Table 2.
Plasmids and PCR primers.
| Plasmid | Description | Ref. | |
|---|---|---|---|
| pMV158GFP | Mobilizable, Gram-positive vector encoding gfp | Nieto and Espinosa (2003) | |
| pSMART-LCKan | Low copy, transcription-free cloning vector | Lucigen Corp., Middleton, WI, USA | |
| pGEM-T Easy | PCR cloning vector | Promega Corp., Madison, WI, USA | |
| pDL276 | Shuttle vector for low %G + C Gram-positive bacteria | Dunny et al. (1991) | |
| pCAMP17 | Phage T12 derived integration vector with a cloning site under the control of the CAMP promoter | Gase et al. (1999) | |
| pWM448 | SpyCIM1 int clone into the CAMP promoter in pCAMP17 | This work | |
| pWM481 | SpyCIM5 int pseudogene and upstream (primers M5int5 and M5int4) fused to gfp in pSMART-LCKan | This work | |
| pWM483 | Insert from pWM481 cloned into pDL276 | This work | |
| pWM485 | SpyCIM5int pseudogene (primers M5int1 and M5int4) fused to gfp in pSMART-LCKan | This work | |
| pWM486 | SpyCIM5 int pseudogene subregion (primers M5int2 and M5int4) fused to gfp in pSMART-LCKan | This work | |
| pWM487 | SpyCIM5 int pseudogene subregion (primers M5int3 and M5int4) fused to gfp in pSMART-LCKan | This work | |
| pWM488 | Insert from pWM486 cloned into pDL276 | This work | |
| pWM489 | Insert from pWM485 cloned into pDL276 | This work | |
| pWM490 | Insert from pWM487 cloned into pDL276 | This work | |
| Sequence | Primer | Gene target | Product size (bp) |
| PRIMERS FOR PCR | |||
| 5′AGCAGGGATTGAACGCTTTA | attB-L | int-mutL intergenic region (attB) | 655 (strain SF370) |
| 5′TCAATTTCCCCACCAGTAGC | attB-R | 694 (strain Manfredo) | |
| 5′CTTGCCTGCTGAACTCATTG | attP-L | SpyCI attP | 461 |
| 5′CACGCTTTTAGACACACTCA | attP-R | ||
| 5′AAGCGTGAGGTCGTTCAAGT | mutS-L | mutS | 406 |
| 5′AGTGGCTGAGTTCTCGCATT | mutS-R | ||
| 5′GAGGCTTTACCGTCTGTTGC | mutL-L | mutL | 413 |
| 5′CCGGAAACTTCAAAATCCAA | mutL-R | ||
| 5′ATGAGTAAAGGAGAAGAACTTT | gfp-L | gfp ORF | 717 |
| 5′TTATTTGTATAGTTCATCCATGC | gfp-R | ||
| 5′ACAAGCATCACAGGAAGAAC | M5int1 | SpyCIM5 int pseudogene and promoter | Various lengths |
| 5′ATAAGATAACCGCTAGTGATA | M5int2 | ||
| 5′TATTGATCCGTCTGATTTGA | M5int3 | ||
| 5′AATTTTGTTTTTAACACTTTCTAAAC | M5int4 | ||
| 5′CTTACTCCCATTCGGGAACA | M5int5 | ||
| PRIMERS FOR qRT-PCR | |||
| 5′ACTGCCAATCCAAGTCCAAT | SpyCIM1 attP | 96 | |
| 5′TGAGAGGTGTAAACCTAATGAAACC | |||
| 5′TTGAGAAGTGTAAACCTAATGAAACC | SpyCIM5attP | 112 | |
| 5′CAATACCCTGTAAAAACTGCCAAT | |||
| 5′ACACTCGCTGGCCTTTCTACAACTTCA | attB | 65 | |
| 5′CGAGGCCTTCGGCTATTTAT | |||
| 5′CTGCGACAGCAACCTTGTAA | mutS | 108 | |
| 5′GCAACAAGAATGCGGAAAAT | |||
| 5′CCGGAAACTTCAAAATCCAA | mutL | 111 | |
| 5′GACATCAGGAACTGGCGATT | |||
| 5′AGCGTTGTCCGGATTTATTG | 16S rRNA | 126 | |
| 5′CACTCTCCCCTTCTGCACTC | |||