TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristic(s)a | Source or reference |
|---|---|---|
| E. coli strains | ||
| CC118λpir | Δ(ara-leu) araD Δlac74 galE galK phoA20 thi-1 rpsE rpsB argE(Am) recA λpir | 41 |
| DH5α | F− ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 32 |
| DH5α λpir | DH5α lysogenized with λpir | 33 |
| V. fischeri strains | ||
| AKD100 | ES114 Tn7-ermR | 47 |
| AKD200 | ES114 Tn7-camR | 48 |
| CG103 | Wild-type isolate from Cleidopus gloriamaris | 84 |
| CL59 | ES114 luxO(D47E) | 19 |
| DC22 | C8-AHL bioreporter: ES114 ΔainS ΔluxR-luxI, mutant luxR (MJ1-T33A, R67M, S116A, M135I), PluxI-luxCDABEG | 22 |
| DM131 | ES114 flaJ::kanR | 48 |
| EM17 | Wild-type isolate from Euprymna morsei | 85 |
| ES114 | Wild-type isolate from E. scolopes | 31 |
| ES12 | Wild-type isolate from E. scolopes | 86 |
| ES213 | Wild-type isolate from E. scolopes | 86 |
| ES401 | Wild-type isolate from E. scolopes | 85 |
| ET101 | Wild-type isolate from Euprymna tasmanica | 87 |
| H905 | Planktonic isolate, Hawaii | 76 |
| KV1319 | ES114 with 2-bp insertion in celG | 51 |
| KV5005 | ES114 ΔrpoN | 43 |
| JB18 | ES114 litR::ermR | 22 |
| JHK057 | ES114 luxO(V106A) | This study |
| JHK061 | ES114 luxO(T335I) | This study |
| JHK062 | ES114 luxO(V106A) ΔrpoN | This study |
| JHK063 | ES114 luxO(T335I) ΔrpoN | This study |
| JHK065 | ES114 luxO(V106A) luxU::pEVS122 | This study |
| JHK066 | ES114 luxO(T335I) luxU::pEVS122 | This study |
| JHK068 | ES114 luxU::pEVS122 | This study |
| JHK069 | ES114 luxO(D47E) luxU::pEVS122 | This study |
| JHK070 | ES114 luxO(V106G) | This study |
| JHK073 | ES114 luxO(V106A) litR::ermR | This study |
| JHK074 | ES114 luxO(A91D) | This study |
| JHK075 | ES114 luxO(P41L) | This study |
| JHK076 | ES114 luxO(A91D) luxU::pEVS122 | This study |
| JHK077 | ES114 luxO(P41L) luxU::pEVS122 | This study |
| JHK087 | ES114 luxO(V106G) luxU::pEVS122 | This study |
| JHK105 | ES114 luxO(T335I) Δqrr | This study |
| NL60 | ES114 ΔainS | 4 |
| PMF8 | ES114 litR::kanR | 23 |
| PP3 | Planktonic isolate, Hawaii | 76 |
| TIM305 | ES114 Δqrr | 24 |
| TIM306 | ES114 ΔluxO | 24 |
| TME014F5 | ES114 mtsA::mini-Tn5 ermR | This study |
| TME014B6 | ES114 znuC2::mini-Tn5 ermR | This study |
| TME021D9 | ES114 yfhD::mini-Tn5 ermR | This study |
| VFS002F6 | ES114 vf0295::mini-Tn5 ermR | C. Whistler |
| VFS002F6-Tb | ES114 vf0295::mini-Tn5 luxO(A91D) ermR | C. Whistler |
| VFS012E9 | ES114 yfbQ::mini-Tn5 ermR | C. Whistler |
| VFS012E9-T | ES114 yfbQ::mini-Tn5 luxO(P41L) ermR | C. Whistler |
| VFS014F5-T | ES114 mtsA::mini-Tn5 luxO(V106A) ermR | C. Whistler |
| VFS014B6 | ES114 znuC2::mini-Tn5 ermR | C. Whistler |
| VFS014B6-T | ES114 znuC2::mini-Tn5 luxO(V106G) ermR | C. Whistler |
| VFS021D9-Tc | ES114 yfhD::mini-Tn5 luxO(T335I) ermR | C. Whistler |
| VLS2 | Wild-type isolate from E. scolopes | 84 |
| WH1 | Planktonic isolate, Massachusetts | 84 |
| WTTG0 | ES114 luxO(T107R) | This study |
| WTTG1 | ES114 luxO(F94C) | This study |
| WTTG2 | ES114 luxO(R114P) | This study |
| WTTG3 | ES114 luxO(H324R) | This study |
| WTTG4 | ES114 luxO(P98Q) | This study |
| WTTG5 | ES114 luxO(R113L) | This study |
| WTTG12 | ES114 luxO(P98L) | This study |
| WTTG17 | ES114 luxO(V108G) | This study |
| WTTG20 | ES114 luxO(V106G) | This study |
| WTTG21 | ES114 luxO(V106G) | This study |
| WTTG22 | ES114 luxO(H319R) | This study |
| WTTG24 | ES114 luxO(L205F) | This study |
| Plasmidsd | ||
| pAKD701 | Promoterless lacZ, pES213 R6Kγ oriTRP4 kanR | 37 |
| pEVS79 | pBC SK (+) oriT camR | 40 |
| pEVS104 | Conjugative helper plasmid, R6Kγ oriTRP4 kanR | 40 |
| pEVS122 | R6Kγ oriTRP4 ermR lacZα | 33 |
| pEVS170 | Mini-Tn5-ermR R6Kγ oriTRP4 kanR | 50 |
| pHK10 | PainS-lacZ reporter in pAKD701, pES213 R6Kγ oriTRP4 kanR | This study |
| pHK11 | mtsA in pVSV104, kanR | This study |
| pHK12 | PainS-gfp Pcon--mCherry in pJLS27, pES213 R6Kγ oriTRP4 kanR camR | This study |
| pHK20 | Pqrr-lacZ reporter in pAKD701, pES213 R6Kγ oriTRP4 kanR | 22 |
| pHK29 | znuC2 in pVSV104, kanR | This study |
| pHK45 | ES114 luxO in pVSV105, camR | This study |
| pHK70 | luxO(V106A) in pVSV105, camR | This study |
| pHK71 | luxO(D47E) in pVSV105, camR | This study |
| pHK73 | luxO(T335I) in pVSV105, camR | This study |
| pHK77 | luxO(V106A) and flanking sequence in pEVS79, camR | This study |
| pHK78 | luxO(T335I) and 1.5 kbp flanking sequence in pEVS79, camR | This study |
| pHK79 | 228-bp luxU fragment in pEVS122, ermR | This study |
| pHK80 | luxO(V106G) and flanking sequence in pEVS79, camR | This study |
| pHK82 | luxO(V106G) in pVSV105, camR | This study |
| pHK83 | luxO(A91D) in pVSV105, camR | This study |
| pHK84 | luxO(P41L) in pVSV105, camR | This study |
| pHK85 | luxO(P41L) and flanking sequence in pEVS79, camR | This study |
| pHK86 | luxO(A91D) and flanking sequence in pEVS79, camR | This study |
| pHK87 | luxO(T335I) and 1.1-kbp flanking sequence in pEVS79, camR | This study |
| pJLB95 | litR::ermR (opposite), ColE1 camR | This study |
| pJLS27 | Promoterless gfp, Pcon-mCherry pES213 R6Kγ oriTRP4 kanR | 38 |
| pLosTfoX | tfoX in pEVS79, camR | 42 |
| pMSM28 | ΔrpoN allele, R6Kγ oriTRP4 camR | 43 |
| pMulTfoX | tfoX in pVSV104, kanR | 42 |
| pTM267 | kan gfp PtetA-mCherry in pVSV105, camR | 24 |
| pTM268 | Pqrr-gfp PtetA-mCherry in pVSV105, camR | 24 |
| pVSV104 | Shuttle vector; pES213 R6Kγ oriTRP4 kanR lacZα | 39 |
| pVSV105 | Shuttle vector; pES213 R6Kγ oriTRP4 camR lacZα | 39 |
| Oligonucleotidese | ||
| pr_HK03 | GGGGCATGCAGAACCAAGACCTGCTCGTGCTAA | This study |
| pr_HK04 | GGCGCTAGCCATCAGTTGTTGAAGTAAATTAAAATTCTGCG | This study |
| pr_HK05 | CATGGTACCATATAGCCGTCTAGATGTAAACATTTCCAAACCG | This study |
| pr_HK06 | CATCCTAGGTTATGGCGCCTCTGTTAAATTAGTACTTTGTTTT | This study |
| pr_HK07 | CATGGTACCAAAATTAATTGTTATGTTATAACATAACAATTAAATAGCC | This study |
| pr_HK08 | CATCCTAGGCATCCAACATTCAGTACACTCCC | This study |
| pr_HK29 | GGCTCTAGACATCAGTTGTTGAAGTAAATTAAAATTCTGCG | This study |
| pr_HK73 | CATGGCATGCATATACCTATTGCAGGGAGCGTGC | This study |
| pr_HK74 | CATGGGTACCCAGCGATTTGATTAACATACTGACTCACGATAG | This study |
| pr_HK93 | CATGGGATCCAGACTATTTATGTCTCAGCCACACC | This study |
| pr_HK94 | CATGGGTACCATCGCCAAATCATGATTGG | This study |
| pr_HK95 | CATGGGATCCTCGGAAGTTGCAGAAGAAGG | This study |
| pr_HK96 | CATGGGTACCTGGTTCCACAGGCCGTATAC | This study |
| pr_HK101 | CATGGGATCCGGGACTATCGTGAGTCAGTA | This study |
| pr_HK102 | CATGGGATCCTGCCATTGTTGCAAGCTTATCT | This study |
| pr_HK110 | CATGGGATCCTGCAAATTGCGTTTTGCG | This study |
| pr_HK111 | CATGGGTACCGCTGTCAAACAAGCGGATTTAATA | This study |
a
Genes for drug resistance are as follows: camR, chloramphenicol resistance; ermR, erythromycin resistance; kanR, kanamycin resistance (aph).
b
Strains designated with the prefix “VFS” and the suffix “-T” were isolated from a mapped-transposon mutant library and represent the translucent (T) luxO*-bearing derivatives of the original transposon mutant parent. Strains designated with “WTTG” were isolated in a series of independent static culture survival experiments.
c
VFS021D9-T lacked the Kan resistance gene from the transposon delivery vector but displayed some Kan resistance.
d
All alleles cloned in this study are from V. fischeri strain ES114. Replication origins of the vectors are listed as R6Kγ, ColE1, oriV, and/or pES213. Plasmids based on pES213 are stable and do not require antibiotic selection for maintenance (39).
e
All oligonucleotides are shown 5′ to 3′. Restriction enzyme recognition sequences are underlined.