TABLE 2.
Site-directed mutations introduced at the −35 element (−40 to −25), the −10 element (−18 to −5), and the downstream regulatory elementa
| Plasmid | Sequences of the fecA promoter and the mutant fecA promoters | Relative fluorescence units (% of wild-type)
|
|
|---|---|---|---|
| NB + 50 μM dipyridyl | NB + 1 mM citrate | ||
| −35 element | |||
| pGFPA′ | GTAAGGAAAATAATTC | 403 (2) | 17,170 (100) |
| pGFP40 | TTAAGGAAAATAATTC | 363 (2) | 13,471 (78) |
| pGFP39 | GGAAGGAAAATAATTC | 247 (1) | 10,816 (61) |
| pGFP03 | GTCAGGAAAATAATTC | 669 (4) | 7,555 (44) |
| pGFP37 | GTACGGAAAATAATTC | 391 (2) | 16,101 (93) |
| pGFP36 | GTAATGAAAATAATTC | 958 (6) | 13,661 (79) |
| pGFP06 | GTAAGTAAAATAATTC | 704 (4) | 9,787 (57) |
| pGFP07 | TTAAGGCAAATAATTC | 1,287 (8) | 13,907 (81) |
| pGFP08 | TTAAGGACAATAATTC | 1,013 (6) | 11,847 (69) |
| pGFP09 | GTAAGGAACATAATTC | 635 (4) | 7,383 (43) |
| pGFP10 | GTAAGGAAACTAATTC | 446 (3) | 6,353 (37) |
| pGFP11 | GTAAGGAAAAGAATTC | 481 (3) | 7,039 (41) |
| pGFP29C | GTAAGGAAAATCATTC | 369 (2) | 6,122 (35) |
| pGFP388 | GTAAGGAAAATAGTTC | 351 (2) | 6,705 (39) |
| pGFP14 | GTAAGGAAAATAAGTC | 601 (4) | 9,272 (54) |
| pGFP26 | GTAAGGAAAATAATGC | 293 (2) | 6,745 (39) |
| pGFP25 | GTAAGGAAAATAATTA | 1,071 (6) | 8,035 (46) |
| pGFP3530 | GTAAGTAAAAGAATTC | 482 (3) | 5,736 (32) |
| pGFP3534 | GTAAGTCAAATAATTC | 287 (2) | 5,199 (29) |
| pGFP3230 | GTAAGGAACAGAATTC | 369 (2) | 3,764 (21) |
| pGFP3231 | GTAAGGAACCTAATTC | 262 (2) | 6,633 (37) |
| −10 element | |||
| pGFPA′ | CGATTGTCCTTTTT | 400 (2) | 17,926 (100) |
| pGFP19 | AGATTGTCCTTTTT | 244 (1) | 16,935 (94) |
| pGFP18 | CTATTGTCCTTTTT | 211 (1) | 12,934 (72) |
| pGFP17 | CGCTTGTCCTTTTT | 244 (1) | 13,639 (76) |
| pGFP16 | CGAGTGTCCTTTTT | 348 (2) | 7,147 (39) |
| pGFP27 | CGATGGTCCTTTTT | 430 (2) | 3,764 (21) |
| pGFP28 | CGATTTTCCTTTTT | 36 (0) | 179 (1) |
| pGFP28C | CGATTCTCCTTTTT | 235 (1) | 8,097 (45) |
| pGFP28A | CGATTATCCTTTTT | 525 (3) | 8,502 (47) |
| pGFP29 | CGATTGGCCTTTTT | 322 (2) | 2,151 (12) |
| pGFP30 | CGATTGTACTTTTT | 502 (3) | 4,660 (26) |
| pGFP31 | CGATTGTCATTTTT | 54 (0) | 717 (4) |
| pGFP32 | CGATTGTCCGTTTT | 72 (0) | 1,255 (7) |
| pGFP8G | CGATTGTCCTTGTT | 186 (1) | 11,228 (62) |
| pGFP7G | CGATTGTCCTTTGT | 208 (1) | 5,563 (31) |
| pGFP6G | CGATTGTCCTTTTG | 470 (3) | 8,869 (49) |
| Downstream regulatory element | |||
| pGFP41 | CTCTCGTTCGACTCATAGCT | 1,470 (8) | 15,954 (89) |
| pGFP44 | TTCCCGTTCGACTCATAGCT | 1,308 (7) | 14,162 (79) |
| pGFP48 | TTCTCGTCCGACTCATAGCT | 968 (5) | 11,831 (66) |
| pGFP49 | TTCTCGTTTGACTCATAGCT | 89 (0) | 896 (5) |
| pGFP50 | TTCTCGTTCTACTCATAGCT | 36 (0) | 179 (1) |
| pGFP50C | TTCTCGTTCCACTCATAGCT | 153 (1) | 9,793 (54) |
| pGFP50A | TTCTCGTTCAACTCATAGCT | 211 (1) | 220 (1) |
| pGFP51 | TTCTCGTTCGCCTCATAGCT | 107 (0) | 1,254 (7) |
| pGFP57 | TTCTCGTTCGACTCATCGCT | 609 (3) | 8,066 (45) |
| pGFP60 | TTCTCGTTCGACTCATAGCC | 753 (4) | 10,397 (58) |
a
The plasmids carry gfp fused to either the wild-type or mutated fecA promoter. Values (in Miller units) were determined for E. coli AA93 (Δfec) pSV66 (fecIRA) transformed with plasmids carrying the wild-type or mutant promoter fused to gfp. The bold letters indicate the changed nucleotides. The first base of each sequence given for the downstream regulatory element is at position +1.