Article Figures & Data
Figures
- Fig. 1.
Physical and genetic map of the (a) NifL- and the (b) NifA-coding regions from A. vinelandii. NifL is divided into a sensory domain and a kinase-like domain. NifA is divided into a regulatory domain, a catalytic domain, and a DNA-binding domain. The hatched regions in NifL and NifA correspond to regions used to construct fusion proteins with GAL4 domains. Numbers correspond to amino acid residue numbering. N and C represent the amino and carboxyl termini, respectively.
- Fig. 2.
The nucleotide sequence and predicted amino acid sequence (represented in single letter code) of fusion protein junctions in pBG502, pBG503, pBG504, and pBG505 are shown. The nucleotide sequences corresponding to NifA and NifL are shown in bold. (A) Fusion junction of the carboxyl-terminal sequence of the GAL4 DNA-binding domain and the catalytic domain of NifA present in pBG502. (B) Fusion junction of the carboxyl-terminal sequence of the GAL4 DNA-binding domain and the kinase-like domain of NifL present in pBG503. (C) Fusion junction of the carboxyl-terminal sequence of the GAL4-activation domain and the catalytic domain of NifA present in pBG504. (D) Fusion junction of the carboxyl-terminal sequence of GAL4-activation domain and the kinase-like domain of NifL present in pBG505.
- Fig. 3.
Colony filter-lift assay to identify the specificity of interaction between NifL and NifA. In these experiments, the strains ofS. cerevisiae CG1945 carrying different combinations of plasmids were grown on SD plates lacking Trp and Leu and were transferred to a Whatman no. 5 paper filter. Cells were permeabilized by freeze-thaw treatment of the filters (freezing in liquid nitrogen and allowing them to thaw at room temperature). The filters carrying the cells were then placed over filters presoaked with Z-buffer–X-Gal solution according to the protocols of the MATCHMAKER yeast two-hybrid assay. β-Galactosidase activity was detected only when S. cerevisiae CG1945 contained pBG503 (GAL4 BD:NifL) and pBG504 (GAL4 AD:NifA).
Tables
- Table 1.
Bacterial strains, yeast strains, and plasmids used in this study
Strain or plasmid Relevant characteristics and description Source or reference A. vinelandiiOP Wild-type, nitrogen-fixing, soil bacterium Laboratory stock Escherichia coliTG1 K-12 Δ(lac-pro) supE thi hsd-5/F′ traD36 proA+B+ lacIqlacZΔM15 Amersham Life Sciences, Inc. E. coliINVαF′ F′ endA1 recA1 hsdR17(rK− mK+) Δ(lac)U169 φ80lacΔM15 Δ(lacZYA-argF)U169 λ− supE44 thi-1 relA1 gyrA96 Invitrogen Corp. S. cerevisiae CG1945 MATa ura3-52 his3-200 lys2-801 ade2-101 leu2-3 trp1-901 112 gal4-542 gal180-538 cyhr2 LYS2::GAL1UAS-GAL1TATA-HIS3 URA3::GAL417-mers(×3)-CYC1TATA-lacZ CLONTECH Laboratories, Inc. pVA3-1 Murine p53(72–390) in pAS2-1, TRP1, Ampr (9.4 kb) CLONTECH Laboratories, Inc. pTD1-1 SV40 large T antigen (84–708) in pACT2, LEU2, Ampr (9.9 kb) CLONTECH Laboratories, Inc. pCR 2.1 Ampr Kanr (3.9 kb); used for direct cloning of PCR products Invitrogen Corp. pBG500 Derivative of pCR 2.1 in which 815-bp DNA fragment that encodes the central domain (catalytic domain, Fig. 1b) of NifA was cloned; this fragment was generated by PCR amplification using the primers described in the text; this fragment could be released by digesting with EcoRI and could be cloned into the EcoRI site of pAS2-1 or pGAD424 to generate in-frame fusions with GAL4 DNA BD or AD, respectively This work pBG501 Derivative of pCR 2.1 in which 789-bp DNA fragment that encodes the central domain (catalytic domain, Fig. 1a) of the NifL was cloned; this fragment was generated by PCR amplification using the primers described in the text; this fragment could be released by digesting with EcoRI and could be cloned into theEcoRI site of pAS2-1 or pGAD424 to generate in-frame fusions with GAL4 DNA BD or AD, respectively This work pAS2-1 GAL4(1–147) DNA-BDTRP1 AmprCYHs2 (8.5 kb); unique cloning sites include NdeI, NcoI,SfiI, EcoRI, XmaI/SmaI,BamHI, SalI, and PstI CLONTECH Laboratories, Inc. pGAD424 GAL4(768–881)AD LEU2 Ampr hemagglutinin epitope tag (8.1 kb); unique cloning sites include SfiI, NcoI,XmaI/SmaI, BamHI, EcoRI,SacI, and XhoI CLONTECH Laboratories, Inc. pBG502 pAS2-1 derivative in which the EcoRI fragment that encodes the catalytic domain of the NifA (obtained byEcoRI digestion of pBG500) was cloned to generate the in-frame GAL4 BD-NifA translation fusion This work pBG503 pAS2-1 derivative in which the EcoRI fragment that encodes the kinase-like domain of the NifL (obtained byEcoRI digestion of pBG501) was cloned to generate the in-frame GAL4 BD-NifL translation fusion This work pBG504 pGAD424 derivative in which the EcoRI fragment that encodes the catalytic domain of the NifA (obtained byEcoRI digestion of pBG500) was cloned to generate the in-frame GAL4 AD-NifA translation fusion This work pBG505 pGAD424 derivative in which the EcoRI fragment that encodes the kinase-like domain of the NifL (obtained byEcoRI digestion of pBG501) was cloned to generate the in-frame GAL4 AD-NifL translation fusion This work - Table 2.
Results of the liquid β-galactosidase assay with ONPG as substrate to demonstrate NifL-NifA interactions
Plasmid to which GAL4 BD was translationally fused Plasmid to which GAL4 AD was translationally fused β-Galactosidase activity (Miller units)a Growth on SD plates lacking Leu, Trp, and His/3ATb Interacting peptides pBG502 (GAL4 BD:NifA) pBG505 (GAL4 AD:NifL) 5.60 ± 0.22 ++ NifA and NifL pBG503 (GAL4 BD:NifL) pBG504 (GAL4 AD:NifA) 5.71 ± 0.37 ++ NifA and NifL pAS2-1 (GAL4 BD) pBG504 (GAL4 AD:NifA) 0.63 −− None pAS2-1 (GAL4 BD) pBG505 (GAL4 AD:NifL) 0.53 −− None pBG502 (GAL4 BD:NifA) pGAD424 (GAL4 AD) 0.50 −− None pBG503 (GAL4 BD:NifL) pGAD424 (GAL4 AD) 0.71 −− None pVA3-1 (GAL4 BD:p53) pTD1-1(GAL4 AD:SV40 large T antigen) 35.9 ± 2.61 ++ p53 and large T antigen pAS2-1 (GAL4 BD) pABCT2 (GAL4 AD) 1.13 −− None ↵a The β-galactosidase activity assay was performed as described in the MATCHMAKER two-hybrid system (20). Briefly, cells from 1.5-ml samples of exponential culture were collected by centrifugation and resuspended in 300 μl of Z-buffer (20). A 100-μl aliquot of the resuspended cells was lysed by quick freeze-thaw (treatment with liquid nitrogen followed by thawing at 37°C). To measure the β-galactosidase activity in the cell lysate, a 0.7-ml sample of the Z-buffer–β-mercaptoethanol solution was added to each tube followed by 0.16 ml of Z-buffer–ONPG (4 mg of ONPG per 1 ml of Z-buffer). The time of ONPG addition was recorded, and the tubes were incubated at 30°C with shaking. When yellow color was visible, 400 μl of 1 M NaCO3 was added to each tube to terminate the reaction, and the time was recorded. The tubes were then centrifuged for 10 min at 10,000 × g to remove cellular debris, and the optical density at 420 nm was recorded. β-Galactosidase units were defined as the amount of enzyme which hydrolyzes 1 μmol of ONPG to o-nitrophenol andd-galactose per min.
↵b S. cerevisiae CG1945 was transformed with plasmid DNA by utilizing the YEASTMAKER yeast transformation system (22) (CLONTECH Laboratories, Inc.). Transformants were selected on appropriate nutrition-deficient SD medium selection plates by incubating at 30°C for 2 to 3 days. Single colonies from each transformation experiment were purified and were used to determine growth characteristics on SD media lacking His.
