Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245
Summary Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole‐3‐acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimula...
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| Published in | Environmental microbiology Vol. 7; no. 11; pp. 1839 - 1846 |
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| Main Authors | , , , |
| Format | Journal Article |
| Language | English |
| Published |
Oxford, UK
Blackwell Science Ltd
01.11.2005
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| Subjects | |
| Online Access | Get full text |
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| Abstract | Summary
Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole‐3‐acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole‐3‐pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC. |
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| AbstractList | Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC. Summary Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole‐3‐acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole‐3‐pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC. Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC.Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC. |
| Author | Rothballer, Michael Hartmann, Anton Fekete, Agnes Schmid, Michael |
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Vande Broek, A., Gysegom, P., Ona, O., Hendrickx, N., Prinsen, E., Van Impe, J., and Vanderleyden, J. (2005) Transcriptional analysis of the Azospirillum brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence involved in auxin responsive expression. Mol Plant Microbe Interact 18: 311-323. Amann, R.I., Zarda, B., Stahl, D.A., and Schleifer, K.H. (1992) Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 58: 3007-3011. Prinsen, E., Costacurta, A., Michiels, K., Vanderleyden, J., and Van Onckelen, H. (1993) Azospirillum brasilense indole-3-acetic acid biosynthesis: evidence for a non-tryptophan dependent pathway. Mol Plant Microbe Interact 6: 609-615. Kessler, B., De Lorenzo, V., and Timmis, K.N. (1992) A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233: 293-301. Schloter, M., and Hartmann, A. (1998) Endophytic and surface colonization of wheat roots (Triticum aestivum) by different Azospirillum brasilense strains studied with strain-specific monoclonal antibodies. Symbiosis 25: 159-179. Umalia-Garcia, M., Hubell, D.H., Gaskins, M., and Dazzo, F. (1980) Association of Azospirillum with grass roots. Appl Environ Microbiol 39: 219-226. Rothballer, M., Schmid, M., and Hartmann, A. (2003) In situ localization and PGPR-effect of Azospirillum brasilense strains colonizing roots of different wheat varieties. Symbiosis 34: 261-279. Stoffels, M., Castellanos, T., and Hartmann, A. (2001) Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster. Syst Appl Microbiol 24: 83-97. Tien, T.M., Gaskins, M.H., and Hubbell, D.H. (1979) Plant growth substances produced by Azospirillum brasilense and their effect on growth of pearl millet (Pennisetum americanum L.). Appl Environ Microbiol 37: 1016. Zimmer, W., Wesche, M., and Timmermans, L. (1998) Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus. Curr Microbiol 36: 327-331. Ramos, H.J., Roncato-Maccari, L.D., Souza, E.M., Soares-Ramos, J.R., Hungria, M., and Pedrosa, F.O. (2002) Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector. J Biotechnol 97: 243-252. Kristensen, C.S., Eberl, L., Sanches-Romero, J.M., Givskov, M., Molin, S., and De Lorenzo, V. (1995) Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4. J Bacteriol 177: 52-58. Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris, M.A., Roop, R.M., II, and Peterson, K.M. (1995) Four new derivatives of the broad-host-range vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176. Vanstockem, M., Michiels, K., Vanderleyden, J., and Van Gool, A. (1987) Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physical analysis of Tn5 and Tn5-Mob insertion mutants. Appl Environ Microbiol 53: 410-415. Patriquin, D.G., Döbereiner, J., and Jain, D.K. (1983) Sites and processes of the association between diazotrophs and grasses. Can J Microbiol 29: 900-915. Brandl, M.T., Quinones, B., and Lindow, S.E. (2001) Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces. Proc Natl Acad Sci USA 98: 3454-3459. Vande Broek, A., Lambrecht, M., Eggermont, K., and Vanderleyden, J. (1999) Auxins upregulate expression of the indole-3-pyruvate decarboxylase gene in Azospirillum brasilense. J Bacteriol 181: 1338-1342. Huffman, J.L., and Brennan, R.G. (2002) Prokaryotic transcription regulators: more than just the helix-turn-helix motif. Curr Opin Struct Biol 12: 98-106. Okon, Y., and Kapulnik, Y. (1986) Development and function of Azospirillum-inoculated roots. Plant and Soil 90: 3-16. Baldani, V.L.D., Baldani, J.I., and Döbereiner, J. (1987) Inoculation of field grown wheat with Azospirillum brasilense spp. Biol Fertil Soils 4: 37-40. Manz, W., Amann, R., Ludwig, W., Wagner, M., and Schleifer, K.-H. (1992) Phylogenetic oligodeoxynucleotide probes for the major subclass of Proteobacteria: problems and solutions. Syst Appl Microbiol 15: 593-600. Fallik, E., Okon, Y., and Fischer, M. (1988) Growth response of maize roots to Azospirillum interaction: effect of soil organic matter content, number of rhizosphere bacteria and timing of inoculation. Soil Biol Biochem 20: 45. O'Hara, G.W., Davey, M.R., and Lucas, J.A. (1983) Associations between the nitrogen-fixing bacterium Azospirillum barsilense and excised plant roots. Z Pflanzenphysiologie 113: 1-13. Amann, R.I., Krumholz, L., and Stahl, D.A. (1990) Fluorescent-oligonucleotide probing of whole cells for determinative and environmental studies in microbiology. J Bacteriol 172: 762-770. Hartmann, A., Singh, M., and Klingmüller, W. (1983a) Isolation and characterization of Azospirillum mutants excreting high amounts of indoleacetic acid. Can J Microbiol 29: 916-923. Dobbelaere, S., Croonenborghs, A., Amber, T., Ptacek, D., Vanderleyden, J., Dutto, P., et al. (2001) Responses of agronomically important crops to inoculation with Azospirillum. Aust J Plant Physiol 28: 1-9. Daims, H., Bruhl, A., Amann, R., Schleifer, K.H., and Wagner, M. (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22: 434-444. Egener, T., Hurek, T., and Reinhold-Hurek, B. (1998) Use of green fluorescent protein to detect expression of nif genes of Azoarcus sp. BH72, a grass-associated diazotroph, on rice roots. Mol Plant Microbe Interact 11: 71-75. Bar, T., and Okon, Y. (1992) Induction of indole-3-acetic acid synthesis and possible toxicity of tryptophan in Azospirillum brasilense Sp7. Symbiosis 13: 191-198. Tarrand, J.J., Krieg, N.R., and Döbereiner, J. (1978) A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. 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| References_xml | – reference: Daims, H., Bruhl, A., Amann, R., Schleifer, K.H., and Wagner, M. (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22: 434-444. – reference: Schloter, M., and Hartmann, A. (1998) Endophytic and surface colonization of wheat roots (Triticum aestivum) by different Azospirillum brasilense strains studied with strain-specific monoclonal antibodies. Symbiosis 25: 159-179. – reference: Baldani, V.L.D., Baldani, J.I., and Döbereiner, J. (1987) Inoculation of field grown wheat with Azospirillum brasilense spp. Biol Fertil Soils 4: 37-40. – reference: Vande Broek, A., Gysegom, P., Ona, O., Hendrickx, N., Prinsen, E., Van Impe, J., and Vanderleyden, J. (2005) Transcriptional analysis of the Azospirillum brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence involved in auxin responsive expression. Mol Plant Microbe Interact 18: 311-323. – reference: Vanstockem, M., Michiels, K., Vanderleyden, J., and Van Gool, A. (1987) Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physical analysis of Tn5 and Tn5-Mob insertion mutants. Appl Environ Microbiol 53: 410-415. – reference: Amann, R.I., Zarda, B., Stahl, D.A., and Schleifer, K.H. (1992) Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 58: 3007-3011. – reference: Pereg Gerk, L.P., Gilchrist, K., and Kennedy, I.R. (2000) Mutants with enhanced nitrogenase activity in hydroponic Azospirillum brasilense-wheat associations. Appl Environ Microbiol 66: 2175-2184. – reference: Stoffels, M., Castellanos, T., and Hartmann, A. (2001) Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster. Syst Appl Microbiol 24: 83-97. – reference: Umalia-Garcia, M., Hubell, D.H., Gaskins, M., and Dazzo, F. (1980) Association of Azospirillum with grass roots. Appl Environ Microbiol 39: 219-226. – reference: Egener, T., Hurek, T., and Reinhold-Hurek, B. (1998) Use of green fluorescent protein to detect expression of nif genes of Azoarcus sp. BH72, a grass-associated diazotroph, on rice roots. Mol Plant Microbe Interact 11: 71-75. – reference: Kessler, B., De Lorenzo, V., and Timmis, K.N. (1992) A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233: 293-301. – reference: Patriquin, D.G., Döbereiner, J., and Jain, D.K. (1983) Sites and processes of the association between diazotrophs and grasses. Can J Microbiol 29: 900-915. – reference: Fallik, E., Okon, Y., and Fischer, M. (1988) Growth response of maize roots to Azospirillum interaction: effect of soil organic matter content, number of rhizosphere bacteria and timing of inoculation. Soil Biol Biochem 20: 45. – reference: Bar, T., and Okon, Y. (1992) Induction of indole-3-acetic acid synthesis and possible toxicity of tryptophan in Azospirillum brasilense Sp7. Symbiosis 13: 191-198. – reference: Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris, M.A., Roop, R.M., II, and Peterson, K.M. (1995) Four new derivatives of the broad-host-range vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176. – reference: Kristensen, C.S., Eberl, L., Sanches-Romero, J.M., Givskov, M., Molin, S., and De Lorenzo, V. (1995) Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4. J Bacteriol 177: 52-58. – reference: Rothballer, M., Schmid, M., and Hartmann, A. (2003) In situ localization and PGPR-effect of Azospirillum brasilense strains colonizing roots of different wheat varieties. Symbiosis 34: 261-279. – reference: Prinsen, E., Costacurta, A., Michiels, K., Vanderleyden, J., and Van Onckelen, H. (1993) Azospirillum brasilense indole-3-acetic acid biosynthesis: evidence for a non-tryptophan dependent pathway. Mol Plant Microbe Interact 6: 609-615. – reference: Manz, W., Amann, R., Ludwig, W., Wagner, M., and Schleifer, K.-H. (1992) Phylogenetic oligodeoxynucleotide probes for the major subclass of Proteobacteria: problems and solutions. Syst Appl Microbiol 15: 593-600. – reference: Murthy, M.G., and Ladha, J.K. (1987) Differential colonization of Azospirillum lipoferum on roots of two varieties of rice. Biol Fertil Soils 4: 3-7. – reference: Okon, Y., and Kapulnik, Y. (1986) Development and function of Azospirillum-inoculated roots. Plant and Soil 90: 3-16. – reference: Burdman, S., Okon, Y., and Jurkevitch, E. 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Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the... Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial... |
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| SubjectTerms | Azospirillum brasilense Azospirillum brasilense - genetics Base Sequence Gene Expression Regulation, Bacterial Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Molecular Sequence Data Plasmids - genetics Promoter Regions, Genetic - genetics Pyruvate Decarboxylase - genetics Pyruvate Decarboxylase - metabolism Sequence Alignment Sequence Analysis, DNA Species Specificity Triticum - microbiology Triticum aestivum |
| Title | Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245 |
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