1887

Abstract

strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially ) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that was expressed at a relatively high level. Individual knockout mutants of all three genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2Δ showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three genes from strain CJ2 were cloned into : the and genes were successfully overexpressed. The subunit mass of the GDOs were ~36–39 kDa, and their structures were deduced to be dimeric. The values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in sp. JS666 and SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the allele of strain CJ2 did not share high sequence identity with its homologues in sp. JS666 and SP-6.

Funding
This study was supported by the:
  • , MOST/KOSEF
  • , 21C Frontier Microbial Genomics and Application Center Program , (Award MG05-0104-4-0)
  • , Technology Development Program for Agriculture and Forestry (TDPAF) of the Ministry for Agriculture, Forestry and Fisheries
  • , National Science Foundation , (Award DEB-0841999)
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2011-10-01
2020-11-24
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References

  1. Adams M. A., Singh V. K., Keller B. O., Jia Z. ( 2006). Structural and biochemical characterization of gentisate 1,2-dioxygenase from Escherichia coli O157:H7. Mol Microbiol 61:1469–1484 [CrossRef][PubMed]
    [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. ( 1990). Basic local alignment search tool. J Mol Biol 215:403–410[PubMed] [CrossRef]
    [Google Scholar]
  3. Andersson D. I., Hughes D. ( 2009). Gene amplification and adaptive evolution in bacteria. Annu Rev Genet 43:167–195 [CrossRef][PubMed]
    [Google Scholar]
  4. Bayly R. C., Barbour M. G. ( 1984). The degradation of aromatic compounds by the meta and gentisate pathway: Biochemistry and regulation. Microbial Degradation of Aromatic Compounds253–294 Gibson D. T. New York: Marcel Dekker;
    [Google Scholar]
  5. Bradford M. M. ( 1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [CrossRef][PubMed]
    [Google Scholar]
  6. Chain P. S. G., Denef V. J., Konstantinidis K. T., Vergez L. M., Agulló L., Reyes V. L., Hauser L., Córdova M., Gómez L. et al. & other authors ( 2006). Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility. Proc Natl Acad Sci U S A 103:15280–15287 [CrossRef][PubMed]
    [Google Scholar]
  7. Chapman P. J. ( 1972). An outline of reaction sequences used for the bacterial degradation of phenolic compounds. Degradation of Synthetic Organic Molecules in the Biosphere17–55 Chapman P. J., Dagley S. Washington, DC: National Academy of Sciences;
    [Google Scholar]
  8. Chen J., Li W., Wang M., Zhu G., Liu D., Sun F., Hao N., Li X., Rao Z., Zhang X. C. ( 2008). Crystal structure and mutagenic analysis of GDOsp, a gentisate 1,2-dioxygenase from Silicibacter pomeroyi . Protein Sci 17:1362–1373 [CrossRef][PubMed]
    [Google Scholar]
  9. Crawford R. L., Hutton S. W., Chapman P. J. ( 1975). Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis . J Bacteriol 121:794–799[PubMed]
    [Google Scholar]
  10. Dereeper A., Guignon V., Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J.-F., Guindon S., Lefort V. et al. & other authors ( 2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36:Web ServerW465–W469 [CrossRef][PubMed]
    [Google Scholar]
  11. Feng Y., Khoo H. E., Poh C. L. ( 1999). Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869. Appl Environ Microbiol 65:946–950[PubMed]
    [Google Scholar]
  12. Fuenmayor S. L., Wild M., Boyes A. L., Williams P. A. ( 1998). A gene cluster encoding steps in conversion of naphthalene to gentisate in Pseudomonas sp. strain U2. J Bacteriol 180:2522–2530[PubMed]
    [Google Scholar]
  13. Gevers D., Vandepoele K., Simillion C., Van de Peer Y. ( 2004). Gene duplication and biased functional retention of paralogs in bacterial genomes. Trends Microbiol 12:148–154 [CrossRef][PubMed]
    [Google Scholar]
  14. Ghosal D., You I.-S. ( 1988). Gene duplication in haloaromatic degradative plasmids pJP4 and pJP2. Can J Microbiol 34:709–715 [CrossRef][PubMed]
    [Google Scholar]
  15. Grund E., Denecke B., Eichenlaub R. ( 1992). Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4. Appl Environ Microbiol 58:1874–1877[PubMed]
    [Google Scholar]
  16. Hirano S., Morikawa M., Takano K., Imanaka T., Kanaya S. ( 2007). Gentisate 1,2-dioxygenase from Xanthobacter polyaromaticivorans 127W. Biosci Biotechnol Biochem 71:192–199 [CrossRef][PubMed]
    [Google Scholar]
  17. Hohnstock A. M., Stuart-Keil K. G., Kull E. E., Madsen E. L. ( 2000). Naphthalene and donor cell density influence field conjugation of naphthalene catabolism plasmids. Appl Environ Microbiol 66:3088–3092 [CrossRef][PubMed]
    [Google Scholar]
  18. Irgens R. L., Gosink J. J., Staley J. T. ( 1996). Polaromonas vacuolata gen. nov., sp. nov., a psychrophilic, marine, gas vacuolate bacterium from Antarctica. Int J Syst Bacteriol 46:822–826 [CrossRef][PubMed]
    [Google Scholar]
  19. Jeon C. O., Park W., Padmanabhan P., DeRito C., Snape J. R., Madsen E. L. ( 2003). Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proc Natl Acad Sci U S A 100:13591–13596 [CrossRef][PubMed]
    [Google Scholar]
  20. Jeon C. O., Park W., Ghiorse W. C., Madsen E. L. ( 2004). Polaromonas naphthalenivorans sp. nov., a naphthalene-degrading bacterium from naphthalene-contaminated sediment. Int J Syst Evol Microbiol 54:93–97 [CrossRef][PubMed]
    [Google Scholar]
  21. Jeon C. O., Park M., Ro H.-S., Park W., Madsen E. L. ( 2006). The naphthalene catabolic (nag) genes of Polaromonas naphthalenivorans CJ2: evolutionary implications for two gene clusters and novel regulatory control. Appl Environ Microbiol 72:1086–1095 [CrossRef][PubMed]
    [Google Scholar]
  22. Johnson G. R., Olsen R. H. ( 1997). Multiple pathways for toluene degradation in Burkholderia sp. strain JS150. Appl Environ Microbiol 63:4047–4052[PubMed]
    [Google Scholar]
  23. Jones R. M., Britt-Compton B., Williams P. A. ( 2003). The naphthalene catabolic (nag) genes of Ralstonia sp. strain U2 are an operon that is regulated by NagR, a LysR-type transcriptional regulator. J Bacteriol 185:5847–5853 [CrossRef][PubMed]
    [Google Scholar]
  24. Kalogeraki V. S., Winans S. C. ( 1997). Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 188:69–75 [CrossRef][PubMed]
    [Google Scholar]
  25. Kamaya Y., Fukaya Y., Suzuki K. ( 2005). Acute toxicity of benzoic acids to the crustacean Daphnia magna . Chemosphere 59:255–261 [CrossRef][PubMed]
    [Google Scholar]
  26. Kivisaar M. ( 2009). Degradation of nitroaromatic compounds: a model to study evolution of metabolic pathways. Mol Microbiol 74:777–781 [CrossRef][PubMed]
    [Google Scholar]
  27. Kulakov L. A., Chen S., Allen C. C. R., Larkin M. J. ( 2005). Web-type evolution of rhodococcus gene clusters associated with utilization of naphthalene. Appl Environ Microbiol 71:1754–1764 [CrossRef][PubMed]
    [Google Scholar]
  28. Lack L. ( 1959). The enzymic oxidation of gentisic acid. Biochim Biophys Acta 34:117–123 [CrossRef][PubMed]
    [Google Scholar]
  29. Lee S. H., Kim J. M., Lee H. J., Jeon C. O. ( 2011). Screening of promoters from rhizosphere metagenomic DNA using a promoter-trap vector and flow cytometric cell sorting. J Basic Microbiol 51:52–60 [CrossRef][PubMed]
    [Google Scholar]
  30. Liu Y., Yao T., Jiao N., Kang S., Zeng Y., Huang S. ( 2006). Microbial community structure in moraine lakes and glacial meltwaters, Mount Everest. FEMS Microbiol Lett 265:98–105 [CrossRef][PubMed]
    [Google Scholar]
  31. Loy A., Beisker W., Meier H. ( 2005). Diversity of bacteria growing in natural mineral water after bottling. Appl Environ Microbiol 71:3624–3632 [CrossRef][PubMed]
    [Google Scholar]
  32. Maeda M., Chung S.-Y., Song E., Kudo T. ( 1995). Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the Gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem. Appl Environ Microbiol 61:549–555[PubMed]
    [Google Scholar]
  33. Mattes T. E., Alexander A. K., Richardson P. M., Munk A. C., Han C. S., Stothard P., Coleman N. V. ( 2008). The genome of Polaromonas sp. strain JS666: insights into the evolution of a hydrocarbon- and xenobiotic-degrading bacterium, and features of relevance to biotechnology. Appl Environ Microbiol 74:6405–6416 [CrossRef][PubMed]
    [Google Scholar]
  34. McBeth D. L., Shapiro J. A. ( 1984). Reversal by DNA amplifications of an unusual mutation blocking alkane and alcohol utilization in Pseudomonas putida . Mol Gen Genet 197:384–391 [CrossRef][PubMed]
    [Google Scholar]
  35. Ogawa N., Miyashita K. ( 1995). Recombination of a 3-chlorobenzoate catabolic plasmid from Alcaligenes eutrophus NH9 mediated by direct repeat elements. Appl Environ Microbiol 61:3788–3795[PubMed]
    [Google Scholar]
  36. Ohmoto T., Sakai K., Hamada N., Ohe T. ( 1991). Salicylic acid metabolism through a gentisate pathway by Pseudomonas sp. TA-2. Agric Biol Chem 55:1733–1737 [CrossRef]
    [Google Scholar]
  37. Park W., Jeon C. O., Cadillo H., DeRito C., Madsen E. L. ( 2004). Survival of naphthalene-degrading Pseudomonas putida NCIB 9816-4 in naphthalene-amended soils: toxicity of naphthalene and its metabolites. Appl Microbiol Biotechnol 64:429–435 [CrossRef][PubMed]
    [Google Scholar]
  38. Park M., Jeon Y., Jang H. H., Ro H.-S., Park W., Madsen E. L., Jeon C. O. ( 2007a). Molecular and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase from Polaromonas naphthalenivorans CJ2. Appl Environ Microbiol 73:5146–5152 [CrossRef][PubMed]
    [Google Scholar]
  39. Park M., Jeon Y., Madsen E. L., Jeon C. O. ( 2007b). Protection of Polaromonas naphthalenivorans CJ2 from naphthalene toxicity by extracellular polysaccharide capsules. J Appl Biol Chem 50:41–45
    [Google Scholar]
  40. Penning T. M., Burczynski M. E., Hung C. F., McCoull K. D., Palackal N. T., Tsuruda L. S. ( 1999). Dihydrodiol dehydrogenases and polycyclic aromatic hydrocarbon activation: generation of reactive and redox active o-quinones. Chem Res Toxicol 12:1–18 [CrossRef][PubMed]
    [Google Scholar]
  41. Pumphrey G. M., Madsen E. L. ( 2007). Naphthalene metabolism and growth inhibition by naphthalene in Polaromonas naphthalenivorans strain CJ2. Microbiology 153:3730–3738 [CrossRef][PubMed]
    [Google Scholar]
  42. Ramos J. L., Duque E., Gallegos M.-T., Godoy P., Ramos-Gonzalez M. I., Rojas A., Terán W., Segura A. ( 2002). Mechanisms of solvent tolerance in Gram-negative bacteria. Annu Rev Microbiol 56:743–768 [CrossRef][PubMed]
    [Google Scholar]
  43. Rangnekar V. M. ( 1988). Variation in the ability of Pseudomonas sp. strain B13 cultures to utilize meta-chlorobenzoate is associated with tandem amplification and deamplification of DNA. J Bacteriol 170:1907–1912[PubMed]
    [Google Scholar]
  44. Reams A. B., Neidle E. L. ( 2003). Genome plasticity in Acinetobacter: new degradative capabilities acquired by the spontaneous amplification of large chromosomal segments. Mol Microbiol 47:1291–1304 [CrossRef][PubMed]
    [Google Scholar]
  45. Reams A. B., Neidle E. L. ( 2004). Gene amplification involves site-specific short homology-independent illegitimate recombination in Acinetobacter sp. strain ADP1. J Mol Biol 338:643–656 [CrossRef][PubMed]
    [Google Scholar]
  46. Reber H. ( 1973). Comparative studies with two pseudomonads on the sequential degradation of aromatic substances metabolized via different pathways. Arch Mikrobiol 89:305–315 [CrossRef][PubMed]
    [Google Scholar]
  47. Ritalahti K. M., Amos B. K., Sung Y., Wu Q., Koenigsberg S. S., Löffler F. E. ( 2006). Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Appl Environ Microbiol 72:2765–2774 [CrossRef][PubMed]
    [Google Scholar]
  48. Sambrook J., Janssen K. J. ( 2001). Molecular cloning: a laboratory manual, 3rd edn. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  49. Schäfer A., Tauch A., Jäger W., Kalinowski J., Thierbach G., Pühler A. ( 1994). Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum . Gene 145:69–73 [CrossRef][PubMed]
    [Google Scholar]
  50. Schell M. A., Wender P. E. ( 1986). Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. J Bacteriol 166:9–14[PubMed]
    [Google Scholar]
  51. Schweigert N., Zehnder A. J., Eggen R. I. ( 2001). Chemical properties of catechols and their molecular modes of toxic action in cells, from microorganisms to mammals. Environ Microbiol 3:81–91 [CrossRef][PubMed]
    [Google Scholar]
  52. Sikkema J., de Bont J. A., Poolman B. ( 1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59:201–222[PubMed]
    [Google Scholar]
  53. Stanier R. Y., Palleroni N. J., Doudoroff M. ( 1966). The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43:159–271[PubMed] [CrossRef]
    [Google Scholar]
  54. Teeling H., Meyerdierks A., Bauer M., Amann R., Glöckner F. O. ( 2004). Application of tetranucleotide frequencies for the assignment of genomic fragments. Environ Microbiol 6:938–947 [CrossRef][PubMed]
    [Google Scholar]
  55. Vaillancourt F. H., Labbe G. M., Drouin N. M., Fortin P. D., Eltis L. D. ( 2002). The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates. J Biol Chem 277:2019–2027 [CrossRef][PubMed]
    [Google Scholar]
  56. Vaillancourt F. H., Bolin J. T., Eltis L. D. ( 2006). The ins and outs of ring-cleaving dioxygenases. Crit Rev Biochem Mol Biol 41:241–267 [CrossRef][PubMed]
    [Google Scholar]
  57. Yagi J. M., Sims D., Brettin T., Bruce D., Madsen E. L. ( 2009). The genome of Polaromonas naphthalenivorans strain CJ2, isolated from coal tar-contaminated sediment, reveals physiological and metabolic versatility and evolution through extensive horizontal gene transfer. Environ Microbiol 11:2253–2270 [CrossRef][PubMed]
    [Google Scholar]
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