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Journal of Bacteriology, April 2002, p. 1832-1842, Vol. 184, No. 7
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.7.1832-1842.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

tRNA Is the Source of Low-Level trans-Zeatin Production in Methylobacterium spp.{dagger},{ddagger}

Robbin L. Koenig,§, Roy O. Morris, and Joe C. Polacco*

Department of Biochemistry, University of Missouri—Columbia, Columbia, Missouri 65211

Received 26 September 2001/ Accepted 19 December 2001

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.

* Corresponding author. Mailing address: Department of Biochemistry, 117 Schweitzer Hall, University of Missouri, Columbia, MO 65211. Phone: (573) 882-4789. Fax: (573) 882-5635. E-mail: polaccoj{at}missouri.edu.

{dagger} Contribution 13,196 of the Missouri Agricultural Experiment Station Journal Series.

{ddagger} For a commentary on this article, see page 1818 in this issue.

§ Present address: Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70458.

Journal of Bacteriology, April 2002, p. 1832-1842, Vol. 184, No. 7
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.7.1832-1842.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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