Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmidGenotype or constructionSource or reference
Strains
E. coli
  TG1supE hsdΔ5 thi Δ(lac-proAB) F′ (traD36 proAB+lacIqlacZΔM15)6
  BL21(DE3)hsdS gal (λcIts857ind1 Sam7 nin5 lacUV5-T7 gene 139
  S17-1 λpirTpr SmrrecA thi pro(r m+) RP4::2 Tc::Mu::Km Tn7λpir30
  C118 λpirΔ(ara-leu) araD ΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA1 λpir phage lysogen16
P. putida
  PaW85Tn46523
  PKS54Tn4652 rpoS::KmrThis work
  PKSRpoSP. putida PKS54 rpoS under control of Ptac promoter and lacIqrepressor; Tcr
Plasmidsa
 pBluescript KS(+)Cloning vector (Apr)Stratagene
 pBlcrpoS-IpBluescript KS(+) containing 1-kb PCR-amplifiedrpoS cloned into EcoRV site
 pUC4KCloning vector containing Kmr gene from transposon Tn903 (AprKmr)34, 46
 pBlcrpoS-Kmr2.5-kbrpoS-Kmr sequence containingXbaI-EcoRI fragment from pUC4K cloned intoEco72I site in rpoS geneThis work
 pUTmini.Tn5 luxABDelivery plasmid for mini-Tn5 luxAB (AprTetr)8
 pUTrpoS-Kmr2.5-kb rpoS-Kmrsequence-containing XbaI-EcoRI fragment from pBlcrpoS-Kmr cloned into pUTmini-Tn5 luxABThis work
 pBlcrpoS-IIpBluescript KS(+) containing 900-bp PCR-amplified rpoS cloned intoEcoRV siteThis work
 pET24dProtein expression vector (Kmr)Stratagene
 pET24d-rpoSpET24d containing rpoS gene from pBlcrpoS-II inserted intoNcoI and HindIII sitesThis work
 pKTlacZCloning vector (Apr)18
 pBRlacItacPtac promoter andlacIq repressor in 2.2-kbNruI-EcoRI fragment from plasmid pMMB208 cloned into EcoRV-EcoRI-cleaved pBR322b
 pMir61rpoSgene of P. putida KT2440 in pUN19Ø36
 pBRlacItac-rpoSrpoS in 1-kbXhoI-HindIII fragment from plasmid pMir61 cloned into SalI-SmaI-cleaved pBRlacItacThis work
 pUC18NotpUC18 with NotI restriction site in multicloning region (Kmr)16
 pUCptac-rpoS3.2-kb rpoS expression cassettePtac-rpoS-lacIq from pBRlacItac-rpoS inserted into BamHI-KpnI-cleaved pUC18NotThis work
 pUTptac-rpoSPtac-rpoS-lacIq from pUCptac-rpoS inserted into NotI-cleaved pUTmini-Tn5 luxABThis work
  • a The strategy of cloning of the fusion promoters and their mutant forms into promoter probe vector pKTlacZ is shown in Materials and Methods and in Fig. 1 and 2.

  • b —, Hõrak and Kivisaar, submitted.