Agrobacterium tumefaciens BlcR represses transcription of the blcABC operon, which is involved in metabolism of γ-butyrolactone, and this repression is alleviated by succinate semialdehyde (SSA). BlcR exists as a homodimer, and the blcABC promoter DNA contains two BlcR-binding sites (IR1 and IR2) that correspond to two BlcR dimers. In this study, we established an in vivo system to examine the SSA-responsive control of BlcR transcriptional regulation. The endogenous blcR, encoded in the pAtC58 plasmid of A. tumefaciens C58, was not optimal for investigating the effect of SSA on BlcR repression, probably due to the SSA degradation mediated by the pAt-encoded blcABC. We therefore introduced blcR (and the blcABC promoter DNA, separately) exogenously into a strain of C58 cured of pAtC58 (and pTiC58). We applied this system to interrogate BlcR–DNA interactions and to test predictions from our prior structural and biochemical studies. This in vivo analysis confirmed the previously mapped SSA-binding site and supported a model by which DNA coordinates formation of a BlcR tetramer. In addition, we identified a specific lysine residue (K59) as an important determinant for DNA binding. Moreover, based on isothermal titration calorimetry analysis, we found IR1 to play the dominant role in binding to BlcR, relative to IR2. Together, these in vivo results expand the biochemical findings and provide new mechanistic insights into BlcR–DNA interactions.
CarlierA., ChevrotR., DessauxY., FaureD.(2004). The assimilation of γ-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal. Mol Plant Microbe Interact 17:951–957 [View Article] [PubMed]
ChaiY., TsaiC. S., ChoH., WinansS. C.(2007). Reconstitution of the biochemical activities of the AttJ repressor and the AttK, AttL, and AttM catabolic enzymes of Agrobacterium tumefaciens. J Bacteriol 189:3674–3679 [View Article] [PubMed]
GuazzaroniM. E., KrellT., FelipeA., RuizR., MengC., ZhangX., GallegosM. T., RamosJ. L.(2005). The multidrug efflux regulator TtgV recognizes a wide range of structurally different effectors in solution and complexed with target DNA: evidence from isothermal titration calorimetry. J Biol Chem 280:20887–20893 [View Article] [PubMed]
GuazzaroniM. E., GallegosM. T., RamosJ. L., KrellT.(2007a). Different modes of binding of mono- and biaromatic effectors to the transcriptional regulator TTGV: role in differential derepression from its cognate operator. J Biol Chem 282:16308–16316 [View Article] [PubMed]
GuazzaroniM. E., KrellT., Gutiérrez del ArroyoP., VélezM., JiménezM., RivasG., RamosJ. L.(2007b). The transcriptional repressor TtgV recognizes a complex operator as a tetramer and induces convex DNA bending. J Mol Biol 369:927–939 [View Article] [PubMed]
JiangH., KendrickK. E.(2000). Characterization of ssfR and ssgA, two genes involved in sporulation of Streptomyces griseus. J Bacteriol 182:5521–5529 [View Article] [PubMed]
KrellT., Molina-HenaresA. J., RamosJ. L.(2006). The IclR family of transcriptional activators and repressors can be defined by a single profile. Protein Sci 15:1207–1213 [View Article] [PubMed]
LuD., FilletS., MengC., AlguelY., KloppsteckP., BergeronJ., KrellT., GallegosM. T., RamosJ., ZhangX.(2010). Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators. Genes Dev 24:2556–2565 [View Article] [PubMed]
Molina-HenaresA. J., KrellT., Eugenia GuazzaroniM., SeguraA., RamosJ. L.(2006). Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors. FEMS Microbiol Rev 30:157–186 [View Article] [PubMed]
Romero-SteinerS., ParalesR. E., HarwoodC. S., HoughtonJ. E.(1994). Characterization of the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate. J Bacteriol 176:5771–5779 [PubMed]
SunnarborgA., KlumppD., ChungT., LaPorteD. C.(1990). Regulation of the glyoxylate bypass operon: cloning and characterization of iclR. J Bacteriol 172:2642–2649 [PubMed]
TempéJ., PetitA., HolstersM., MontaguM., SchellJ.(1977). Thermosensitive step associated with transfer of the Ti plasmid during conjugation: possible relation to transformation in crown gall. Proc Natl Acad Sci U S A 74:2848–2849 [View Article] [PubMed]
TraagB. A., KelemenG. H., Van WezelG. P.(2004). Transcription of the sporulation gene ssgA is activated by the IclR-type regulator SsgR in a whi-independent manner in Streptomyces coelicolor A3(2). Mol Microbiol 53:985–1000 [View Article] [PubMed]
TsoiT. V., PlotnikovaE. G., ColeJ. R., GuerinW. F., BagdasarianM., TiedjeJ. M.(1999). Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates. Appl Environ Microbiol 65:2151–2162 [PubMed]
YamazakiH., OhnishiY., HorinouchiS.(2003). Transcriptional switch on of ssgA by A-factor, which is essential for spore septum formation in Streptomyces griseus. J Bacteriol 185:1273–1283 [View Article] [PubMed]
ZhangH. B., WangL. H., ZhangL. H.(2002). Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A 99:4638–4643 [View Article] [PubMed]