Abstract
Conjugative transfer of Agrobacterium Ti plasmids is regulated by TraR, a quorum-sensing activator. Quorum dependence requires TraM, which binds to and inactivates TraR. In this study, we showed that TraR and TraM form a 151-kDa stable complex composed of two TraR and two TraM dimers both in vitro and in vivo. When interacted with TraR bound to tra box DNA, wild-type TraM formed a nucleoprotein complex of 77 kDa composed of one dimer of each protein and DNA. The complex converted to the 151-kDa species with concomitant release of DNA with a half-life of 1.6 h. TraR in the complex still retained tightly bound autoinducer. From these results, we conclude that TraM interacts in a two-step process with DNA-TraR to form a large, stable antiactivation complex. Mutagenesis identified residues of TraR important for interacting with TraM. These residues form two patches, possibly defining the binding interfaces. Consistent with this interpretation, comparison of the trypsin-digested polypeptides of TraR and of TraM with that of the TraR-TraM complex revealed that a tryptic site at position 177 of TraR around these patches is accessible on free TraR but is blocked by TraM in the complex. From these genetic and structural considerations, we constructed three-dimensional models of the complex that shed light on the mechanism of TraM-mediated inhibition of TraR and on TraM-mediated destabilization of the TraR-DNA complex.
Highlights
Agrobacterium tumefaciens, the causative agent of plant tumors called crown galls, utilizes a LuxR/LuxI-type quorum-sensing system to regulate the conjugative transfer of its tumor-inducing (Ti) plasmids (1, 2)
Mutagenesis identified residues of TraR important for interacting with TraM. These residues form two patches, possibly defining the binding interfaces. Consistent with this interpretation, comparison of the trypsin-digested polypeptides of TraR and of TraM with that of the TraR-TraM complex revealed that a tryptic site at position 177 of TraR around these patches is accessible on free TraR but is blocked by TraM in the complex
Strain NTS2(pKMA1, pKKTM), which co-expresses both TraR and TraM, yielded a TraR-TraM complex (Fig. 2, B and C, lanes 1 and 2) that elutes at the same position as the ϳ151-kDa complex, as assessed by gel filtration chromatography followed by SDS-PAGE
Summary
Bacterial Strains and Growth Media—Bacteria used in this study included Escherichia coli BL21(DE3) (T7 promoter expression host) (Novagen) and Agrobacterium tumefaciens strain NTL4, a Ti plasmid-cured derivative of C58 (19). Gel Filtration Chromatography of Purified Proteins—Samples containing TraR, TraM, the 18-bp tra box DNA (ACGTGCAGATCTGCACGT), or their mixtures were chromatographed on Superdex 200HR using an AKTA fast protein liquid chromatography system as described previously (15). Interactions between purified TraM and TraR proteins on membrane strips or separated by SDS-PAGE were assessed by Far Western analysis using anti-TraM antiserum as described previously (12). The broken cell preparation was extracted with ethyl acetate three times as described previously (25), and the resulting extracts containing 3-oxo-C8-HSL were combined and concentrated to dryness using a Vacufuge. Quantification of tra Box DNA Contained in Protein Samples— A series of 5-l samples of fractions eluted from gel filtration columns were spotted onto a sheet of Parafilm (American National Can). The polypeptides in the digests were analyzed on a Micromass Q-TOF Ultima, a hybrid quadrupole time-of-flight mass spectrometer by the Mass Spectrometry Laboratory in the school of Chemical Science at the University of Illinois at Urbana-Champaign
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
