Table 1.
Strains, plasmids and oligonucleotides
| Name | Descriptiona,b,c | Source |
|---|---|---|
| Strains | ||
| JCB387 | Δnir Δlac | (33) |
| RPB104 Δhns | RPB104MG1655 with C-terminal SPA-tagged rpoS | (23) |
| RPB104 hns::kan | This work | |
| Plasmids | ||
| pRW50 | low copy number lac fusion vector with EcoRI/HindIII cloning site and TetR | (34) |
| pSR | pBR322-derived vector with EcoRI/HindII cloning site upstream of λoop terminator. | (20) |
| Encodes AmpR. | ||
| pSR Δ45-9A-10T | pSR carrying an optimised derivative of the cbpA promoter | (17) |
| Primers for generating random DNA sequences | ||
| Random F | GGCTGCGAATTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN | This work |
| NNNNNAGGAGGATGCGGACTATG | ||
| Random R | CGCCCGAAGCTTcatagtccgcatcctcct | This work |
| Primers for generating synthetic promoter sequences | ||
| -10 F | GGCTGCGAATTCgaccggcgagcttcgcagtcagctgactataattgccgcgcgca | This work |
| -10 R | CGCCCGAAGCTTcatagtccgcatcctcctgcgcgcggcaattatagtcagctgac | This work |
| -10/-35TT F | GGCTGCGAATTCgaccttcgagcttcgcagtcagctgactataattgccgcgcgca | This work |
| -10/ATi F | GGCTGCGAATTCgaccggcgagcttcgctatttattgactataattgccgcgcgca | This work |
| -10/ATi R | CGCCCGAAGCTTcatagtccgcatcctcctgcgcgcggcaattatagtcaataaat | This work |
| -10/ATi/-35TT F | GGCTGCGAATTCgaccttcgagcttcgctatttattgactataattgccgcgcgca | This work |
| -10/ATii F | GGCTGCGAATTCgaccggcgagcttcgcagaattttgactataattgccgcgcgca | This work |
| -10/ATii R | CGCCCGAAGCTTcatagtccgcatcctcctgcgcgcggcaattatagtcagctgac | This work |
| -10/ATii/-35TT F | GGCTGCGAATTCgaccttcgagcttcgcagaattttgactataattgccgcgcgca | This work |
| -10 general R | GCCCGAAGCTTCatagtccgcatcctcctgcgcgcggcaattatagtc | This work |
| Primers for introducing an AT-rich spacer of random sequence | ||
| -10/ATR F | GGCTGCGAATTCgaccggcgagcttcgcwwwwwwwtgactataattgccgcgcgc | This work |
| -10/ATR/-35TT F | GGCTGCGAATTCgaccttcgagcttcgcwwwwwwwtgactataattgccgcgcgc | This work |
| Primers for amplifying intragenic promoters | ||
| wzxB 1.1 F | GGCTGCGAATTCacgttactttatctttactatctgc | This work |
| wzxB 1.1 R | GCCCGAAGCTTCCTCCTttgtaagaacacttggtcctgaaaa | This work |
| yigG 1.2 F | GGCTGCGAATTCtactccattatctcgtcatcaacatg | This work |
| yigG 1.2 R | GCCCGAAGCTTCCTCCTcattgcctgaacaggcaaaatcttc | This work |
| yqiI 2.2 F | GGCTGCGAATTCataagttacaccgaaagtataagag | This work |
| yqiI 2.2 R | GCCCGAAGCTTCCTCCTgaatattttatgaatgttttctg | This work |
| ygaQ 1.1 F | GGCTGCGAATTCcggttacacaatactaacttatttaac | This work |
| ygaQ 1.1 R | GCCCGAAGCTTCCTCCTtgaaaaatcaatggcgcttaaatcatc | This work |
| wcaD F | GGCTGCGAATTCTcaaacagtttggtatcaaaacg | This work |
| wcaD R | GCCCGAAGCTTCATAGTCCGCATCCTCCTcccctgaaaacgatccgg | This work |
| lpxD F | GGCTGCGAATTCAccagtgccagattgcacataacg | This work |
| lpxD R | GCCCGAAGCTTCATAGTCCGCATCCTCCTtcaggctgcccgccataatgacg | This work |
| Overlapping primers replacing AT-rich spacers with a GC-rich spacer | ||
| wcaD GC fwd | GGCTGCGAATTCtcaaacagtttggtatcaaacttcgcagtcagcttgctatgat | This work |
| wcaD GC rev | AGCCCGAAGCTTcctcctcccctgaaaacgatccggataatattatccctgcgagaat | This work |
| catagcaagctgactgcgaagtttgat | This work | |
| lpxD GC fwd | GGCTGCGAATTCaccagtgccagattgcaccttcgcagtcagctgacgacaat | This work |
| lpxD GC rev | AGCCCGAAGCTTcctccttcaggctgcccgccataatgacgccaccggcaaccgccgt | This work |
| attgtcgtcagctgactgcgaaggtgcaa | This work | |
| Synthetic DNA fragments to replace AT-rich spacers with GC-rich spacer | ||
| wzxB1.1 gc_Spacer | CTTGAGTCCACGCTAGATCTGGCTGCGAATTCAcgttactttatctttactatctgctg | This work |
| ctttggcaatactctgagttgctgtgagattgaaacttcgcagtcagctgactatcatatatagcatagtcg | ||
| cttggcaaaaaccgaatataccgaaattttcaggaccaagtgttcttacaaaggaggAAGCTTCGG | ||
| GCTTGTCAGTGCGCAAAAAGAT | ||
| yigG1.2 gc_spacer | CTTGAGTCCACGCTAGATCTGGCTGCGAATTctactccattatctcgtcatcaacatga | This work |
| attgccagcgactccgtgatagtggtttcatctatatacttcgcagtcagcttggtacattagcagtatatatc | ||
| atctctatcatcacaatgatagccgaagattttgcctgttcaggcaatgaggaggAAGCTTCGGGCTT | ||
| GTCAGTGCGCAAAAAGAT | ||
| yqiI2.2 gc_spacer | CTTGAGTCCACGCTAGATCTGGCTGCGAATTCataagttacaccgaaagtataagagtt | This work |
| ttgattataaaagtcttgacctcttcgcagtcagctgactatatttgcccatgcagatgggtattcttctcctggag | ||
| atgggcctggtagtgcattattacagaaaacattcataaaatattcaggaggAAGCTTCGGGCTTGT | ||
| CAGTGCGCAAAAAGAT | ||
| ygaQ1.1 gc_spacer | CTTGAGTCCACGCTAGATCTGGCTGCGAATTCcggttacacaatactaacttatttaac | This work |
| ccaaaatatcataaaaaagccgttatgaatttcgcagtcagcttggtaacttgtcagttggatgaacaacaa | ||
| atgtcatcactgctttatgaaagagatgatttaagcgccattgatttttcaaggaggAAGCTTCGGGCTT | ||
| GTCAGTGCGCAAAAAGAT |
aN is either A, C, G or T incorporated into the oligonucleotide at random but supplied at a defined % of each nucleotide. Used to generate the DNA fragment library described in Figure 1.
bW is A or T, with an equal likelihood of either base being incorporated.
cSynthetic promoter -10 elements are underlined and key base changes introduced by oligonucleotides are in bold.