生物技术通报 ›› 2021, Vol. 37 ›› Issue (6): 136-146.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1385
黄景晓(), 尚俊康, 陈慧敏, 沈嘉旻, 黎圆圆, 喻玉立, 倪进东(), 林伯坤()
收稿日期:
2020-11-13
出版日期:
2021-06-26
发布日期:
2021-07-08
作者简介:
黄景晓,女,硕士研究生,研究方向:传染病预防控制;E-mail: 基金资助:
HUANG Jing-xiao(), SHANG Jun-kang, CHEN Hui-min, SHEN Jia-min, LI Yuan-yuan, YU Yu-li, NI Jin-dong(), LIN Bo-kun()
Received:
2020-11-13
Published:
2021-06-26
Online:
2021-07-08
摘要:
随着抗生素耐药问题的日益加重,使得致病菌的生物防治愈发困难,新型抗菌剂的研发迫在眉睫。使用噬菌体控制致病菌重新获得了极大关注。为获取具有防治沙门氏菌潜力的噬菌体,采用双层琼脂平板法从河水中分离出一株烈性噬菌体PSM6,在肠炎沙门氏菌的菌苔可形成直径约1.5 mm-2 mm的透亮均匀的噬菌斑;具有宽裂解谱,可裂解多种血清型沙门氏菌、部分大肠杆菌和宋内志贺氏菌;最佳感染复数为0.01;繁殖活性强,潜伏期约20 min,爆发量约56 PFU/cell;在40-60℃、pH=5-10时,效价较为稳定。基因组核酸为双链DNA,基因组大小为90 730 bp,G+C含量为39.57%,有133个开放阅读框,含有holin-lysin裂解系统,未发现毒力因子相关基因和抗生素抗性基因。透射电镜观察和基因组测序均显示PSM6属于肌尾噬菌体科。
黄景晓, 尚俊康, 陈慧敏, 沈嘉旻, 黎圆圆, 喻玉立, 倪进东, 林伯坤. 一株烈性沙门氏菌噬菌体的生物学特性及基因组分析[J]. 生物技术通报, 2021, 37(6): 136-146.
HUANG Jing-xiao, SHANG Jun-kang, CHEN Hui-min, SHEN Jia-min, LI Yuan-yuan, YU Yu-li, NI Jin-dong, LIN Bo-kun. Biological Characterization and Genome Analysis of a Lytic Phage Infecting Salmonella[J]. Biotechnology Bulletin, 2021, 37(6): 136-146.
宿主菌 Host bacterium | 宿主菌编号 Host No. | PSM6裂解活性 Infectivity of PSM6 |
---|---|---|
Salmonella Typhimurium | J25、J29、J45、J47、J49 | ++ |
Salmonella Derby | J07 | ++ |
Salmonella Derby | J23 | + |
Salmonella Rissen | J13、J14、J20、J26 | - |
Salmonella Corvallis | J15、J30 | ++ |
Salmonella Acina | J18 | - |
Salmonella London | J19、J24 | ++ |
Salmonella Agona | J22、J58 | - |
Escherichia coli | ATCC25922、DH5α、BL21 | ++ |
Shigella Sonnei | CMCC(B)51592 | + |
Shigella Flexneri | CMCC(B)51572 | - |
Shigella Dysenteriae | CMCC(B)51105 | - |
Shigella | ATCC14029 | - |
表1 噬菌体PSM6的裂解范围
Table 1 Host range of phages PSM6
宿主菌 Host bacterium | 宿主菌编号 Host No. | PSM6裂解活性 Infectivity of PSM6 |
---|---|---|
Salmonella Typhimurium | J25、J29、J45、J47、J49 | ++ |
Salmonella Derby | J07 | ++ |
Salmonella Derby | J23 | + |
Salmonella Rissen | J13、J14、J20、J26 | - |
Salmonella Corvallis | J15、J30 | ++ |
Salmonella Acina | J18 | - |
Salmonella London | J19、J24 | ++ |
Salmonella Agona | J22、J58 | - |
Escherichia coli | ATCC25922、DH5α、BL21 | ++ |
Shigella Sonnei | CMCC(B)51592 | + |
Shigella Flexneri | CMCC(B)51572 | - |
Shigella Dysenteriae | CMCC(B)51105 | - |
Shigella | ATCC14029 | - |
图2 电镜下PSM6的形态 A:单个的噬菌体PSM6;B:聚集的噬菌体PSM6
Fig.2 Morphology characterization of phage PSM6 under transmission electron microscopy A:Single phage PSM6. B:Clustered phage PSM6
感染 复数 MOI | 噬菌体效价 Titer of phage (PFU·mL-1) | 宿主菌浓度 Concentration of host bacteria (CFU·mL-1) | 培养3 h后的噬菌体效价 The titer of phage after 3 h culture (PFU·mL-1) |
---|---|---|---|
100 | 1.0×105 | 1.0×103 | (9.4±0.2)×108 |
10 | 1.0×105 | 1.0×104 | (2.2±0.5)×108 |
1 | 1.0×105 | 1.0×105 | (6.2±0.6)×107 |
0.1 | 1.0×105 | 1.0×106 | (8.1±0.5)×108 |
0.01 | 1.0×105 | 1.0×107 | (1.2±0.6)×1010 |
0.001 | 1.0×105 | 1.0×108 | (4.7±0.3)×109 |
表2 噬菌体PSM6的最佳感染复数测定结果
Table 2 Optimal multiplicity of infection(MOI)of phages PSM6
感染 复数 MOI | 噬菌体效价 Titer of phage (PFU·mL-1) | 宿主菌浓度 Concentration of host bacteria (CFU·mL-1) | 培养3 h后的噬菌体效价 The titer of phage after 3 h culture (PFU·mL-1) |
---|---|---|---|
100 | 1.0×105 | 1.0×103 | (9.4±0.2)×108 |
10 | 1.0×105 | 1.0×104 | (2.2±0.5)×108 |
1 | 1.0×105 | 1.0×105 | (6.2±0.6)×107 |
0.1 | 1.0×105 | 1.0×106 | (8.1±0.5)×108 |
0.01 | 1.0×105 | 1.0×107 | (1.2±0.6)×1010 |
0.001 | 1.0×105 | 1.0×108 | (4.7±0.3)×109 |
图6 噬菌体PSM6的核酸类型判断 M1:10 Kb DNA 分子量标准;1:PSM6的核酸;2:DNase I消化PSM6核酸;3:Mung-Bean Nuclease消化PSM6核酸;4:RNase A消化PSM6核酸
Fig.6 Nucleic acid type of phage PSM6 M1:10 Kb DNA molecular weight standard. 1:PSM6 nucleic acid. 2:DNase I PSM6 nucleic acid. 3:Mung-Bean Nuclease PSM6 nucleic acid. 4:RNase A digests PSM6 nucleic acid
噬菌体名称 Phage name | 大小Genome length/bp | GC含量 GC content/% | 编码区 Coding region | 编码区平均大小 Coding region length/bp | 编码区占百分比 Encoding area percent/% | 转运RNA Transfer RNA | GenBank登录号 Accession No. |
---|---|---|---|---|---|---|---|
PSM6 | 90730 | 39.57% | 133 | 601.8 | 88.2 | 25 | This study |
Salmonella phage vB_SPuM_SP116 | 87510 | 38.29% | 126 | 604.1 | 87.0 | 22 | KP010413.1 |
Escherichia phage vB_EcoM_AYO145A | 87372 | 39.00% | 131 | 592.6 | 88.9 | 20 | KR014248.1 |
Escherichia phage VpaE1_ev108 | 87749 | 38.92% | 129 | 596.1 | 87.6 | 26 | LR597658.1 |
Escherichia phage VpaE1_ev035 | 88002 | 38.96% | 132 | 591.4 | 88.0 | 26 | LR699048.1 |
Escherichia phage NBEco005 | 88272 | 38.84% | 130 | 593.6 | 87.4 | 19 | MN994499.1 |
Shigella phage Z31 | 89355 | 38.88% | 132 | 594.7 | 87.9 | 26 | MN655999.1 |
Salmonella phage D1-2 | 86878 | 38.73% | 128 | 577.6 | 85.1 | 23 | MN481367.1 |
表3 噬菌体PSM6基因组和其近缘噬菌体的比较
Table 3 Comparative analysis of PSM6 and its closely related phage
噬菌体名称 Phage name | 大小Genome length/bp | GC含量 GC content/% | 编码区 Coding region | 编码区平均大小 Coding region length/bp | 编码区占百分比 Encoding area percent/% | 转运RNA Transfer RNA | GenBank登录号 Accession No. |
---|---|---|---|---|---|---|---|
PSM6 | 90730 | 39.57% | 133 | 601.8 | 88.2 | 25 | This study |
Salmonella phage vB_SPuM_SP116 | 87510 | 38.29% | 126 | 604.1 | 87.0 | 22 | KP010413.1 |
Escherichia phage vB_EcoM_AYO145A | 87372 | 39.00% | 131 | 592.6 | 88.9 | 20 | KR014248.1 |
Escherichia phage VpaE1_ev108 | 87749 | 38.92% | 129 | 596.1 | 87.6 | 26 | LR597658.1 |
Escherichia phage VpaE1_ev035 | 88002 | 38.96% | 132 | 591.4 | 88.0 | 26 | LR699048.1 |
Escherichia phage NBEco005 | 88272 | 38.84% | 130 | 593.6 | 87.4 | 19 | MN994499.1 |
Shigella phage Z31 | 89355 | 38.88% | 132 | 594.7 | 87.9 | 26 | MN655999.1 |
Salmonella phage D1-2 | 86878 | 38.73% | 128 | 577.6 | 85.1 | 23 | MN481367.1 |
蛋白编号 Protein No. | 蛋白功能预测 Protein function prediction | 蛋白编号 Protein No. | 蛋白功能预测 Protein function prediction | |
---|---|---|---|---|
GENE8 | D-alanyl-D-alanine carboxypeptidase | GENE65 | Minor tail protein | |
GENE10 | Holin | GENE66 | Deoxynucleotide monophosphate kinase | |
GENE11 | Tail protein | GENE68 | DNA helicase activity | |
GENE17 | Lysin | GENE71 | Exodeoxyribonuclease | |
GENE18 | Putative membrane protein | GENE72 | NAD synthetase | |
GENE20 | Terminase | GENE74 | Protein disulfide oxidoreductase | |
GENE21 | Structural protein | GENE77 | Ribonucleotide reductase | |
GENE24 | Peptidase family S49 | GENE79 | Ribonucleotide reductase | |
GENE25 | Structural protein | GENE80 | Protein disulfide oxidoreductase | |
GENE26 | Phage major capsid protein E | GENE84 | Ribonucleoside-triphosphate reductase | |
GENE31 | Structural protein | GENE86 | Ribonucleoside-triphosphate reductase-activating protein | |
GENE33 | Tape measure chaperone | GENE92 | Ribose-phosphate pyrophosphokinase | |
GENE35 | Tape measure chaperone | GENE93 | Nicotinate-nucleotide diphosphorylase | |
GENE39 | Baseplate assembly protein | GENE106 | rIIa protein | |
GENE41 | Baseplate assembly protein | GENE107 | rIIB lysis inhibitor | |
GENE45 | Tail fiber protein | GENE108 | Lysin,lytic transglycosylase | |
GENE47 | Holin | GENE109 | Polynucleotide kinase | |
GENE48 | Thymidylate synthase | GENE111 | i-spannin | |
GENE49 | Dihydrofolate reductase | GENE113 | O-spanin | |
GENE53 | Transcriptional regulatory protein | GENE114 | Tail assembly protein | |
GENE56 | DNA ligase | GENE117 | Phosphatase | |
GENE61 | DNA polymerase family A | GENE123 | Tail sheath monomer | |
GENE62 | Endonuclease activity | GENE124 | Tail tube protein | |
GENE63 | DNA polymerase family A |
表4 噬菌体PSM6编码蛋白功能预测
Table 4 Prediction of PSM6 protein-coding regions
蛋白编号 Protein No. | 蛋白功能预测 Protein function prediction | 蛋白编号 Protein No. | 蛋白功能预测 Protein function prediction | |
---|---|---|---|---|
GENE8 | D-alanyl-D-alanine carboxypeptidase | GENE65 | Minor tail protein | |
GENE10 | Holin | GENE66 | Deoxynucleotide monophosphate kinase | |
GENE11 | Tail protein | GENE68 | DNA helicase activity | |
GENE17 | Lysin | GENE71 | Exodeoxyribonuclease | |
GENE18 | Putative membrane protein | GENE72 | NAD synthetase | |
GENE20 | Terminase | GENE74 | Protein disulfide oxidoreductase | |
GENE21 | Structural protein | GENE77 | Ribonucleotide reductase | |
GENE24 | Peptidase family S49 | GENE79 | Ribonucleotide reductase | |
GENE25 | Structural protein | GENE80 | Protein disulfide oxidoreductase | |
GENE26 | Phage major capsid protein E | GENE84 | Ribonucleoside-triphosphate reductase | |
GENE31 | Structural protein | GENE86 | Ribonucleoside-triphosphate reductase-activating protein | |
GENE33 | Tape measure chaperone | GENE92 | Ribose-phosphate pyrophosphokinase | |
GENE35 | Tape measure chaperone | GENE93 | Nicotinate-nucleotide diphosphorylase | |
GENE39 | Baseplate assembly protein | GENE106 | rIIa protein | |
GENE41 | Baseplate assembly protein | GENE107 | rIIB lysis inhibitor | |
GENE45 | Tail fiber protein | GENE108 | Lysin,lytic transglycosylase | |
GENE47 | Holin | GENE109 | Polynucleotide kinase | |
GENE48 | Thymidylate synthase | GENE111 | i-spannin | |
GENE49 | Dihydrofolate reductase | GENE113 | O-spanin | |
GENE53 | Transcriptional regulatory protein | GENE114 | Tail assembly protein | |
GENE56 | DNA ligase | GENE117 | Phosphatase | |
GENE61 | DNA polymerase family A | GENE123 | Tail sheath monomer | |
GENE62 | Endonuclease activity | GENE124 | Tail tube protein | |
GENE63 | DNA polymerase family A |
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