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. 1995 Jun;61(6):2257–2261. doi: 10.1128/aem.61.6.2257-2261.1995

Identification of grass-associated and toluene-degrading diazotrophs, Azoarcus spp., by analyses of partial 16S ribosomal DNA sequences.

T Hurek 1, B Reinhold-Hurek 1
PMCID: PMC167497  PMID: 7793946

Abstract

The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thauera selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N2O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Amann R. I., Krumholz L., Stahl D. A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990 Feb;172(2):762–770. doi: 10.1128/jb.172.2.762-770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  3. Dazzo F. B., Hubbell D. H. Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association. Appl Microbiol. 1975 Dec;30(6):1017–1033. doi: 10.1128/am.30.6.1017-1033.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DeLong E. F., Wickham G. S., Pace N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989 Mar 10;243(4896):1360–1363. doi: 10.1126/science.2466341. [DOI] [PubMed] [Google Scholar]
  5. Ferguson R. L., Buckley E. N., Palumbo A. V. Response of marine bacterioplankton to differential filtration and confinement. Appl Environ Microbiol. 1984 Jan;47(1):49–55. doi: 10.1128/aem.47.1.49-55.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fries M. R., Zhou J., Chee-Sanford J., Tiedje J. M. Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats. Appl Environ Microbiol. 1994 Aug;60(8):2802–2810. doi: 10.1128/aem.60.8.2802-2810.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hurek T., Burggraf S., Woese C. R., Reinhold-Hurek B. 16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs. Appl Environ Microbiol. 1993 Nov;59(11):3816–3824. doi: 10.1128/aem.59.11.3816-3824.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hurek T., Reinhold-Hurek B., Turner G. L., Bergersen F. J. Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp. strain BH72. J Bacteriol. 1994 Aug;176(15):4726–4733. doi: 10.1128/jb.176.15.4726-4733.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hurek T., Reinhold-Hurek B., Van Montagu M., Kellenberger E. Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J Bacteriol. 1994 Apr;176(7):1913–1923. doi: 10.1128/jb.176.7.1913-1923.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones J. G. Studies on freshwater bacteria: effect of medium composition and method on estimates of bacterial population. J Appl Bacteriol. 1970 Dec;33(4):679–686. doi: 10.1111/j.1365-2672.1970.tb02250.x. [DOI] [PubMed] [Google Scholar]
  11. Laguerre G., Bossand B., Bardin R. Free-living dinitrogen-fixing bacteria isolated from petroleum refinery oily sludge. Appl Environ Microbiol. 1987 Jul;53(7):1674–1678. doi: 10.1128/aem.53.7.1674-1678.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Larimer F. W., Perry J. R., Hardigree A. A. The REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis. J Bacteriol. 1989 Jan;171(1):230–237. doi: 10.1128/jb.171.1.230-237.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Macy J. M., Rech S., Auling G., Dorsch M., Stackebrandt E., Sly L. I. Thauera selenatis gen. nov., sp. nov., a member of the beta subclass of Proteobacteria with a novel type of anaerobic respiration. Int J Syst Bacteriol. 1993 Jan;43(1):135–142. doi: 10.1099/00207713-43-1-135. [DOI] [PubMed] [Google Scholar]
  14. Reinhold-Hurek B., Hurek T., Claeyssens M., van Montagu M. Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph. J Bacteriol. 1993 Nov;175(21):7056–7065. doi: 10.1128/jb.175.21.7056-7065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Reinhold-Hurek B., Shub D. A. Self-splicing introns in tRNA genes of widely divergent bacteria. Nature. 1992 May 14;357(6374):173–176. doi: 10.1038/357173a0. [DOI] [PubMed] [Google Scholar]
  16. Reinhold B., Hurek T., Niemann E. G., Fendrik I. Close association of azospirillum and diazotrophic rods with different root zones of kallar grass. Appl Environ Microbiol. 1986 Sep;52(3):520–526. doi: 10.1128/aem.52.3.520-526.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  18. Trebesius K., Amann R., Ludwig W., Mühlegger K., Schleifer K. H. Identification of Whole Fixed Bacterial Cells with Nonradioactive 23S rRNA-Targeted Polynucleotide Probes. Appl Environ Microbiol. 1994 Sep;60(9):3228–3235. doi: 10.1128/aem.60.9.3228-3235.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  20. Winship P. R. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 1989 Feb 11;17(3):1266–1266. doi: 10.1093/nar/17.3.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Young J. P., Downer H. L., Eardly B. D. Phylogeny of the phototrophic rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J Bacteriol. 1991 Apr;173(7):2271–2277. doi: 10.1128/jb.173.7.2271-2277.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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