TABLE 1.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Description or sequencea | Reference or source |
|---|---|---|
| E. coli strains | ||
| Nissle 1917 clbA-lux | Luciferase fusion of the gene clbA, Kanr | 22 |
| Nissle 1917 clbR-lux | Luciferase fusion of the gene clbR, Kanr | 22 |
| Nissle 1917 clbB-lux | Luciferase fusion of the gene clbB, Kanr | 22 |
| Nissle 1917 clbQ-lux | Luciferase fusion of the gene clbQ, Kanr | 22 |
| Nissle 1917 clbA-lux ΔryhB | ryhB mutant strain, Kanr Cmr | This study |
| Nissle 1917 clbA-lux ΔryhB+p-ryhB | ryhB mutant strain carrying p-ryhB, Kanr Cmr Ampr | This study |
| Nissle 1917 clbA-lux Δfur | fur mutant strain, Kanr Cmr | This study |
| Nissle 1917 clbA-lux Δfur+p-fur | fur mutant strain carrying p-fur, Kanr Cmr Ampr | This study |
| M1/5 | Colibactin producer, commensal strain | 20 |
| M1/5 ΔryhB | ryhB mutant strain, Cmr | This study |
| M1/5 ΔryhB+p-ryhB | ryhB mutant strain carrying p-ryhB, Cmr Ampr | This study |
| M1/5 Δfur | fur mutant strain, Kanr | This study |
| M1/5 Δfur+p-fur | fur mutant strain carrying p-fur, Kanr Ampr | This study |
| Nissle 1917 | Colibactin producer, probiotic strain | 16 |
| Nissle 1917 ΔclbA+pBAD33-clbA | This study | |
| Nissle 1917 ΔclbA+pBAD33-clbA | clbA mutant strain carrying pBAD33-clbA, Kanr | This study |
| Nissle 1917 ΔclbA+pBAD33-clbAΔclbR | clbA mutant strain carrying pBAD33-clbAΔclbR, Kanr | This study |
| Nissle 1917 ΔryhB | ryhB mutant strain, Cmr | This study |
| Nissle 1917 ΔryhB+p-ryhB | ryhB mutant strain carrying p-ryhB, Cmr Ampr | This study |
| Nissle 1917 Δfur | fur mutant strain, Kanr | This study |
| Nissle 1917 Δfur+p-fur | fur mutant strain carrying p-fur, Kanr Ampr | This study |
| SP15 | Colibactin producer, pathogenic strain | 35 |
| SP15 ΔryhB | ryhB mutant strain, Cmr | This study |
| SP15 ΔryhB+p-ryhB | ryhB mutant strain carrying p-ryhB, Cmr Ampr | This study |
| SP15 Δfur | fur mutant strain, Kanr | This study |
| SP15 Δfur+p-fur | fur mutant strain carrying p-fur, Kanr Ampr | This study |
| MG1655 ΔryhB | ryhB mutant strain, Cmr | Gift from C. M. Dozois |
| Bw25113 Δfur | fur mutant strain, Kanr | Gift from C. M. Dozois |
| Plasmids | ||
| p-ryhB | High-copy-no. pSC-A plasmid carrying the ryhB gene; Ampr Kanr | This study |
| p-fur | High-copy-no. pSC-A plasmid carrying the fur gene; Ampr Kanr | This study |
| pBAD33-T7-Venus | High-copy-no. pBAD33 plasmid containing the Venus gene coding for a variant of the yellow fluorescent protein of Aequorea victoria | This study |
| pBAD33-T7-clbA | High-copy-no. pBAD33-T7 plasmid carrying the Venus fusion of the clbA gene | This study |
| pBAD33-T7-clbAΔclbR | High-copy-no. pBAD33-T7 plasmid carrying the Venus fusion of the clbA gene and mutated clbR | This study |
| Primers | ||
| Fur-P1 | AAAATCCTGGAAGTTCTTCAGGAGCCGGACAACCATCACGTCAGTGCGGAGTGTAGGCTGGAGCTGCTTC | This study |
| Fur-P2 | TTCGCGGCAATCGCCTTCGGCACAGTGACCGTAAAGATAGAGACTGTGGTCATATGAATATCCTCCTTAG | This study |
| Fur_F | CGCCCTAAAGAAAGCTGGCC | This study |
| Fur_R | CCTTCGTGCGCATGTTCATC | This study |
| Fur_F_Compl | CTGTAAGCTGTGCCACGTTTT | This study |
| Fur_R _compl | CTGAGAGCTGTAACTCTCGCTTTTC | This study |
| CMD1171_ST (ryhB::cat) | TTTGGGGTAAATGTCCCTTTC | 29 |
| CMD1172_ST (ryhB::cat) | GTGCGCATAACGAACACAAG | 29 |
| RyhB_F_Compl | CCTCTCGAGAAAGCGGACGTGGTTCCTAC | This study |
| RyhB_R_Compl | GTACTCGAGTGTTTCTGCGTGGCGTATTAC | 29 |
| Venus-XbaI | GCGCTCTAGATGGTGTCTATCACTAAAGATCAAATC | This study |
| Venus-fus-as | GCGCGTCGACTTACTTGTACAGCTCGTCCATG | This study |
| T7-term-fw | AAGGGGATCCGGCTGCTAAC | This study |
| T7-term-rev | GCGCAATTGCGGATATAGTTCCTCCTTTCAG | This study |
| pBAD33-Venus-Bam-rev | GCGCGCGGATCCGGCTGCTAACAAAG | This study |
| pBAD33-Venus-fw | P-TGGTGTCTATCACTAAAGATCAAATC | This study |
| clbAp-BamHI-fw | GCGCGGATCCAACCATCACCTTATTATCGG | This study |
| clbAp-rev1-1 | TTAGATAATCTCATTCCTGTTAGC | This study |
| clbAp-DelclbR-fw2 | CCGTTATCTAAGACAAGTATTGCGCATG | This study |
| clbAp-DelclbR-rev2 | ACTTGTCTTAGATAACGGGTTTTTTTCTTTG | This study |
| fyuA-up | CAACGCGCAGGCCTTTAC | This study |
| fyuA-rev | GCGTGCTTTCGTCTTGCTG | This study |
| clbC-up | CGAGTCAAATGCGCCATCAC | This study |
| clbC-rev | GGACCGCCATACCAATAATG | This study |
| JPN41 | CAGATACACAGATACCATTC | 14 |
| JPN42 | TCAATGAGGAAGAAATAAAAC | 14 |
a
Restriction sites are underlined. A P in front of the sequence indicates a phosphate residue.