/Fax wangwu@jiangnan.edu.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 2270 微生物学通报 Microbiol. China 2017, Vol.44, No.10 two strains was the strongest when initial acetic acid concentration was 1%; and their average acid production rate reached g/(l h) during 48 hours fermentation. At the same time, enzyme activity levels of ADH (alcohol dehydrogenase) and ALDH (acetaldehyde dehydrogenase) also rise to the highest of and 9.77 U/mg on average. And the relative transcriptional level of the respiratory chain related enzyme synthesis genes were also increased compared with initial acid of 0. The enzyme activity and acid production capacity decreased gradually when initial acetic acid concentration increased to 2% and 3%. The adh, cyt bd, cyt o and fapa gene on respiratory chain maintained a relatively high transcriptional level while the transcriptional level of rest genes decreased. [Conclusion] It is identified that under high-intensity acetic acid conditions, the transcriptional level of adh gene is increased spontaneously, which activates the substrate phosphorylation and pump acetic acid out. At the same time, the aldh gene s expression is inhibited, which causing the reducing of acid production and maintaining rather low concentration of acetic acid. In addition, the level of other efflux enzymes in the respiratory chain will also increase, such as cyt bd and cyt o, which enhance the substrate phosphorylation in metabolic process and accelerate the release of energy. Keywords: Acetobacter pasteurianus, Respiratory chains, Metabolic changes, Initial acetic acid concentration, Real-time quantitative PCR (Acetobacter pasteurianus A. pasteurianus) [1-2] (ADH) (ALDH) H + [3 5] [6] 3% Takemura [7] ADH ADH Beppu [8] ADH ALDH 98.6 g/l 40% ADH ALDH [9] TCA TCA Matsushita [10] [11] ( ABC ) [12] 1.01 (A. pasteurianus CICC 20001) A. pasteurianus ATCC % 2% 3%

3 : Acetobacter pasteurianus 材料与方法 1.1 材料主要试剂和仪器 : PCR RNase-free ddh 2 O Trizol PCR () (1) 50 mmol/l KPB (ph 5.8) 22.8 g 100 ml 1 mol/l 13.6 g 100 ml 1 mol/l 1 mol/l 5 ml 1 mol/l 45 ml ml (2) Mcllvaine (ph 4.0) 2.76 g 1.29 g 3.72 g 100 ml (3) 0.1 mol/l 3.29 g 100 ml 4 C (4) -Dupanol 5 g Fe 2 (SO 4 ) 3 nh 2 O 3 g Dupanol () 95 ml 85% ml Stepone plus PCR ABI 菌种 :(1) 1.01 CICC (2) ATCC ATCC 培养基 :(1) (g/l) Pa 20 min 60 C 30 g/l (2) (g/l) Pa 20 min 30 g/l 荧光定量 PCR 引物 : Primer Premier 表 1 荧光定量 PCR 引物 Table 1 Oligo-nucleotides for qrt-pcr Genes Primers Primers sequence (5 3 ) 16S rrna 16S-F 16S-R CCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG adh adh-f adh-r TATGACGCCACGAACGGTAG AGCCCACTTCAACAGCAACAT aldh aldh-f aldh-r AGTGGATGCTCGGAAGAATGA CTGTGTGCTGAATGACGTCGT ucb ucb-f ucb-r AATCTGAATGGCTGGTGGAAT CTGGCGTTCTATGGCTTCAA nadh Nadh-F Nadh-R ATGGATGGCACCAAGTTTGT CCGTAGACTGGCGAGAAATAA cyt bd Cyt bd-f Cyt bd-r TTGTTCAGGGCGTAAAAGTTG CTCCCAACAAAGCATAACCAC cyt o Cyt o-f Cyt o-r GGGTCTGGCTTGTGGTATCTT GTGCCATCAGTTTACGGGTGT cyt c Cyt c-f Cyt c-r GTATCCCTTGGAATCCTGAACA TCCGCAACTTATGGCGTATC cyt d1 Cyt d1-f Cyt d1-r AGGACCTTCAGGAAAGTGGC CTTGGTTGTGCGATGGTTTAC cyt d2 Cyt d2-f Cyt d2-r CATTCTGCTATCTGCCTTGGA ACGGTACATCGTTCTCGGTTT fapa fapa-f fapa-r ATCTTCCTTGAAACCGACCTG GGCTCTGTTTCAGCAACTCAA fapb fapb-f fapb-r GTTCTACCGAAGGCTCTAAAGTG CCTGCGTAAAGCGGAAAA

4 2272 微生物学通报 Microbiol. China 2017, Vol.44, No 培养方法菌种活化 : 1 ml 50 ml ( ) 30 C 170 r/min 24 h 3 ml 600 nm OD 摇瓶发酵 : 10% ( ) 500 ml 90 ml 0 1% 2% 3% 30 C 170 r/min 平板培养 : 30 C 60 h 转接方法 : 30 C 170 r/min 24 h 1% 24 h 1% 2% 3% 1.3 分析方法菌体浓度 : OD 600 (CFU/mL) 3 OD 600 (CFU/mL) OD 600 ( 1) 菌体产酸测定 : 1 ml 150 ml % 0.1 mol/l NaOH 30 s V (ml) (g/l)=c NaOH V 60 C NaOH NaOH (mol/l) V NaOH (ml) 蛋白浓度测定 : Bradford 1 g/l 图 1 醋酸杆菌菌体浓度与吸光度 OD 600 的线性关系 Figure 1 Relationship between cell concentrations and the OD 600 value 5.0 ml G-250 () 5 min 595 nm OD 595 OD 乙醇 ( 乙醛 ) 脱氢酶活力测定 :(1) 4 C r/min 10 min 50 mmol/l (ph 5.8) (KPB) 2 (0.1 g /3 ml KPB) 240 W 2 s/2 s 10 min 图 2 Bradford 法蛋白质浓度测定标准曲线 Figure 2 Calibration curve of protein concentrations with Bradford method

5 : Acetobacter pasteurianus 2273 (2) Wood [13] 0.5 ml Mcllvaine (ph 4.0) 1.0 mol/l () 0.1 ml 0.1 ml 10% Triton X ml 10 ml 25 C 5 min 0.1 mol/l 0.2 ml 25 C 5 min ( )-Dupanol 0.5 ml 25 C 20 min 3.5 ml 660 nm 25 C ph min l μmol (U/mL)=A 660 1/4 1/5 1/(mL) (U/mg)= (U/mL)/ (mg/ml) 1.4 荧光定量 PCR 0.1 g UNlQ-10 Trizol RNA RNA DNA DNase I AMV FirstStrand cdna Synthesis Kit cdna 5 μl RNA (0.2 g/l) 1 μl Random Primerp(dN) 6 (0.2 g/l) 5 μl RNase-free ddh 2 O 16S rrna cdna (50 mg/l) (10 μl) 2 SYBR Green qpcr Master Mix 5 μl 10 μmol/l Primer F 0.2 μl 10 μmol/l Primer R 0.2 μl ddh 2 O 3.6 μl (cdna) 1 μl 95 C 10 min 95 C 10 s 60 C 30 s C 97 C 5 2 ΔΔC t ΔC t =C t C t ΔΔC t =ΔC t ΔC t =2 ΔΔC t C t C t C t 1.5 数据处理 3 OriginPro8.5 Adobe Illustrator CS6 3 P 结果与分析 2.1 不同初始醋酸浓度对菌体生长的影响 [5] (A. pasteurianus ATCC A. pasteurianus CICC 20001) CFU/mL 3% CFU/mL 28.3% 29.8% 10 h 2.2 初始醋酸对乙醇呼吸链产酸和酶活的影响不同初始醋酸浓度下的产酸变化 : ph [14] 4 Wang [15] 1%A. pasteurianus ATCC A. pasteurianus CICC g/l 48 h g/(l h) 2% 3% 3% g/l 48 h g/(l h)

6 2274 微生物学通报 Microbiol. China 2017, Vol.44, No.10 图 3 A. pasteurianus ATCC (A) 和 A. pasteurianus CICC (B) 在不同培养条件下的生长曲线 Figure 3 Growth curves of A. pasteurianus ATCC (A) and A. pasteurianus CICC (B) under different culture conditions 图 4 A. pasteurianus ATCC (A) 和 A. pasteurianus CICC (B) 在不同培养条件下的产酸曲线 Figure 4 Acid production curves of A. pasteurianus ATCC (A) and A. pasteurianus CICC (B) under different culture conditions 醋酸发酵过程中乙醇呼吸链的酶活变化 : ADH ALDH [4] 5 12 h ADH ALDH 5 1% ADH (ALDH)A. pasteurianus ATCC A. pasteurianus CICC (11.03) (8.51) U/mg (9.77) U/mg 3% ADH (ALDH) 6.97 (4.39) 6.25 (3.82) U/mg 6.61 (4.11) U/mg ADH ALDH

Activity of ADH and ALDH in two strains under

3 不同初始醋酸浓度对乙醇呼吸链若干酶活与基因转录水平的影响 0 1% 2% 3% 2.3.1 乙醇呼吸链代谢途径相关酶合成基因转录水平分析 : A.

(ALDH) Q Q H + Q ( o Figure 6 图 6 细胞膜呼吸链的模型

7 : Acetobacter pasteurianus 2275 图 5 两种菌株在不同初始醋酸浓度条件下的 ADH 和 ALDH 的活性水平 Figure 5 Activity of ADH and ALDH in two strains under different initial acetic acid concentrations 2.3 不同初始醋酸浓度对乙醇呼吸链若干酶活与基因转录水平的影响 0 1% 2% 3% 乙醇呼吸链代谢途径相关酶合成基因转录水平分析 : A. pasteurianus ATCC [5,16] 6 (PQQ)(ADH) (ALDH) Q Q H + Q ( o Figure 6 图 6 细胞膜呼吸链的模型 Model of cell membrane s respiratory chain

2276 微生物学通报 Microbiol. China 2017, Vol.44, No.10 bd ) H 2 O [17-18] H + (ATPase) ATP ( 2) A. pasteurianus ATCC 33445 A. pasteurianus CICC 20001 0 1% 2% 3% PCR 7 1%A. pasteurianus ATCC 33445 A. pasteurianus CICC 20001 1% 2% 3% A.

23 adh [7] adh aldh cyt o cyt bd H + ATPase ADP ATP 表 2 巴氏醋酸杆菌呼吸链相关基因及其编码蛋白 Table 2 Respiratory chain related genes and their coding proteins of A.

8 2276 微生物学通报 Microbiol. China 2017, Vol.44, No.10 bd ) H 2 O [17-18] H + (ATPase) ATP ( 2) A. pasteurianus ATCC A. pasteurianus CICC % 2% 3% PCR 7 1%A. pasteurianus ATCC A. pasteurianus CICC % 2% 3% A. pasteurianus ATCC cyt o fapa cyt bd adh A. pasteurianus CICC cyt o fapa cyt bd adh adh [7] adh aldh cyt o cyt bd H + ATPase ADP ATP 表 2 巴氏醋酸杆菌呼吸链相关基因及其编码蛋白 Table 2 Respiratory chain related genes and their coding proteins of A. pasteurianus Gene Encoding protein adh PQQ-alcohol dehydrogenase aldh PQQ-aldehyde dehydrogenase cyt c cytochrome c oxidase cyt d1 cytochrome d ubiquinol oxidase subunit I cyt d2 cytochrome d ubiquinol oxidase subunit II nadh NADH dehydrogenase cytbd cytochrome bdubiquinol oxidase subunit II cyt o cytochorome o oxidase ucb Ubiquinol-cytochrome b reductase fapa F1F0 ATPase subunit a fapb F1F0 ATPase subunit b 图 7 A. pasteurianus ATCC (A) 和 A. pasteurianus CICC (B) 上与呼吸链相关的耐酸基因在不同培养条件下的相对转录水平 Figure 7 Relative transcription levels of relative levels of respiratory chain-related acid-resistant genes under different culture conditions in A. pasteurianus ATCC (A) and A. pasteurianus CICC (B)

9 : Acetobacter pasteurianus 2277 adh aldh cyt bd cyt o ATP 乙醇呼吸链上 adh/aldh 相对转录水平与酶活之间的关系 : 12 h A. pasteurianus ATCC A. pasteurianus CICC A. pasteurianus ATCC A. pasteurianus CICC adh ADH 2% ADH ADH A. pasteurianus ATCC A. pasteurianus CICC aldh A. pasteurianus ATCC A. pasteurianus CICC aldh ALDH 2% aldh ALDH aldh ALDH 表 3 adh 基因的转录水平与酶活之间的关系 Table 3 The relationship between adh transcriptional level and enzyme activity Initial acetic acid concentration (%) Strain Transcriptional level (times) Enzyme activity (U/mg) 0 A. pasteurianus ATCC A. pasteurianus CICC A. pasteurianus ATCC A. pasteurianus CICC A. pasteurianus ATCC A. pasteurianus CICC A. pasteurianus ATCC A. pasteurianus CICC 表 4 aldh 基因的转录水平与酶活之间的关系 Table 4 The relationship between aldh transcriptional level and enzyme activity Initial acetic acid concentration (%) Strain Transcriptional level (times) Enzyme activity (U/mg) 0 A. pasteurianus ATCC A. pasteurianus CICC A. pasteurianus ATCC A. pasteurianus CICC A. pasteurianus ATCC A. pasteurianus CICC A. pasteurianus ATCC A. pasteurianus CICC

10 2278 微生物学通报 Microbiol. China 2017, Vol.44, No.10 3 结论与讨论 Wang [15] adh aldh adh aldh cyt bd cyt o fapa ATP [17,19] A. pasteurianus CICC A. pasteurianus ATCC cyt o cyt bd adh ADH ABC [12] [20 21] (GroEL/S DnaJ/K) Inoki [22] Liu [23] A. pasteurianus CICC A. pasteurianus ATCC adh aldh cyt o cyt bd fapa Acetobacter pasteurianus 参考文献 [1] Bartowsky EJ, Henschke PA. Acetic acid bacteria spoilage of bottled red wine a review[j]. International Journal of Food Microbiology, 2008, 125(1): [2] Sengun IY, KarabiyikliS. Importance of acetic acid bacteria in food industry[j]. Food Control, 2011, 22(5): [3] Gullo M, Giudici P. Acetic acid bacteria in traditional balsamic vinegar: phenotypic traits relevant for starter cultures selection[j]. International Journal of Food Microbiology, 2008, 125(1): [4] Chinnawirotpisan P, Theeragool G, Limtong S, et al. Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianus SKU1108[J]. Journal of Bioscience and Bioengineering, 2003, 96(6): [5] Yakushi T, Matsushita K. Alcohol dehydrogenase of acetic acid bacteria: structure, mode of action, and applications in biotechnology[j]. Applied Microbiology and Biotechnology, 2010, 86(5): [6] Zheng Y, Jiang CY, Chen XJ, et al. Effects of acetic acid on the growth and metabolic activity of Acetobacter pasteurianus[j]. Modern Food Science & Technology, 2014, 30(9): (in Chinese),,,. [J]., 2014, 30(9): [7] Takemura H, Horinouchi S, Beppu T. Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability[j]. Journal of Bacteriology, 1991, 173(22): [8] Beppu T. Genetic organization of Acetobacter for acetic acid fermentation[j]. Antonievan Leeuwenhoek, 1993, 64(2): [9] Qi ZL, Yang HL, Xia XL, et al. Analysis of energetic metabolism of Acetobacter pasteurianus during high acidic vinegar fermentation[j]. Microbiology China, 2013, 40(12): (in Chinese),,,. [J]., 2013, 40(12): [10] Matsushita K, Toyama H, Adachi O. Respiratory chains inacetic acid bacteria: membraneboundperiplasmic sugar and alcohol respirations[a]//zannoni D. Respiration in Archaea and Bacteria: Diversity of Prokaryotic Respiratory Systems[M]. Netherlands: Springer, 2004, 36: [11] Matsushita K, Inoue T, Adachi O, et al. Acetobacteraceti possesses a proton motive force-dependent efflux system for acetic acid[j]. Journal of Bacteriology, 2005, 187(13): [12] Nakano S, Fukaya M, Horinouchi S. Putative ABC transporter responsible for acetic acid resistance in Acetobacteraceti[J]. Applied and Environmental Microbiology, 2006, 72(1): [13] Shinagawa E, Toyama H, Matsushita K, et al. A novel type of formaldehyde-oxidizing enzyme from the membrane of Acetobacter sp. SKU 14[J]. Bioscience, Biotechnology, and Biochemistry, 2006, 70(4): [14] Nakano S, Fukaya M. Analysis of proteins responsive to acetic acid in Acetobacter: molecular mechanisms conferring acetic acid resistance in acetic acid bacteria[j]. International Journal of Food Microbiology, 2008, 125(1): [15] Wang B, Shao YC, Chen T, et al. Global insights into acetic acid resistance mechanisms and genetic stability of Acetobacter pasteurianus strains by comparative genomics[j]. Scientific

11 : Acetobacter pasteurianus 2279 Reports, 2015, 5: [16] Azuma Y, Hosoyama A, Matsutani M, et al. Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus[j]. Nucleic Acids Research, 2009, 37(17): [17] González B, Martínez S, Chávez JL, et al. Respiratory system of Gluconacetobacter diazotrophicus PAL5 evidence for a cyanide-sensitive cytochrome bb and cyanide-resistant cytochrome ba quinol oxidases[j]. Biochimicaet Biophysica Acta (BBA)-Bioenergetics, 2006, 1757(12): [18] Chávez-Pacheco JL, Contreras-Zentella M, Membrillo-Hernández J, et al. The quinohaemoprotein alcohol dehydrogenase from Gluconacetobacter xylinus: molecular and catalytic properties[j]. Archives of Microbiology, 2010, 192(9): [19] Matsushita K, Toyama H, Adachi O. Respiratory chains and bioenergetics of acetic acid bacteria[j]. Advances in Microbial Physiology, 1994, 36: [20] Ishikawa M, Okamoto-Kainuma A, MatsuiK, et al. Cloning and characterization of clpb in Acetobacter pasteurianus NBRC 3283[J]. Journal of Bioscience and Bioengineering, 2010, 110(1): [21] Okamoto-Kainuma A, Yan W, Fukaya M. Cloning and characterization of the dnakj operon in Acetobacteraceti[J]. Journal of Bioscience and Bioengineering, 2004, 97(5): [22] Inoki K, Zhu TQ, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival[j]. Cell, 2003, 115(5): [23] Liu LP, Cash TP, Jones RG, et al. Hypoxia-induced energy stress regulates mrna translation and cell growth[j]. Molecular Cell, 2006, 21(4): 征稿简则 ( et al.) : [1] Marcella C, Claudia E, Pier GR, et al. Oxidation of cystine to cysteic acid in proteins by peroyacids as monitored by immobilized ph gradients[j]. Electrophoresis, 1991, 12(5): [2] Wang BJ, Liu SJ. Perspectives on the cultivability of environmental microorganisms[j]. Microbiology China, 2013, 40(1): 6-17 (in Chinese),. [J]., 2013, 40(1): 6-17 [3] Shen T, Wang JY. Biochemistry[M]. Beijing: Higher Education Press, 1990: 87 (in Chinese),. [M]. :, 1990: 87 [4] Liu X. Diversity and temporal-spatial variability of sediment bacterial communities in Jiaozhou Bay[D]. Qingdao: Doctoral Dissertation of Institute of Oceanology, Chinese Academy of Sciences, 2010 (in Chinese). [D]. :, () Foundation item: *Corresponding author: Tel: ; Fax: ; Received: January 01, 20xx; Accepted: March 01, 20xx; Published online ( March 31, 20xx (No. ) * Tel Fax 20xx xx ( 20xx ( p.2406)

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇