Analysis of A Plasmid from Lactobacillus acidophilus and the Construction of Shuttle Vectors
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摘要: 为分析嗜酸乳杆菌内源性质粒pDX，并基于该质粒构建大肠杆菌-嗜酸乳杆菌穿梭载体。本研究从嗜酸乳杆菌XW118中分离获得内源性的质粒pDX，对其进行序列测定及功能分析，然后利用质粒pDX的复制子构建了能在大肠杆菌和乳酸菌中穿梭的载体，并研究嗜酸乳杆菌的内源性质粒启动子在大肠杆菌中的活性和穿梭载体在乳酸菌中宿主的范围、转化效率和传代稳定性。结果显示嗜酸乳杆菌质粒pDX大小为2062 bp，GC含量为38.2%，包含4个开放阅读框（Open Reading Frame，ORF），推定为滚环复制质粒。质粒pDX在嗜酸乳杆菌中的拷贝数最高为31.05。本研究构建了基于质粒pDX复制子和启动子的大肠杆菌-乳酸菌穿梭载体。该载体可转化多种类型的乳酸菌，转化效率在0.24×102~2.75×103 CFU/μg（质粒量）之间，质粒丢失率在25.45%~48.36%之间。本研究通过构建获得能在大肠杆菌-乳酸菌穿梭的载体，并获得了在大肠杆菌中具有活性的乳酸菌内源性质粒的启动子，为乳酸菌基因工程和大肠杆菌代谢工程提供了新的操作途径。Abstract: To analyze the endogenous plasmid pDX of Lactobacillus acidophilus, and construct the Escherichia coli-Lactobacillus acidophilus shuttle vector based on the plasmid. In this study, the endogenous plasmid pDX was isolated from Lactobacillus acidophilus XW118, its sequence determination and functional analysis were performed, and then the promotor of plasmid pDX was used to construct a vector that could shuttle between Escherichia coli and lactic acid bacteria. The activity of the endogenous plasmid replicon of L. acidophilus in E. coli and the host range, transformation efficiency and passage stability of the shuttle vector in lactic acid bacteria were studied. The results showed that the size of Lactobacillus acidophilus plasmid pDX was 2062 bp, the GC content was 38.2%, and the plasmid contained four open reading frames (ORFs), which was presumed to be a rolling loop replication plasmid. The copy number of plasmid pDX in L. acidophilus was 31.05. In this study, an E. coli-lactic acid bacteria shuttle vector based on the replicon and promoter of plasmid pDX was constructed. The transformation efficiency of the vector was 0.24×102 to 2.75×103 CFU/μg (plasmid weight), and the plasmid loss rate was 25.45% to 48.36%. In this study, a vector capable of shuttling between E. coli and lactic acid bacteria was constructed, and the promoter of the endogenous plasmid of lactic acid bacteria with activity in E. coli was obtained, which provided a new operational approach for genetic engineering of lactic acid bacteria and metabolic engineering of E. coli.
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Key words:
- shuttle vector /
- Lactobacillus acidophilus /
- plasmid /
- lactic acid bacteria /
- transformation efficiency
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图 3 质粒 pDX的拷贝数与嗜酸乳杆菌生长曲线之间的关系
Figure 3. The relationship between the copy number of plasmid pDX and the growth curve of Lactobacillus acidophilus
表 1 质粒pDX的Rep蛋白Motifs保守序列比对
Table 1. Conservated sequence alignment of Rep protein Motifs of plasmid pDX
家族 基序Ⅰ 基序Ⅱ 基序Ⅲ pDX FLTLTVKN QHLHVLLF TAKYEVKSAD pLD1 FLTLTVKN QHLHVLVF TAKYEVKSAD p200 FLTLTVKN QHLHVLLF TAKYEVKSAD pLR1 FLTLTVKN QHLHVLLF TAKYEVKSAD pM4 FLTLTVEN HHMHVLLF TAKYQVKSKD pC194 FLTLTTPN PHFHVLIA MAKYSGKDSD 保守序列 FLTLT**N *H*HVL** *AKY**K**D 注：表中*代表非保守位点。 
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表 2 穿梭质粒pDXAO4在不同宿主中的转化效率及稳定性
Table 2. Transformation efficiency and stability of shuttle plasmid pDXAO4 in different hosts
菌株 转化效率（CFU/μg） 质粒丢失率（%） 嗜酸乳杆菌 （2.75±0.12）×103 27.63±2.43 乳脂乳球菌 （5.37±0.26）×102 25.45±1.95 乳酸乳球菌乳酸亚种 （0.24±0.03）×102 48.36±3.28 植物乳杆菌 （1.06±0.09）×103 35.72±2.19 鼠李糖乳杆菌 （2.53±0.17）×102 42.94±2.82 副干酪乳杆菌 （0.87±0.04）×102 39.16±3.07 乳酸片球菌 none none 注：none表示无转化子。
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[1] GE Q F, YANG B, LIU R, et al. Antioxidant activity of Lactobacillus plantarum NJAU-01 in an animal model of aging[J]. BMC Microbiology,2021,21(1):182−190. doi: 10.1186/s12866-021-02248-5 [2] ZAFAR H, SAIER M H J. Comparetive genomics of the transport proteins of ten Lactobacillus strains[J]. Gene(Basel),2020,21(10):1234−1241. [3] TAWEECHOTIPATR M, LYER C, SPINLER J K, et al. Lactobacillus saerimneri and Lactobacillus ruminis: Novel human-derived probiotic strains with immunomodulatory activities[J]. FEMS Microbiology Letters,2009,293(1):65−72. doi: 10.1111/j.1574-6968.2009.01506.x [4] 汤回花, 李宏, 陶慧玲, 等. 潜在益生乳酸菌分离和鉴定研究进展[J]. 中国酿造,2021,40(11):21−25. [TANG H H, LI H, TAO H L, et al. Research progress in isolation and identification of potential probiotic lactic acid bacteria[J]. China Brewing,2021,40(11):21−25. doi: 10.11882/j.issn.0254-5071.2021.11.004TANG H H, LI H, TAO H L, et al. Research progress in isolation and identification of potential probiotic lactic acid bacteria[J]. China Brewing, 2021, 40(11): 21−25. doi: 10.11882/j.issn.0254-5071.2021.11.004 [5] 乔荣更, 贾宇, 张红星, 等. 可抑制口腔致病菌的乳酸菌筛选及其抑菌特性研究[J]. 食品与发酵工业,2021,47(11):75−81. [QIAO R G, JIA Y, ZHANG H X, et al. Research progress in isolation and identification of potential probiotic lactic acid bacteria[J]. Food and Fermentation Industries,2021,47(11):75−81. doi: 10.13995/j.cnki.11-1802/ts.026417QIAO R G, JIA Y, ZHANG H X, et al. Research progress in isolation and identification of potential probiotic lactic acid bacteria[J]. Food and Fermentation Industries, 2021, 47(11): 75−81. doi: 10.13995/j.cnki.11-1802/ts.026417 [6] YAMADA N, IWAMOTO C, KANO H, et al. Evaluation of purine utilization by Lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine[J]. Nucleosides Nucleotides Nucleic Acids, 2016, 35(10−12): 670−676. [7] 秦文飞, 宋馨, 夏永军, 等. 乳酸菌在肠道定植的影响因素及研究方法[J]. 食品科学,2021,42(23):275−283. [QIN W F, SONG X, XIA Y J, et al. Factors affecting intestinal colonization of lactic acid bacteria and research methods for it[J]. Food Science,2021,42(23):275−283. doi: 10.7506/spkx1002-6630-20200811-140QIN W F, SONG X, XIA Y J, et al. Factors affecting intestinal colonization of lactic acid bacteria and research methods for it[J]. Food Science, 2021, 42(23): 275−283. doi: 10.7506/spkx1002-6630-20200811-140 [8] 张振东, 王玉荣, 向凡舒, 等. 老年人肠道乳酸菌的分离与鉴定及其来源发酵乳杆菌的遗传进化分析[J]. 食品科学,2021,42(22):105−112. [ZHANG Z D, WANG Y R, XIANG F S, et al. Isolation and identification of lactic acid bacteria from the elderly gut and genetic evolution of isolated Lactobacillus fermentum[J]. Food Science,2021,42(22):105−112.ZHANG Z D, WANG Y R, XIANG F S, et al. Isolation and identification of lactic acid bacteria from the elderly gut and genetic evolution of isolated Lactobacillus fermentum[J]. Food Science, 2021, 42(23): 275−283. [9] 蒋俊艳, 万晓强, 王苏, 等. 复合乳酸菌胶囊辅助四联疗法治疗幽门螺杆菌阳性消化性溃疡的疗效分析[J]. 现代消化及介入诊疗,2019,24(7):801−803. [JIANG J Y, WAN X Q, WANG S, et al. Efficacy analysis of compound lactic acid bacteria capsules assisted quadruple therapy in the treatment of Helicobacter pylori positive peptic ulcer[J]. Modern Digestion and Intervention,2019,24(7):801−803. doi: 10.3969/j.issn.1672-2159.2019.07.028JIANG J Y, WAN X Q, WANG S, et al. Efficacy analysis of compound lactic acid bacteria capsules assisted quadruple therapy in the treatment of Helicobacter pylori positive peptic ulcer[J]. Modern Digestion and Intervention, 2019, 24(7): 801−803. doi: 10.3969/j.issn.1672-2159.2019.07.028 [10] MARIYA I P, GREGOR R, JESSICA A H. Lacticaseibacillus rhamnosus GR-1, a. k. a. Lactobacillus rhamnosus GR-1: Past and future perspectives[J]. Trends in Microbiology,2021,29(8):747−761. doi: 10.1016/j.tim.2021.03.010 [11] CHOI G H, LEE N K, PAIK H D. Optimization of medium composition for biomass production of Lactobacillus plantarum 200655 using response surface methodology[J]. The Journal of Microbiology and Biotechnology,2021,31(5):717−725. doi: 10.4014/jmb.2103.03018 [12] NAWAZ M, WANG J, ZHOU A P, et al. Characterization and transfer of antibiotic resistance in lactic acid bacteria from fermented food products[J]. Current Microbiology,2011,62(3):1081−1089. doi: 10.1007/s00284-010-9856-2 [13] OHTSU T, TAKAGI A, UEMURA N, et al. The ameliorating effect of Lactobacillus gasseri OLL2716 on functional dyspepsia in helicobacter pylori-uninfected individuals: A randomized controlled study[J]. Digestion,2017,96(2):92−102. doi: 10.1159/000479000 [14] WAN I I, ALI M H, TECK L, et al. Dietary postbiotic Lactobacillus plantarum improves serum and ruminal antioxidant activity and upregulates hepatic antioxidant enzymes and ruminal barrier function in post-weaning lambs[J]. Antioxidants,2020,9(3):250−259. doi: 10.3390/antiox9030250 [15] MILLION-WEAVER S, CAMPS M. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?[J]. Plasmid,2014,75(9):27−36. [16] YANG X M, DONG N, CHAN E W C, et al. Carbapenem resistance-encoding and virulence-encoding conjugative plasmids in Klebsiella pneumoniae[J]. Trends in Microbiology,2021,29(1):65−83. doi: 10.1016/j.tim.2020.04.012 [17] 张广峰, 张楠笛, 赵婷婷, 等. 一株食窦魏斯氏菌的分离鉴定及其质粒的序列分析[J]. 新宝登录入口(中国)有限公司,2018,39(8):100−105. [ZHANG G F, ZHANG N D, ZHAO T T, et al. Phylogenetic analysis of natural plasmids encoding the replication initiation protein in Lactobacillus plantarum[J]. Science and Technology of Food Industry,2018,39(8):100−105. doi: 10.13386/j.issn1002-0306.2018.08.019ZHANG G F, ZHANG N D, ZHAO T T, et al. Phylogenetic analysis of natural plasmids encoding the replication initiation protein in Lactobacillus plantarum[J]. Science and Technology of Food Industry, 2018, 39(8): 100−105. doi: 10.13386/j.issn1002-0306.2018.08.019 [18] 孙大庆, 李洪飞, 杨健, 等. 短乳杆菌天然质粒分类[J]. 食品科学,2018,39(10):173−178. [SUN D Q, LI H F, YANG J, et al. Classification of natural plasmids inLactobacillus brevis[J]. Food Science,2018,39(10):173−178. doi: 10.7506/spkx1002-6630-201810027SUN D Q, LI H F, YANG J, et al. Classification of Natural Plasmids in Lactobacillus brevis[J]. Food Science, 2018, 39(10): 173−178. doi: 10.7506/spkx1002-6630-201810027 [19] 王瑶, 韦云路, 李平兰. 乳酸菌食品级质粒及应用的研究进展[J]. 食品科学,2017,38(13):269−276. [WANG Y, WEI Y L, LI P L, et al. A review of research on lactic acid bacteria food-grade vectors and their applications[J]. Food Science,2017,38(13):269−276. doi: 10.7506/spkx1002-6630-201713044WANG Y, WEI Y L, LI P L, et al. A review of research on lactic acid bacteria food-grade vectors and their applications[J]. Food Science, 2017, 38(13): 269−276. doi: 10.7506/spkx1002-6630-201713044 [20] 方来杉, 赖强, 钟泽民, 等. 植物乳杆菌内源性质粒序列分析及其表达载体的构建[J]. 食品科学,2020,41(4):118−124. [FANG L S, LAI Q, ZHONG Z M, et al. Sequence analysis of an endogenous plasmid in Lactobacillus plantarum and construction of a shuttle expression vector using it[J]. Food Science,2020,41(4):118−124. doi: 10.7506/spkx1002-6630-20181130-361FANG L S, LAI Q, ZHONG Z M, et al. Sequence analysis of an endogenous plasmid in Lactobacillus plantarum and construction of a shuttle expression vector using it[J]. Food Science, 2020, 41(4): 118−124. doi: 10.7506/spkx1002-6630-20181130-361 [21] 袁林, 曾静, 郭建军, 等. 粪肠球菌内源性质粒的序列分析及其穿梭载体的构建[J]. 新宝登录入口(中国)有限公司,2021,42(23):141−149. [YUAN L, ZENG J, GUO J J, et al. Sequence analysis of an endogenous plasmid from Enterococcus faecalis and the construction of shuttle vectors[J]. Science and Technology of Food Industry,2021,42(23):141−149. doi: 10.13386/j.issn1002-0306.2021040175YUAN L, ZENG J, GUO J J, et al. Sequence analysis of an endogenous plasmid from Enterococcus faecalis and the construction of shuttle vectors[J]. Science and Technology of Food Industry, 2021, 42(23): 141−149. doi: 10.13386/j.issn1002-0306.2021040175 [22] DE ANDRADE E W V, DUPONT S, BENEY L. Osmoporation is a versatile technique to encapsulate fisetin using the probiotic bacteria Lactobacillus acidophilus[J]. Applied Microbiology and Biotechnology,2022,106(3):1031−1044. doi: 10.1007/s00253-021-11735-8 [23] MENG L, LI S, LIU G F, et al. The nutrient requirements of Lactobacillus acidophilus LA-5 and their application to fermented milk[J]. Journal of Dairy Science,2021,104(1):138−150. doi: 10.3168/jds.2020-18953 [24] 李思函, 王莹, 李艾黎, 等. 含嗜酸乳杆菌软质豆酪制作工艺优化及品质分析[J]. 新宝登录入口(中国)有限公司,2019,40(15):106−112. [LI S H, WANG Y, LI A L, et al. Processing technology optimization and quality analysis of L. acidophilus soft soy cheese[J]. Science and Technology of Food Industry,2019,40(15):106−112. doi: 10.13386/j.issn1002-0306.2019.15.018LI S H, WANG Y, LI A L, et al. Processing technology optimization and quality analysis of L. acidophilus soft soy cheese[J]. Science and Technology of Food Industry, 2019, 40(15): 106−112. doi: 10.13386/j.issn1002-0306.2019.15.018 [25] DEL G S, GIRALDO R, RUIZ-ECHEVARR M J. Replication and control of circular bacterial plasmid[J]. Microbiology and Molecular Biology Reviews,1998,62(2):434−464. doi: 10.1128/MMBR.62.2.434-464.1998 [26] DEL SOLAR G H, PUYET A, ESPINOSA M. Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1[J]. Nucleic Acids Research,1987,15(14):5561−5580. doi: 10.1093/nar/15.14.5561 [27] LI R Y, ZHAI Z Y, YIN S, et al. Characterization of a rolling-circle replication plasmid pLR1 from Lactobacillus plantarum LR1[J]. Current Microbiology,2009,58(2):106−110. doi: 10.1007/s00284-008-9280-z [28] 孙大庆, 李洪飞, 杨健. 植物乳杆菌编码复制起始蛋白天然质粒的系统进化分析[J]. 微生物学通报,2017,44(5):1047−1055. [SUN D Q, LI H F, YANG J. Phylogenetic analysis of natural plasmids encoding the replication initiation protein in Lactobacillus plantarum[J]. Microbiology China,2017,44(5):1047−1055. doi: 10.13344/j.microbiol.china.160940SUN D Q, LI H F, YANG J. Phylogenetic analysis of natural plasmids encoding the replication initiation protein in Lactobacillus plantarum[J]. Microbiology China, 2017, 44(5): 1047−1055. doi: 10.13344/j.microbiol.china.160940 [29] 杨帆, 苏卜利, 王永红, 等. 启动子对重组大肠杆菌合成番茄红素能力的影响[J]. 食品与发酵工业,2020,46(17):27−32. [YANG F, SU B L, WANG Y H, et al. Effects of promoters for lycopene production in engineering Escherichia coli[J]. Food and Fermentation Industries,2020,46(17):27−32. doi: 10.13995/j.cnki.11-1802/ts.023250YANG F, SU B L, WANG Y H, et al. Effects of promoters for lycopene production in engineering Escherichia coli[J]. Food and Fermentation Industries, 2020, 46(17): 27−32. doi: 10.13995/j.cnki.11-1802/ts.023250 [30] 姚智绚, 张晓娟, 段艳婷, 等. 原子力显微镜力谱研究大肠杆菌启动子与RNA聚合酶的相互作用[J]. 微生物学报,2018,58(1):109−121. [YAO Z X, ZHANG X J, DUAN Y T, et al. Atomic force microscope based force spectroscopy as tool to study the interaction between Escherichia coli promoter and RNA polymerase[J]. Acta Microbiologica Sinica,2018,58(1):109−121. doi: 10.13343/j.cnki.wsxb.20170046YAO Z X, ZHANG X J, DUAN Y T, et al. Atomic force microscope based force spectroscopy as tool to study the interaction between Escherichia coli promoter and RNA polymerase[J]. Acta Microbiologica Sinica, 2018, 58(1): 109−121. doi: 10.13343/j.cnki.wsxb.20170046 -
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