Article Figures & Data
Figures
- FIG. 1.
Map of the traI-trb operon, bisR triR gene region. Open reading frames are depicted by block arrows. Plasmids carrying different parts of the region are denoted by lines, and the positions of lacZ fusions are indicated by arrowheads. Restriction sites are marked as follows: H, HindIII; P, PstI; E, EcoRI.
- FIG. 2.
AHL production by bisR and triR mutants. Extracts of AHLs were separated by TLC, and the AHLs produced were visualized using C. violaceum CV026 as described previously (24). Lanes 1 to 3 depict extracts of AHLs which were isolated after 24 h of growth of strains A34, A549 (bisR::Tn5), and A627 (triR::Tn5), respectively. Lanes 4 to 6 depict extracts from the same strains after 48 h growth. The migration positions of various chemically synthesized standards are indicated on the left of the TLC.
- FIG. 3.
Growth inhibition tests indicate bisR represses cinI expression. Colonies of different AHL-producing bacteria were inoculated onto a lawn of the indicator strain A34 in a small bacteriocin-type test. The results of inoculation onto the lawn with strains 8401 and A34 (A), strains A549 and A627 (B), and the E. coli DH5α strain carrying pIJ7986 (cinR, cinI, and bisR) or pIJ7750 (cinR and cinI) (C) are shown.
- FIG. 4.
Assay of cinI-lacZ expression in a bisR mutant. Expression of cinI throughout growth in TY medium was assayed using pIJ7910 (cinI-lacZ), which had been introduced into the wild-type A34 strain (filled squares) and the bisR mutant A549 (triangles). For comparison, the expression of cinI-lacZ in strain 8401/pIJ7910 (derivative of A34 lacking pRL1JI) is also shown (diamonds). The growth of strain A34 is shown (open squares), and the growth rates of the other strains were similar. LSD, least significant difference.
- FIG. 5.
Assays of triR-lacZ expression in various mutants. Expression of triR was assayed using pIJ7878 (triR-lacZ) introduced into the wild-type strain A34 (filled diamonds), the rhiR mutant A160 (filled squares), the bisR mutant A549 (open squares), the triR mutant A627 (filled triangles), and the cinI mutant A664 (open triangles). OD600s measured using strain A34 are represented by open diamonds, and the growth rates of all the strains were similar.
- FIG. 6.
Growth inhibition tests indicate that growth sensitivity to 3OH-C14:1-HSL requires the presence of other AHLs. (A) The indicator strain was A34. (B) The indicator strain was A34/pIJ7867 (bisR and triR). (A and B) Strains 8401 and A34 were inoculated on top. (C and D) The indicator strain was 8401/pIJ7867 (bisR and triR). (C) Strain 8401 was inoculated on top. (D) Both 8401 and A34 were inoculated on top. (E) The indicator strain was 8401/pIJ7867/pIJ9071 (bisR triR traI) and strain 8401 was inoculated on top. (F) The indicator strain was A. tumefaciens C58.00/pIJ7867, and 8401 and A34 were inoculated on top. A similar result to that seen in panel F was obtained if the indicator strain was ANU265 carrying pIJ7867 (data not shown).
- FIG. 7.
Growth inhibition assays using purified AHLs. The assays were similar to those described for Fig. 3, except AHLs were added to the plate instead of AHL-producing colonies. In the three plates, the indicator strain was 8401/pIJ7867 (bisR triR). (Plate 1) 3OH-C14:1-HSL was added to the middle of the agar. (Plate 2) Both 3OH-C14:1-HSL and 3O-C8-HSL were added to the middle of the agar. (Plate 3) Both 3OH-C14:1-HSL and C8-HSL were added to the middle of the agar.
- FIG. 8.
Analysis of proteins induced by growth-inhibitory AHL extract. Following growth of strain A34/pIJ7867 to an OD600 of 0.1 in TY medium, AHL extracts from 8401 (containing 3OH-C14:1-HSL) (lane 2) and the cinI mutant A552 (lacking 3OH-C14:1-HSL) (lane 1) were added. After 2.5 h of incubation, the cells were disrupted and the proteins were separated by SDS-PAGE and stained with Coomassie blue. Lane 3 shows size markers (in kDa). The proteins indicated by arrows were induced by the extract from strain 8401.
- FIG. 9.
Model for regulatory interactions between the cinI cinR quorum-sensing genes and the bisR, triR, and traI genes. Bold arrows designate genes, and the contiguous arrows between traI and the trb genes indicate that these probably constitute an operon.
Tables
- TABLE 1.
Strains and plasmids
Strain or plasmid Description Source or reference R. leguminosarum strains 8401 Strain lacking pSym 22 A34 Derivative of 8401 carrying pRL1JI 12 A73 Derivative of 8401, Rifr A160 Derivative of 8401 carrying rhiR::Tn5 11 A549 Derivative of A34 carrrying bisR::Tn5 This work A552 Derivative of 8401 carrying cinR1::Tn5 23 A621 Derivative of A34 carrying cinR1::Tn5 23 A627 Derivative of A34 carrying triR::Tn5 This work A664 Derivative of A34 carrying cinI3::spc 23 A682 Derivative of A34 carrying trbE::Tn5-lacZ This work Rhizobium sp. strain ANU265 Plasmid-cured derivative of broad-host-range Rhizobium sp. strain NGR234 26 A. tumefaciens C58.00 Strain lacking the AT and Ti plasmids 39 C. violaceum CV026 AHL biosensor strain 24 Plasmids pIJ1891 pLAFR3-based cloning vector 14 pIJ7500 EcoRI fragment carrying bisR::Tn5 from A549 This work pIJ7542 pLAFR1 cosmid carrying bisR and triR This work pIJ7546 1.3-kb HindIII fragment from pIJ7545, carrying bisR, in pIJ1891 This work pIJ7750 2.35-kb EcoRV fragment, carrying cinR and cinI, in the HincII site of pUC19 23 pIJ7752 0.3-bp fragment carrying the bisR promoter cloned into pMP220 as a PstI-KpnI fragment; bisR-lacZ This work pIJ7630 10.8-kb EcoRI fragment from pIJ7542 carrying bisR and triR cloned in pUC18. This work pIJ7777 Deleted derivative of pIJ7750 carrying cinI but not cinR This work pIJ7786 EcoRI fragment carrying triR::Tn5 from A627 This work pIJ7867 2.2-kb SacI-EcoRI fragment carrying bisR and triR in pKT230 This work pIJ7873 bisR and triR cloned on a PstI fragment in pBBR1-MCS5 This work pIJ7878 triR-lacZ fusion in pMP220 This work pIJ7910 cinI-lacZ in pMP220 23 pIJ7986 1.3-kb HindIII fragment carrying bisR in the HindIII site of pIJ7750; bisR promoter is next to the pLac This work pIJ7987 As pIJ7986, except the 1.3-kb fragment is cloned in the opposite orientation This work pIJ9036 6.5-kb EcoRI fragment carrying traI in pUC18 This work pIJ9071 6.5-kb EcoRI fragment carrying traI in pMP220 This work pBBR1-MCS5 Broad-host-range cloning vector 21 pKT230 IncQ broad-host-range vector 3 pMP220 lacZ promoter vector 35 - TABLE 2.
Effect of 3OH-C14:1-HSL on triR-lacZ expression in mutants
Strain carrying pIJ7878 (triR-lacZ) β-Galactosidase activity (Miller units) No added AHL +3OH-C14:1-HSL A34 (wt)a 5,400 (±170) 22,700 (±500) A549 (bisR) 290 (±33) 320 (±40) A621 (cinR) 400 (±39) 21,800 (±450) A664 (cinI) 620 (±50) 22,100 (±440) ↵ a wt, wild type.
