1887

Abstract

The LysR protein PcaQ regulates the expression of genes encoding products relevant to the degradation of the aromatic acid protocatechuate (3,4-dihydroxybenzoate), and we have previously defined a PcaQ DNA-binding site located upstream of the target operon in . In this work, we show that PcaQ also regulates the expression of the gene cluster, which is predicted to encode an ABC transport system. ABC transport systems have not been shown before to transport protocatechuate, and we have designated this gene cluster . The transcriptional start site of was mapped, and the predicted PcaQ DNA-binding site was located at −73 to −58 relative to this site. Results from electrophoretic mobility shift assays with purified PcaQ and from expression assays indicated that PcaQ activates expression of the transport system in the presence of protocatechuate. To investigate this transport system further, we generated a deletion mutant (predicted to encode the substrate-binding protein) and introduced a polar insertion mutation into , a gene that is predicted to encode a permease. These mutants grew poorly on protocatechuate, presumably because they fail to transport protocatechuate. Genome analyses revealed PcaQ-like DNA-binding sites encoded upstream of ABC transport systems in other members of the α-proteobacteria, and thus it appears likely that these systems are involved in the uptake of protocatechuate.

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2011-09-01
2019-12-06
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References

  1. Akakura R., Winans S. C.. ( 2002;). Mutations in the occQ operator that decrease OccR-induced DNA bending do not cause constitutive promoter activity. . J Biol Chem 277:, 15773–15780. [CrossRef][PubMed]
    [Google Scholar]
  2. Allaway D., Schofield N. A., Leonard M. E., Gilardoni L., Finan T. M., Poole P. S.. ( 2001;). Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes. . Environ Microbiol 3:, 397–406. [CrossRef][PubMed]
    [Google Scholar]
  3. Buchan A., Neidle E. L., Moran M. A.. ( 2004;). Diverse organization of genes of the β-ketoadipate pathway in members of the marine Roseobacter lineage. . Appl Environ Microbiol 70:, 1658–1668. [CrossRef][PubMed]
    [Google Scholar]
  4. Bundy B. M., Collier L. S., Hoover T. R., Neidle E. L.. ( 2002;). Synergistic transcriptional activation by one regulatory protein in response to two metabolites. . Proc Natl Acad Sci U S A 99:, 7693–7698. [CrossRef][PubMed]
    [Google Scholar]
  5. Chaudhry M. T., Huang Y., Shen X. H., Poetsch A., Jiang C. Y., Liu S. J.. ( 2007;). Genome-wide investigation of aromatic acid transporters in Corynebacterium glutamicum. . Microbiology 153:, 857–865. [CrossRef][PubMed]
    [Google Scholar]
  6. Chugani S. A., Parsek M. R., Hershberger C. D., Murakami K., Ishihama A., Chakrabarty A. M.. ( 1997;). Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription. . J Bacteriol 179:, 2221–2227.[PubMed]
    [Google Scholar]
  7. Collier L. S., Nichols N. N., Neidle E. L.. ( 1997;). benK encodes a hydrophobic permease-like protein involved in benzoate degradation by Acinetobacter sp. strain ADP1. . J Bacteriol 179:, 5943–5946.[PubMed]
    [Google Scholar]
  8. Cowie A., Cheng J., Sibley C. D., Fong Y., Zaheer R., Patten C. L., Morton R. M., Golding G. B., Finan T. M.. ( 2006;). An integrated approach to functional genomics: construction of a novel reporter gene fusion library for Sinorhizobium meliloti. . Appl Environ Microbiol 72:, 7156–7167. [CrossRef][PubMed]
    [Google Scholar]
  9. Crooks G. E., Hon G., Chandonia J. M., Brenner S. E.. ( 2004;). WebLogo: a sequence logo generator. . Genome Res 14:, 1188–1190. [CrossRef][PubMed]
    [Google Scholar]
  10. D’Argenio D. A., Segura A., Coco W. M., Bünz P. V., Ornston L. N.. ( 1999;). The physiological contribution of Acinetobacter PcaK, a transport system that acts upon protocatechuate, can be masked by the overlapping specificity of VanK. . J Bacteriol 181:, 3505–3515.[PubMed]
    [Google Scholar]
  11. Estrem S. T., Ross W., Gaal T., Chen Z. W., Niu W., Ebright R. H., Gourse R. L.. ( 1999;). Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase α subunit. . Genes Dev 13:, 2134–2147. [CrossRef][PubMed]
    [Google Scholar]
  12. Finan T. M., Kunkel B., De Vos G. F., Signer E. R.. ( 1986;). Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. . J Bacteriol 167:, 66–72.[PubMed]
    [Google Scholar]
  13. Fisher R. F., Egelhoff T. T., Mulligan J. T., Long S. R.. ( 1988;). Specific binding of proteins from Rhizobium meliloti cell-free extracts containing NodD to DNA sequences upstream of inducible nodulation genes. . Genes Dev 2:, 282–293. [CrossRef][PubMed]
    [Google Scholar]
  14. Fritsch P. S., Urbanowski M. L., Stauffer G. V.. ( 2000;). Role of the RNA polymerase α subunits in MetR-dependent activation of metE and metH: important residues in the C-terminal domain and orientation requirements within RNA polymerase. . J Bacteriol 182:, 5539–5550. [CrossRef][PubMed]
    [Google Scholar]
  15. Gerischer U., Segura A., Ornston L. N.. ( 1998;). PcaU, a transcriptional activator of genes for protocatechuate utilization in Acinetobacter. . J Bacteriol 180:, 1512–1524.[PubMed]
    [Google Scholar]
  16. Goethals K., Van Montagu M., Holsters M.. ( 1992;). Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. . Proc Natl Acad Sci U S A 89:, 1646–1650. [CrossRef][PubMed]
    [Google Scholar]
  17. Harwood C. S., Parales R. E.. ( 1996;). The β-ketoadipate pathway and the biology of self-identity. . Annu Rev Microbiol 50:, 553–590. [CrossRef][PubMed]
    [Google Scholar]
  18. Harwood C. S., Nichols N. N., Kim M. K., Ditty J. L., Parales R. E.. ( 1994;). Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate. . J Bacteriol 176:, 6479–6488.[PubMed]
    [Google Scholar]
  19. Huang J. Z., Schell M. A.. ( 1991;). In vivo interactions of the NahR transcriptional activator with its target sequences. Inducer-mediated changes resulting in transcription activation. . J Biol Chem 266:, 10830–10838.[PubMed]
    [Google Scholar]
  20. Jiménez J. I., Miñambres B., García J. L., Díaz E.. ( 2002;). Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. . Environ Microbiol 4:, 824–841. [CrossRef][PubMed]
    [Google Scholar]
  21. Ledger T., Aceituno F., González B.. ( 2009;). 3-Chlorobenzoate is taken up by a chromosomally encoded transport system in Cupriavidus necator JMP134. . Microbiology 155:, 2757–2765. [CrossRef][PubMed]
    [Google Scholar]
  22. Leveau J. H., Zehnder A. J., van der Meer J. R.. ( 1998;). The tfdK gene product facilitates uptake of 2,4-dichlorophenoxyacetate by Ralstonia eutropha JMP134(pJP4). . J Bacteriol 180:, 2237–2243.[PubMed]
    [Google Scholar]
  23. MacLean A. M.. ( 2008;). Study of saprophytic competence in Sinorhizobium meliloti. PhD thesis, McMaster University;, Hamilton, ON, Canada:.
    [Google Scholar]
  24. MacLean A. M., MacPherson G., Aneja P., Finan T. M.. ( 2006;). Characterization of the β-ketoadipate pathway in Sinorhizobium meliloti. . Appl Environ Microbiol 72:, 5403–5413. [CrossRef][PubMed]
    [Google Scholar]
  25. MacLean A. M., Anstey M. I., Finan T. M.. ( 2008;). Binding site determinants for the LysR-type transcriptional regulator PcaQ in the legume endosymbiont Sinorhizobium meliloti. . J Bacteriol 190:, 1237–1246. [CrossRef][PubMed]
    [Google Scholar]
  26. MacLellan S. R., Smallbone L. A., Sibley C. D., Finan T. M.. ( 2005;). The expression of a novel antisense gene mediates incompatibility within the large repABC family of α-proteobacterial plasmids. . Mol Microbiol 55:, 611–623. [CrossRef][PubMed]
    [Google Scholar]
  27. Maddocks S. E., Oyston P. C.. ( 2008;). Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. . Microbiology 154:, 3609–3623. [CrossRef][PubMed]
    [Google Scholar]
  28. Mauchline T. H., Fowler J. E., East A. K., Sartor A. L., Zaheer R., Hosie A. H., Poole P. S., Finan T. M.. ( 2006;). Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome. . Proc Natl Acad Sci U S A 103:, 17933–17938. [CrossRef][PubMed]
    [Google Scholar]
  29. McFall S. M., Chugani S. A., Chakrabarty A. M.. ( 1998;). Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme. . Gene 223:, 257–267. [CrossRef][PubMed]
    [Google Scholar]
  30. Nichols N. N., Harwood C. S.. ( 1997;). PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida. . J Bacteriol 179:, 5056–5061.[PubMed]
    [Google Scholar]
  31. Oke V., Long S. R.. ( 1999;). Bacterial genes induced within the nodule during the Rhizobium–legume symbiosis. . Mol Microbiol 32:, 837–849. [CrossRef][PubMed]
    [Google Scholar]
  32. Overhage J., Kresse A. U., Priefert H., Sommer H., Krammer G., Rabenhorst J., Steinbüchel A.. ( 1999;). Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199. . Appl Environ Microbiol 65:, 951–960.[PubMed]
    [Google Scholar]
  33. Park W., Jeon C. O., Madsen E. L.. ( 2002;). Interaction of NahR, a LysR-type transcriptional regulator, with the alpha subunit of RNA polymerase in the naphthalene degrading bacterium, Pseudomonas putida NCIB 9816-4. . FEMS Microbiol Lett 213:, 159–165.[PubMed]
    [Google Scholar]
  34. Parke D.. ( 1993;). Positive regulation of phenolic catabolism in Agrobacterium tumefaciens by the pcaQ gene in response to β-carboxy-cis,cis-muconate. . J Bacteriol 175:, 3529–3535.[PubMed]
    [Google Scholar]
  35. Parke D.. ( 1996;). Characterization of PcaQ, a LysR-type transcriptional activator required for catabolism of phenolic compounds, from Agrobacterium tumefaciens. . J Bacteriol 178:, 266–272.[PubMed]
    [Google Scholar]
  36. Parke D., D’Argenio D. A., Ornston L. N.. ( 2000;). Bacteria are not what they eat: that is why they are so diverse. . J Bacteriol 182:, 257–263. [CrossRef][PubMed]
    [Google Scholar]
  37. Parsek M. R., Shinabarger D. L., Rothmel R. K., Chakrabarty A. M.. ( 1992;). Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida. . J Bacteriol 174:, 7798–7806.[PubMed]
    [Google Scholar]
  38. Prentki P., Krisch H. M.. ( 1984;). In vitro insertional mutagenesis with a selectable DNA fragment. . Gene 29:, 303–313. [CrossRef][PubMed]
    [Google Scholar]
  39. Quandt J., Hynes M. F.. ( 1993;). Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. . Gene 127:, 15–21. [CrossRef][PubMed]
    [Google Scholar]
  40. Ross W., Gosink K. K., Salomon J., Igarashi K., Zou C., Ishihama A., Severinov K., Gourse R. L.. ( 1993;). A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. . Science 262:, 1407–1413. [CrossRef][PubMed]
    [Google Scholar]
  41. Rothmel R. K., Shinabarger D. L., Parsek M. R., Aldrich T. L., Chakrabarty A. M.. ( 1991;). Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting. . J Bacteriol 173:, 4717–4724.[PubMed]
    [Google Scholar]
  42. Schell M. A., Poser E. F.. ( 1989;). Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. . J Bacteriol 171:, 837–846.[PubMed]
    [Google Scholar]
  43. Stanier R. Y., Ingraham J. L.. ( 1954;). Protocatechuic acid oxidase. . J Biol Chem 210:, 799–808.[PubMed]
    [Google Scholar]
  44. Tao K., Fujita N., Ishihama A.. ( 1993;). Involvement of the RNA polymerase α subunit C-terminal region in co-operative interaction and transcriptional activation with OxyR protein. . Mol Microbiol 7:, 859–864. [CrossRef][PubMed]
    [Google Scholar]
  45. Wang L., Helmann J. D., Winans S. C.. ( 1992;). The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite. . Cell 69:, 659–667. [CrossRef][PubMed]
    [Google Scholar]
  46. Williams P. A., Shaw L. E.. ( 1997;). mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source. . J Bacteriol 179:, 5935–5942.[PubMed]
    [Google Scholar]
  47. Wong C. M., Dilworth M. J., Glenn A. R.. ( 1991;). Evidence for two uptake systems in Rhizobium leguminosarum for hydroxyl-aromatic compounds metabolized by the 3-oxoadipate pathway. . Arch Microbiol 156:, 385–391. [CrossRef]
    [Google Scholar]
  48. Yuan Z. C., Zaheer R., Finan T. M.. ( 2006;). Regulation and properties of PstSCAB, a high-affinity, high-velocity phosphate transport system of Sinorhizobium meliloti. . J Bacteriol 188:, 1089–1102. [CrossRef][PubMed]
    [Google Scholar]
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