Table 4.
Oligonucleotide sequences used in this study
| Primer | Sequence (5`→ 3`) | Amplicon (bp) | Target gene |
|---|---|---|---|
|
ldi deletion construct |
|
|
|
| (pK19mobsacB |
|
|
|
| ORF25_EcoRI_F |
TCGTAGAATTCCATGCCTGCGCACGCTGATG |
1307 |
ORF25 |
| ORF25_XhoI_ATG_R |
GAGACTCGAGATGTTCAGTCGCATGTCGTCT |
|
|
| ORF27_XhoI_TAA_F |
TATACTCGAGTAAGGGGGACGCGGCGGCCTG |
763 |
ORF27 |
| ORF27_HindIII_R |
TCGTAAAGCTTATGGACGACGGCACATGGA |
|
|
| p27 + _F |
ACGAAGCCGAGCATGCCCAC |
2199 |
encompassing |
| p27 + _R |
AGCAGCAGGCCGACGTGTTC |
|
ORF27 |
| p27mismatch_F |
CGCCCGGTTCGAGGAAGG |
- |
nucleotide |
| p27mismatch_R |
CCCTTCCTCGAACCGGGCG |
|
exchange |
|
geoA deletion construct |
|
|
|
| (pK19mobsacB) |
|
|
|
| ORF2930_XbaI_F |
TCTAGACCACCAGGGCGCATGCTTCAGTT |
1749 |
ORF2930 |
| ORF2930_XhoI_R |
CTCGAGTGAGCAGGGCGCGACTCC |
|
|
| ORF32_XhoI_F |
CTCGAGCATCGTTGAGTGTCTCCTGGTTG |
1712 |
ORF32 |
| ORF32_HindIII_R |
AAGCTTTGGAAACGACATAGGGGACAGGA |
|
|
| Control of ldi or geoA deletion |
|
|
|
|
ldi_F |
CGCCACCACCGAGGACTATTTC |
432 |
ldi |
|
ldi_R |
AGGTGGGCATGCTCGGCTTCGTA |
|
|
| ORF25_401_F |
GAAGGTGCGCGGCAAGGAATA |
2463a/ |
ORF25-27 |
| ORF27_2005_R |
CATGGACAGCGGCACACGGGCAA |
1269b |
|
|
geoA_260_F |
ACCGGGTCGTGCTGTCCTTCAAT |
284 |
geoA |
|
geoA_527_R |
CGCGCCGGTCTGGATGC |
|
|
| ORF30_30967_F |
CCAGACGCCGCCGATGATGAAGAG |
1904a/ |
ORF30-32 |
| ORF32_32822_R |
TATCTGAACAAGCCCGAACTGACC |
740c |
|
|
ldi complementation construct |
|
|
|
| (pBBR1MCS-4) |
|
|
|
|
ldi_EcoRI_F |
TGCGGAATTCATGCGGTTCACATTG |
1206 |
ldi |
|
ldi_BglII_R |
CGCGAGATCTTTATTTCCCTGCGA |
|
|
|
geoA complementation construct |
|
|
|
| (pBBR1MCS-2) |
|
|
|
|
geoA_XbaI_F |
AATCTAGACGCCCTGCTCAGAACAC |
1290 |
geoA |
|
geoA_HindIII_R |
GAGCAAGCTTACCCTGCGCAAGCAGTTC |
|
|
| Control of adjacent gene transcription |
|
|
|
| ORF25_254_F |
CCCACCGGCTTCTCGTAGTC |
535 |
ORF25 |
| ORF25_788_R |
GCAAGGGCCTGGGCGTGATGTC |
|
|
| ORF27_28_F |
CATGGACAGCGGCACACGGGCAA |
350 |
ORF27 |
| ORF27_377_R |
CAGTGGACCTCGCCGTGGAAAT |
|
|
| ORF30_315_F |
CGCGGGCGGGATGGTGAT |
411 |
ORF30 |
| ORF30_725_R |
CGGGCGGCGGGTTCGTT |
|
|
| ORF32_608_F |
CCTGGCGGCCGGACGACAT |
462 |
ORF32 |
| ORF32_1069_R | CGCCGGAAGGGGAAACGAC |
Restriction sites are underlined. Oligonucleotide primers derived from annotated 50 kb contig of C. defragrans 65Phen (Acc. no. FR669447.2) [47]. a wild type; b C. defragrans Δldi, c C. defragransΔgeoA.