Table 1.
Bacterial strains and plasmids used in this study.
| Strain, plasmid, or oligonucleotide | Relevant characteristicsa | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| CC118λpir | Δ(ara-leu) araD Δlac74 galE galK phoA20 thi-1 rpsE rpsB argE(Am) recA λpir | 68 |
| DH5α | F-φ80dlacZΔM15 ∆(lacZYA-argF)U169 deoR supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 63 |
| DH5α λpir | DH5α lysogenized with λpir | 64 |
| JW1644-5 | ∆(araD-araB)567 ∆(lacZ4787(::rrnB-3)) λ- ∆(rnt-730::kanR) rph-1 F- ∆(rhaD-rhaB)568 hsd514 | 52 |
| JW1793-1 | ∆(araD-araB)567 ∆(lacZ4787(::rrnB-3)) λ- ∆(rnd-729::kanR) rph-1 F- ∆(rhaD-rhaB)568 hsd514 | 51 |
| JW3618-2 | ∆(araD-araB)567 ∆(lacZ4787(::rrnB-3)) λ- ∆(rph-749::kanR) rph-1 F- ∆(rhaD-rhaB)568 hsd514 | 51 |
| JW5741-1 | ∆(araD-araB)567 ∆(lacZ4787(::rrnB-3)) λ- ∆(rnr-729::kanR) rph-1 F- ∆(rhaD-rhaB)568 hsd514 | 52 |
| MG1655 | F– λ– ilvG – rfb-50 rph-1 | 65 |
| SK2595 | araD139 galE15 galK16 Δ(ara-leu)7697 spoT1 λ- hsdR2 Δ(codB-lacI)3 mcrA0 relA1 rpsL150 mcrB9999 ∆(rnz-500::kanR) ∆(elaC500::kanR) | 53 |
| V. fischeri | ||
| DC22 | C8-HSL bioreporter: ES114 ∆ainS ∆luxR-luxI, mutant luxR (MJ1-T33A, R67M, S116A, M135I), PluxI-luxCDABEG | 24 |
| DC43 | ES114 ΔainS ΔluxI Pcon-luxR MJ1 PluxI-luxCDABEG | 21 |
| DJ01 | ES114 ΔainS ΔluxI Pcon-luxR ES114 PluxI-luxCDABEG | 21 |
| ES114 | Wild-type isolate from E. scolopes | 36 |
| JB18 | ES114 litR::ermR | 13 |
| JHK003 | ES114 ∆ainR | 13 |
| JHK007 | ES114 ΔainS ΔluxIR PluxI-luxCDABEG | 13 |
| JHK045 | ES114 ΔainS ΔluxI Pcon-luxR ES114 PluxI-luxCDABEG litR::ermR | This study |
| JHK046 | ES114 ΔainS ΔluxIR PluxI-luxCDABEG litR::ermR | This study |
| JHK055 | ES114 ainR trunc (Δ30-815) | This study |
| JHK056 | ES114 ainR nat (Δ36-815) | This study |
| JHK091 | ES114 ΔluxI Pcon-luxR MJ1 PluxI-luxCDABEG litR::ermR | This study |
| JHK099 | ES114 ΔainS ΔluxI Pcon-luxR MJ1 PluxI-luxCDABEG litR::ermR | This study |
| JHK114 | ES114 ΔainR | This study |
| JHK115 | ES114 ainR scar | This study |
| JHK119 | ES114 ainR scar-repaired | This study |
| JHK120 | ES114 ainR mut | This study |
| NL60 | ES114 ∆ainS | 23 |
| NL63 | ES114 ∆ainS luxI | 13 |
| Plasmids b | ||
| pCR-Blunt | PCR product cloning vector; ColE1 oriV kanR | Invitrogen |
| pCR-Blunt-II-TOPO | PCR product cloning vector; ColE1 oriV kanR | Invitrogen |
| pDJ01 | Pcon-luxR ES114 PluxI-luxCDABEG ColE1, R6Kγ, oriT RP4, kanR, camR | 21 |
| pEVS104 | Conjugative helper plasmid; R6Kγ oriT RP4 kanR | 69 |
| pEVS118 | Suicide vector; R6Kγ, oriT RP4, camR | 64 |
| pEVS122 | Suicide vector; R6Kγ, oriT RP4, ermR, lacZα | 64 |
| pHK12 | PainS-gfp Pcon -mCherry in pJLS27; pES213, R6Kγ oriT RP4 kanR, camR | 70 |
| pHK34 | ainR trunc (Δ30-815) in pCR-Blunt; ColE1 oriV kanR | This study |
| pHK37 | ainR trunc (Δ30-815) allele; ColE1 R6Kγ oriV oriT RP4 camR kanR | This study |
| pHK75 | 1500-bp ainR downstream in pEVS122; R6Kγ oriT RP4 ermR lacZα | This study |
| pHK76 | ainR nat (Δ36-815) allele; R6Kγ oriT RP4 ermR lacZα | This study |
| pHK93 | PainS-ainR trunc (Δ36-815) in pCR-Blunt; ColE1 oriV kanR | This study |
| pHK94 | PainS-ainR nat (Δ30-815) in pCR-Blunt; ColE1 oriV kanR | This study |
| pHK95 | PainS-ainR in pCR-Blunt; ColE1 oriV kanR | This study |
| pHK102 | PainS-ainR trunc (Δ30-815) allele; ColE1 R6Kγ oriV oriT RP4 kanR camR | This study |
| pHK103 | PainS-ainR nat (Δ36-815) allele; ColE1 R6Kγ oriV oriT RP4 kanR camR | This study |
| pHK104 | PainS-ainR allele; ColE1 R6Kγ oriV oriT RP4 kanR camR | This study |
| pHK129 | ainSR ClaI-NdeI fragment with mutated IR1 (ainR_IR_conAA) in pCR-Blunt; ColE1 oriV kanR | This study |
| pHK135 | 1,350-bp upstream of ainR in pCR-Blunt; ColE1 oriV kanR | This study |
| pHK136 | ∆ainR, 1,480-bp downstream ainR in pHK135; ColE1 oriV kanR | This study |
| pHK137 | ainR scar in pHK136; ColE1 oriV kanR | This study |
| pHK138 | ∆ainR allele; ColE1 R6Kγ oriT RP4 kanR camR | This study |
| pHK139 | ainR scar allele; ColE1 R6Kγ oriT RP4 kanR camR | This study |
| pHK152 | ainR mut in pHK95; ColE1 oriV kanR | This study |
| pHK153 | ainR mut allele; ColE1 R6Kγ oriT RP4 kanR camR | This study |
| pHK156 | PainS-gfp (truncated) Pcon -mCherry in pJLS27; pES213, R6Kγ oriT RP4 kanR, camR | This study |
| pJLS27 | Promoterless gfp, Pcon -mCherry pES213, R6Kγ oriT RP4 kanR | 26 |
| pJLB95 | litR::ermR (opposite) allele; ColE1 camR ermR | 13 |
| Oligonucleotides c | ||
| pr_HK01 | GGATCTGGCTTTTAAAAAATGCATCATCTGC | This study |
| pr_HK02 | CATCTAGATGACGATGAAGTACAGATATTGGTTTATGAAT | This study |
| pr_HK03 | GGGGCATGCAGAACCAAGACCTGCTCGTGCTAA | 70 |
| pr_HK13 | AGCGCCCAATACGCAAACC | This study |
| pr_HK14 | CCGGCGTGTCAATAATATCACTCTGTACA | This study |
| pr_HK17.2 | CATGGGATCCTAGAGAGCGGATAAAATACCCTACCCAA | This study |
| pr_HK27.3 | CATGGGTACCAGAACCAAGACCTGCTCGTGCTAA | This study |
| pr_HK28.2 | CATGGGATCCTAAGGGTTTACCTTTGTCCGCTCTCTA | This study |
| pr_HK40.1 | CATGGGATCCATAAGTGGTTATAACACCGATAAAAAAATAGCC | This study |
| pr_HK41.4 | CATGGCATGCTGAAGGTGCTTGCTATTACTGATCA | This study |
| pr_HK126 | CATGGGATCCTGAAGGTGCTTGCTATTACTGATC | This study |
| pr_HK136 | CATGGCTAGCTTAACTACTTTACCTAAAGTTTATTTAC | This study |
| pr_HK137 | CATGGCTAGCTAACCACTTATCTACGACCT | This study |
| pr_HK144 | AAAATAAGTATTCCAAATTTCCAA | This study |
| pr_HK146 | AAAGTACTCATAACACCACTACC | This study |
| pr_NL28.3 | GGGCCTAGGCATTTATATAAAACTCACTGA TTTCGAAGTTT | 23 |
| pr_NL29 | GGGGCCTAGGTAACACCGATAAAAAAATAGCCAGAAC | 23 |
| pr_NL35 | GAGTCCGTTAGCAAGGTCACACTTTGTTG | 23 |
| pr_NL63 | GGGCCTAGGCTACTCTTTTATAAATTCATATTGCAGGTTTT | 23 |
| pr_NL89 | AAATCTAAGGGTTTACCTTTGTCCGCTCTC | 24 |
| pr_NL108 | GGCGGAACGATTGGAAATTTGGAATACTTATTTTCAACATC | 24 |
| pr_NL109 | CAGTACTGCATTTCAAAAGACAACCAAAAACTTTGATAGCC | 24 |
| QO | CCAGTGAGCAGAGTGACG | 72 |
| QI | GAGGACTCGAGCTCAAGC | 72 |
| QT | CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT | 72 |
aDrug resistance abbreviations used: camR, chloramphenicol resistance; ermR, erythromycin resistance; and kanR, kanamycin resistance (aph).
bAll alleles cloned in this study are from V. fischeri strain ES114. Replication origin(s) of each vector are listed as R6Kγ, ColE1, oriV and/or pES213. Plasmids based on pES213 are stable and do not require antibiotic selection for maintenance.
cAll oligonucleotides are shown 5′ to 3′. Restriction enzyme recognition sequences are underlined.