Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (4): 297-305.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1068
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WANG Cai-hong(), JIANG Meng-yuan, SHAO Yu-han
Received:
2023-11-14
Online:
2024-04-26
Published:
2024-04-30
Contact:
WANG Cai-hong
E-mail:13520137104@163.com
WANG Cai-hong, JIANG Meng-yuan, SHAO Yu-han. Identification and Functional Study of T6SS Effector Protein PA0423 of Pseudomonas aeruginosa[J]. Biotechnology Bulletin, 2024, 40(4): 297-305.
菌株 Strain | 特点 Characteristic | 来源 Source |
---|---|---|
DH5a | 大肠杆菌基因克隆菌株 | 全式金 |
BL21(DE3)pLysS | 大肠杆菌基因表达菌株 | 全式金 |
PAO1 | 野生型 | 本实验室 |
S17-1 | 接合菌株 | 本实验室 |
PAO1△rets | PAO1的rets敲除突变 | 本实验室 |
PAO1△rets△clpV1 | PAO1的rets和clpV1敲除突变 | 本实验室 |
PAO1△rets△clpV2 | PAO1的rets和clpV2敲除突变 | 本实验室 |
PAO1△rets△clpV3 | PAO1的rets和clpV3敲除突变 | 本实验室 |
PAO1△PA0262 | PAO1的PA0262敲除突变 | 本实验室 |
PAO1△PA0423 | PAO1的PA0423敲除突变 | 本实验室 |
PAO1△PA0423+PME-PA0423 | 包含PME6032-PA0423重组质粒的PAO1的PA0423敲除菌 | 本实验室 |
PAO1△PA0422-0423 | PAO1的PA0422和PA0423敲除突变 | 本实验室 |
Table 1 Strains used in this study
菌株 Strain | 特点 Characteristic | 来源 Source |
---|---|---|
DH5a | 大肠杆菌基因克隆菌株 | 全式金 |
BL21(DE3)pLysS | 大肠杆菌基因表达菌株 | 全式金 |
PAO1 | 野生型 | 本实验室 |
S17-1 | 接合菌株 | 本实验室 |
PAO1△rets | PAO1的rets敲除突变 | 本实验室 |
PAO1△rets△clpV1 | PAO1的rets和clpV1敲除突变 | 本实验室 |
PAO1△rets△clpV2 | PAO1的rets和clpV2敲除突变 | 本实验室 |
PAO1△rets△clpV3 | PAO1的rets和clpV3敲除突变 | 本实验室 |
PAO1△PA0262 | PAO1的PA0262敲除突变 | 本实验室 |
PAO1△PA0423 | PAO1的PA0423敲除突变 | 本实验室 |
PAO1△PA0423+PME-PA0423 | 包含PME6032-PA0423重组质粒的PAO1的PA0423敲除菌 | 本实验室 |
PAO1△PA0422-0423 | PAO1的PA0422和PA0423敲除突变 | 本实验室 |
引物 Primer | 序列 Sequence | 作用 Function |
---|---|---|
PA0423-up-F | ACAGCTATGACATGATTAC GAATTCGCGGGTCGCCTTCGCCGGCG AGCAC | 构建pk18-PA0423 |
PA0423-up-R | GCGTTTTTCGTTTCAGGTATCCGCGGACATTCTCCTTAACGTGGTTGTTG | 构建pk18-PA0423 |
PA0423-down-F | CAACAACCACGTTAAGGAGAATGTCCGCGGATACCTGAAACGAAAAACGC | 构建pk18-PA0423 |
PA0423-down-R | CTGCAGGTCGACTCTAGA GGATCCGGCGCTTGAGGATGATGCCGTTCAC | 构建pk18-PA0423 |
PA0422-0423-up-F | ACAGCTATGACATGATTAC GAATTCGGTGCTGGCGGAAGCCTTCG | 构建pk18-PA0422-0423 |
PA0422-0423-up-R | TTCGTTTCAGGTATCCGCGAAAAACTTCCTTGAAAAGC | 构建pk18-PA0422-0423 |
PA0422-0423-down-F | GCTTTTCAAGGAAGTTTTTCGCGGATACCTGAAACGAA | 构建pk18-PA0422-0423 |
PA0422-0423-down-R | CTGCAGGTCGACTCTAGA GGATCCTGCCGTTCACCTGGGGACGC | 构建pk18-PA0422-0423 |
PME-0423-F | ATTTCACACAGGAAACA GAATTCATGCTGAAGAAGACCCTTGC | 构建PME6032-PA0423-FLAG |
PME-0423-flag-R | ACTGATCCGCTAGTCCGAGGCCTCGAGCTTATCGTCGTCATCCTTGTAAT C | 构建PME6032-PA0423-FLAG |
PME-flag-R | ACTGATCCGCTAGTCCGAGGC CTCGAGCTTATCGTCGTCATCCTTGTAAT C | 构建PME6032-PA0423-FLAG |
RSF-PA0423-F | CATCACCACAGCCAGGATCC GAATTCGATGCTGAAGAAGACCCTTGCCG | 构建RSFduet-PA0423-myc |
RSF-PA0423-R | GTCGAAGGCATTCGCCAGGAGCAGAAACTCATCTCTGAAGAGGATCTG AAGCTTGCGGCCGCATAATGCT | 构建RSFduet-PA0423-myc |
pET-PA0262-F | CATATGGCAGATCTCAATTG GATATCATGCGTCAAA GGGACCTGAA ATTC | 构建pETduet-PA0262 |
pET-PA0262-R | GCAGCGGTTTCTTTACCAGA CTCGAGGTATCCCGTTGGGAAGTTTTTCAG | 构建pETduet-PA0262 |
pET28-PA0423-F | ATGCTGAAGAAGACCCTTGC | 构建pET28-PA423 |
pET28-PA0423-R | CTTATCGTCGTCATCCTTG | 构建pET28-PA423 |
Table 2 Primers used in this study
引物 Primer | 序列 Sequence | 作用 Function |
---|---|---|
PA0423-up-F | ACAGCTATGACATGATTAC GAATTCGCGGGTCGCCTTCGCCGGCG AGCAC | 构建pk18-PA0423 |
PA0423-up-R | GCGTTTTTCGTTTCAGGTATCCGCGGACATTCTCCTTAACGTGGTTGTTG | 构建pk18-PA0423 |
PA0423-down-F | CAACAACCACGTTAAGGAGAATGTCCGCGGATACCTGAAACGAAAAACGC | 构建pk18-PA0423 |
PA0423-down-R | CTGCAGGTCGACTCTAGA GGATCCGGCGCTTGAGGATGATGCCGTTCAC | 构建pk18-PA0423 |
PA0422-0423-up-F | ACAGCTATGACATGATTAC GAATTCGGTGCTGGCGGAAGCCTTCG | 构建pk18-PA0422-0423 |
PA0422-0423-up-R | TTCGTTTCAGGTATCCGCGAAAAACTTCCTTGAAAAGC | 构建pk18-PA0422-0423 |
PA0422-0423-down-F | GCTTTTCAAGGAAGTTTTTCGCGGATACCTGAAACGAA | 构建pk18-PA0422-0423 |
PA0422-0423-down-R | CTGCAGGTCGACTCTAGA GGATCCTGCCGTTCACCTGGGGACGC | 构建pk18-PA0422-0423 |
PME-0423-F | ATTTCACACAGGAAACA GAATTCATGCTGAAGAAGACCCTTGC | 构建PME6032-PA0423-FLAG |
PME-0423-flag-R | ACTGATCCGCTAGTCCGAGGCCTCGAGCTTATCGTCGTCATCCTTGTAAT C | 构建PME6032-PA0423-FLAG |
PME-flag-R | ACTGATCCGCTAGTCCGAGGC CTCGAGCTTATCGTCGTCATCCTTGTAAT C | 构建PME6032-PA0423-FLAG |
RSF-PA0423-F | CATCACCACAGCCAGGATCC GAATTCGATGCTGAAGAAGACCCTTGCCG | 构建RSFduet-PA0423-myc |
RSF-PA0423-R | GTCGAAGGCATTCGCCAGGAGCAGAAACTCATCTCTGAAGAGGATCTG AAGCTTGCGGCCGCATAATGCT | 构建RSFduet-PA0423-myc |
pET-PA0262-F | CATATGGCAGATCTCAATTG GATATCATGCGTCAAA GGGACCTGAA ATTC | 构建pETduet-PA0262 |
pET-PA0262-R | GCAGCGGTTTCTTTACCAGA CTCGAGGTATCCCGTTGGGAAGTTTTTCAG | 构建pETduet-PA0262 |
pET28-PA0423-F | ATGCTGAAGAAGACCCTTGC | 构建pET28-PA423 |
pET28-PA0423-R | CTTATCGTCGTCATCCTTG | 构建pET28-PA423 |
Fig. 1 Detection and validation of PA0423 secretion Expression and secretion levels of PA0423 in wild-type and different mutant PAO1 bacteria were detected by Western blotting. RNAP was used as an internal control
Fig. 3 Protein structure and distribution of PA0423 a: Gene arrangement map of PA0423. b: Structural model of PA0423(AF-Q9I690-F1)by AlphaFold. c: Distribution of small protease S85 family analyzed by MEROPS database
分析 Analysis | 编号 Accession No. | 描述 Description | Interpro 编号 Interpro accession | Interpro 描述 Interpro description | 氨基酸起始Amino acid start | 氨基酸终止 Amino acid stop | 误差 E-value |
---|---|---|---|---|---|---|---|
Hamap | MF_00780 | Protein YceI | IPR023480 | Uncharacterised protein family UPF0312/YceI | 2 | 191 | 45.339172 |
PANTHER | PTHR34406 | Protein YCEI | - | - | 2 | 190 | 6.6E-62 |
SUPERFAMILY | SSF101874 | YceI-like | IPR036761 | Lipid/polyisoprenoid-binding,Yce-like superfamily | 22 | 191 | 2.88E-56 |
Gene3D | G3DSA:2.40128.110 | - | IPR036761 | Lipid/polyisoprenoid-binding,Yce-like superfamily | 24 | 191 | 1.7E-58 |
FunFam | G3DSA:2.40128.110:FF:000001 | UPF0312 protein HMPREF3014_17255 | - | - | 24 | 191 | 3.7E-118 |
SMART | SM00867 | YceI_2 | IPR007372 | Lipid/polyisoprenoid-binding,Yce-like | 25 | 189 | 1.3E-71 |
Pfam | PF04264 | YceI-like domain | IPR007372 | Lipid/polyisoprenoid-binding,Yce-like | 26 | 187 | 2.9E-41 |
Table 3 Function prediction via Interpro database
分析 Analysis | 编号 Accession No. | 描述 Description | Interpro 编号 Interpro accession | Interpro 描述 Interpro description | 氨基酸起始Amino acid start | 氨基酸终止 Amino acid stop | 误差 E-value |
---|---|---|---|---|---|---|---|
Hamap | MF_00780 | Protein YceI | IPR023480 | Uncharacterised protein family UPF0312/YceI | 2 | 191 | 45.339172 |
PANTHER | PTHR34406 | Protein YCEI | - | - | 2 | 190 | 6.6E-62 |
SUPERFAMILY | SSF101874 | YceI-like | IPR036761 | Lipid/polyisoprenoid-binding,Yce-like superfamily | 22 | 191 | 2.88E-56 |
Gene3D | G3DSA:2.40128.110 | - | IPR036761 | Lipid/polyisoprenoid-binding,Yce-like superfamily | 24 | 191 | 1.7E-58 |
FunFam | G3DSA:2.40128.110:FF:000001 | UPF0312 protein HMPREF3014_17255 | - | - | 24 | 191 | 3.7E-118 |
SMART | SM00867 | YceI_2 | IPR007372 | Lipid/polyisoprenoid-binding,Yce-like | 25 | 189 | 1.3E-71 |
Pfam | PF04264 | YceI-like domain | IPR007372 | Lipid/polyisoprenoid-binding,Yce-like | 26 | 187 | 2.9E-41 |
Fig. 4 PA0423 is a H2-T6SS-dependent antibacterial effector protein of P. aeruginosa The PAO1ΔPA0422-0423 strain competed with the attacker strains at 37℃ for 24 h. Values and error bars were SD ± average(n= 3 biological replicates). One-way ANOVA using Dunnett’s test, *P <0.05, **P <0.01, and ***P<0.001; the same below
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