Table 1.
Bacterial strains and plasmids used in this study.
| Strain or plasmid | Descriptiona or sequence | Source, reference, PCR products |
|---|---|---|
| E. coli DH5α | Host for cloning vector | In this lab |
| 05ZYH33 | Virulent Chinese S. suis serotype 2 isolate | In this lab |
| ΔMRP | Gene mrp knockout mutant strain; CmR | This study |
| ΔFhb | Gene fhb knockout mutant strain; CmR | Pian et al., 2012 |
| ΔSsads | Gene ssads knockout mutant strain; CmR | Liu et al., 2014 |
| Δ1538 | Gene 1538 knockout mutant strain; CmR | This study |
| Δ1083 | Gene 1083 knockout mutant strain; CmR | This study |
| Plasmids | Description | |
| pMD18-T | TA cloning vector, lacZ, AmpR | TaKaRa |
| pEASY-T1 | TA cloning vector, lacZ, AmpR | TransGen Biotech |
| pSET1 | S. suis-E. coli shuttle vector, CmR | Takamatsu et al., 2001 |
| pSET4s | Gene replacement vector with MCS of pUC19, SpcR | Takamatsu et al., 2001 |
| pSET4s:: mrp | The gene mrp knockout plasmid, SpcR CmR | This study |
| pSET4s:: 1538 | The gene 1538 knockout plasmid, SpcR CmR | This study |
| pSET4s:: 1083 | The gene 1083 knockout plasmid, SpcR CmR | This study |
| Primers | Sequenceb (5′–3′) | PCR products |
| mrp KOP1 | CCCAAGCTTTGGAACGTGAGCGTTTGGCAAG | The mrp gene and its upstream flanking regions |
| mrp KOP2 | CCTCGGAACCCATCGAATTACGACACACCAAGCAAAACG | |
| mrp KOP5 | CATCAAGCTCTAGTTCGGTGTACTGGTGAGGCTTCATCTG | The mrp gene and its downstream flanking regions |
| mrp KOP6 | CGCGGATCCCTCAAAGGATAGAGAGTTTGGAGCA | |
| mrp-F | GGAGCTGAAGTTGATGCCT | A fragment of mrp gene |
| mrp-R | GGTCGTTCTCCACAATTTCACG | |
| 1538KOP1 | GCAGGATCCAAAGAAGCCTTCACTGCAG | The 1538 gene and its upstream flanking regions |
| 1538KOP2 | CCTCGGAACCCATCGAATTAAGCTTGTGTTGCAATAACGC | |
| 1538KOP5 | CATCAAGCTCTAGTTCGGTGATACCGGTCAAGAAGC | The 1538 gene and its downstream flanking regions |
| 1538KOP6 | GCGGAATTCGTTATGAAGTCTTCGGCGC | |
| 1538-F | GGACTTGATGAGTATAACCG | A fragment of 1538 gene |
| 1538-R | GCCCGTTGTACCGTTTGTAT | |
| 1083KOP1 | GCAGGATCCGTGCAGTTCTTGTAGCTAAGACAGG | The 1083 gene and its upstream flanking regions |
| 1083KOP2 | CCTCGGAACCCATCGAATTAGATGCTTCTGAGCT | |
| 1083KOP5 | CATCAAGCTCTAGTTCGGTGGGTGTTGAATTGGCAG | The 1083 gene and its downstream flanking regions |
| 1083KOP6 | GCGGAATTCCACCGCTTGTAGGTTCC | |
| 1083-F | TGTGTTCGGTATTGGTTTTGCC | A fragment of 1083 gene |
| 1083-R | TGTACCTTCTGCTGATTGG | |
| CM-F | TAATTCGATGGGTTCCGAGG | Chloramphenicol resistant gene |
| CM-R | CACCGAACTAGAGCTTGATG | |
| SPC-F | GTGTTCGTGAATACATGTTATA | Spectinomycin resistant gene |
| SPC-R | GTTTTCTAAAATCTGATTACCA |
a
AmpR, ampicillin resistant; CmR, chloramphenicol resistant; Spcr, spectinomycin resistant; EmR, erythromycin resistant.
b
The underlined sequences are the restriction sites.