Source: UNIVERSITY OF ARIZONA submitted to
COLONIZATION AND NITROGEN FIXATION WITHIN SUGARCANE AND SORGHUM BY GLUCONACETOBACTER DIAZOTROPHICUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0175359
Grant No.
(N/A)
Project No.
ARZT-136768-H-25-180
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Jun 30, 2007
Grant Year
(N/A)
Project Director
Kennedy, C.
Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
PLANT SCIENCE
Non Technical Summary
Cereals and grasses require substantial amounts of nitrogen-containing fertilizers for optimum growth and crop production. Inoculation experiments have shown that a nitrogen-fixing bacterium living inside sugarcane plants, Acetobacter diazotrophicus, can significantly enhance the growth of its host plant compared to uninoculated controls. This is likely to be due to benefits of bacterial nitrogen fixation and also production of the plant growth hormone, auxin. The use of A. diazotrophicus in association with other monocot plants, including cereals, may be beneficial in agriculture.
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1022030108010%
1022030110010%
1022099108010%
1022099110010%
2032030104010%
2032030108010%
2032030110010%
2032099104010%
2032099108010%
2032099110010%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2099 - Sugar crops, general/other; 2030 - Sweet sorghum;

Field Of Science
1080 - Genetics; 1100 - Bacteriology; 1040 - Molecular biology;
Goals / Objectives
Both objectives concern the ability of the bacterium Gluconacetobacter diazotrophicus to colonize and stimulate growth of sugarcane and sweet sorghum. 1. To establish definitively whether fixed nitrogen is transferred from bacteria to plant tissue. 2. To determine whether bacterial production of indole acetic acid is a factor that is beneficial to growth of plants.
Project Methods
1. The methodology for this has been developed by Dr. Kahn, Washington State University, who will work with us to set up the experiments and analyze the results by Mass Spectroscopy in his lab. We propose to set up plants in a large gas-tight containment glove-box, fitted with CO2 monitoring and supply capability, inside a growth chamber custom designed for our needs. Trial runs will precede those to which 15N2 is added to determine the length of time that plants can be grown under these conditions and remain reasonably healthy. Microprogatated sugarcane SP70-1143 will probably be used in these experiments, as this cultivar was successfully used for previous experiments (Sevilla et al, 2001) or possibly sweet sorghum. The chlorophyll in leaves will be extracted with methanol, then converted to pheophytin by adding HCl. Pheophytin is chlorophyll lacking its central magnesium atom but retaining all four N atoms. The technique used for measuring mass is positive ion atmospheric pressure ionization, electrospray mass spectrometry. In experiments using Chlamydomonas as a test organism, Kahn et al. (2002) showed that over the range of 0.4%-98% supplied 15N, the average pheophytin mass shifted 3.9 units, indicating that 15N is incorporated into chlorophyll as efficiently as 14N. Discrete peaks corresponding to the amount of pheophytin at the masses corresponding to molecules with 0,1,2,3, or 4 15N atoms (4,3,2,1 or 0 14N), ranged between 872-876 mass units. Objective 2. Genes specifically involved in IAA production should be identified by the Brazilian consortium currently sequencing the G. diazotrophicus genome, expected to be completed later in early 2003. It is likely that an ipdC gene encoding indole pyruvate decarboxylase will be found. Other potential biosynthetic routes characterized in bacteria include the indole-3-acetamide pathway with enzymes encoded by iaaH and iaaM. The conservation of sequence of IpdC, IaaH and IaaM in other plant-associating bacteria should ensure their being identified from the genome project or by new primer design. Candidate IAA biosynthetic genes will be PCR amplified, cloned and mutagenized. Introducing the mutated alleles into the G. diazotrophicus via suicide vectors is straightforward, using conjugation or electroporation techniques that were previously successful. Mutants will be tested for ability to produce IAA. Those that are significantly deficient in IAA production, screened by the Salkowski reagent test and further by HPLC analysis, will be tested for ability to enhance growth of sorghum and/or sugarcane, as described above. The IAA pathway mutations will be conjoined with the nifD::kan mutation to construct double mutants that are both IAA - and Nif -. The single and double mutation-carrying strains will be tested, as described previously, for ability to benefit the growth of sorghum and/or sugarcane, in order to determine the contribution of both IAA production and nitrogen fixation to plant growth enhancement.

Progress 10/01/03 to 06/30/07

Outputs
OUTPUTS: Sorry, PI passed away. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Sorry, PI passed away.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
The following question was addressed during this reporting period: Is IAA production an important factor in the ability of G. diazotrophicus to stimulate plant growth? G. diazotrophicus is beneficial to sugarcane growth possibly by two mechanisms, one dependent, and one not, on nitrogen fixation by the bacterial partner. To test the hypothesis that IAA production is a factor leading to sugarcane growth enhancement, random Tn5 mutants of G. diazotrophicus strain PAl5 led to the isolation of strain MAd10, which produced very little IAA. The mutation that led to decreased IAA production was not associated with the insertion site of Tn5 in MAd10. To determine the site of the mutation leading to decreased levels of IAA in MAd10, a pLAFR3 library carrying G. diazotrophicus DNA inserts was transferred by conjugation into MAd10, followed by screening of transconjugants for IAA production. In two IAA+ transconjugants, the cosmids isolated shared identical regions in the insert fragments; analysis by subcloning, complementation, and DNA sequencing, indicated that the mutation in MAd10 was located in the ccmC gene, involved in cytochrome c maturation. The ccm operon was sequenced and found to encode Ccm proteins of ~50% identity to those of the ccm operon in Bradyrhizobium japonicum. Insertion of kan cassettes into the G. diazotrophisuc ccm genes cloned on suicide vectors, followed by their reintroduction into the G. diazotrophicus genome, led to the construction of ccm mutants. The mutants were IAA-, producing ~5% of wild-type levels of IAA. Mobilization of the entire ccm operon on a plasmid into these mutants complemented the IAA- phenotype. Therefore cytochrome c is likely to be an essential component of an IAA biosynthetic enzyme with redox functions in G. diazotrophicus. Spectral analysis of cytochrome c, heme-associated peroxidase activities, and membrane-associated respiratory activities in the wild-type and mutant strains showed that the Ccm proteins of G. diazotrophicus are involved in cytochrome c maturation. Growth rates on several media and ability to fix nitrogen were not influenced in the ccm mutant strains; the only phenotype observed was a decrease in IAA production. The effect of a ccmC mutation on plant growth enhancement was examined. Regardless of nitrogen supply, plants inoculated with wild-type PAl5 were larger than uninoculated plants. Plants inoculated with a ccmC mutant, a nifD mutant, or a ccmC nifD double mutant were no larger than uninoculated plants in conditions of both N-deficiency and N-sufficiency. This is in contrast to the results of Sevilla et al (2001) who found that the nifD mutant could enhance sugarcane growth under +N conditions; the reason for this discrepancy is not known, but could be related to the shorter growth period before plants were harvested. Similar experiments will be repeated, possibly with sweet sorghum. These results indicate that both nitrogen fixation and IAA production are factors that allow G. diazotrophicus to benefit plant growth, and that a threshold of input by either factor must be attained before growth enhancement is achieved.

Impacts
Nitrogen-fixing bacteria that live within plants (diazotrophic endophytes)offer avenues to reduce fertilizer needs, particularly in monocots. The diazotrophic endophyte of sugarcane, Gluconacetobacter diazotrophicus, enhances plant growth by supplying fixed nitrogen and also by another mechanism, which is possibly the production of the plant growth hormone indole acetic acid (IAA). Results from this work do not prove that this is a factor but strongly indicate that IAA production by the bacteria benefits the growth of sugarcane.

Publications

  • No publications reported this period


Progress 01/01/00 to 12/31/00

Outputs
Analysis of genes for nitrogen fixation: A major 31 kilobase cluster of nif and associated genes of Acetobacter diazotrophicus, a nitrogen-fixing endophyte of sugarcane, was sequenced and characterized. This cluster represents the largest and most complete assembly of contiguous nif/fix and associated genes so far found in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that 8 transciptional units are present in the cluster. The overall arrangement of genes is most similar to that in Azospirillum brasilense while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus. Phylogenetic analyses of the deduced amino acid sequence of NifH, NifD, and NifK indicated a close relationship of A. diazotrophicus with other members of the alpha-proteobacteria and also with another known sugarcane endophyte, a beta-group member, Herbaspirillum seropedicae. With a few exceptions, the phylogenetic trees inferred from the amino acid sequence of the three nitrogenase subunits were in agreement with the 16S rRNA tree. Plant growth enhancement: The ability of the nitrogen-fixing bacterial endophyte Acetobacter diazotrophicus strain PAl5 to enhance the growth of sugarcane SP70-1143 was evaluated in the growth chamber, greenhouse, and field, by comparing plants inoculated with wild type and a Nif - mutant, MAd3A, in two independent experiments. Both wild type and Nif - mutant strains colonized sugarcane plants equally well and persisted in mature plants. In N-deficient conditions, sugarcane plants inoculated with A. diazotrophicus PAl5 generally grew better and had higher total N content 60 days after planting than plants inoculated with mutant MAd3A or uninoculated plants. These results indicate that the transfer of fixed N from A. diazotrophicus to sugarcane might be a significant mechanism for plant growth promotion in this association. When N was not limiting, growth enhancement was observed in plants inoculated with either wild type or Nif - mutants, suggesting the additional effect of a plant growth-promoting factor provided by A. diazotrophicus. An 15N2 incorporation experiment demonstrated that A. diazotrophicus wild type strain but not the Nif - mutants actively fixed N2 inside sugarcane plants.

Impacts
Inoculation experiments have shown that a nitrogen-fixing bacterium living inside sugarcane plants, Acetobacter diazotrophicus, can significantly enhance the growth of its host plant compared to uninoculated controls. This is likely to be due to benefits of bacterial nitrogen fixation and also production of the plant growth hormone, auxin. The use of A. diazotrophicus in association with other monocot plants, including cereals, may be beneficial in agriculture.

Publications

  • Teixeira KRS, Wulling M, Morgan T, Galler R, Zellermann, EM, Baldani JI, Kennedy C, Meletzus D (1999) Molecular analysis of the chromosomal region encoding the nifA and nifB genes of Acetobacter diazotrophicus FEMS Microbiol Letts 176:301-309
  • Teixeira KRS, Morgan TV, Meletzus D, Galler R, Baldani JI, Kennedy C (1999) Identification, sequencing and structural analysis of a nifA-like gene of Acetobacter diazotrophicus. An Acad Bras Cienc 71:521-530
  • Sevilla M, Kennedy C (2000) Genetic analysis of nitrogen fixation and plant-growth stimulating properties of Acetobacter diazotrophicus, an endophyte of sugarcane. In: Triplett, E. W. (ed) Prokaryotic Nitrogen Fixation. Horizon Scientific Press, Wyndham UK, pp 737-760 Lee S, Meletzus D, Sevilla M, Kennedy C (2000) Characterization of a major nif/fix gene cluster in Acetobacter diazotrophicus, an endophyte of sugarcane. J Bacteriol 182:7088-7091
  • Sevilla M, Kennedy C (2000) Colonization of rice and other cereals by Acetobacter diazotrophicus, an endophyte of sugarcane. In: JK Ladha, PM Reddy (eds) The quest for nitrogen fixation in rice, IRRI Press, Manila, pp 151-165
  • Sevilla M, Burris RH, Gunapala N, Kennedy C (2001) Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and Nif- mutant strains. Mol Plant-Microbe Interactions 14:358-366
  • Lee S, Sevilla M, Meletzus D, Gunapala N, Kennedy C (2000) Characterization of nitrogen fixation genes and plant-growth promoting properties in Acetobacter diazotrophicus, an endophyte of sugarcane. In G Stacey, JN Keen (eds) Plant-Microbe Interactions V. American Phytopathology Press,
  • Rudnick PA, Arcondeguy T, Kennedy C, Kahn D (2001) glnD and mviN are genes of an essential operon in Sinorhizobium meliloti J Bacteriol 183:2682-2685.


Progress 01/01/99 to 12/31/99

Outputs
The ability of the nitrogen-fixing bacterial endophyte Acetobacter diazotrophicus strain PAl5 to enhance the growth of sugarcane SP70-1143 was evaluated in the growth chamber, greenhouse, and field, by comparing plants inoculated with wild type and a Nif - mutant, MAd3A, in three independent experiments. Both wild type and Nif - mutant strains colonized sugarcane plants equally well and persisted in mature plants. In N-deficient conditions, sugarcane plants inoculated with A. diazotrophicus PAl5 generally grew better and had higher total N content 60 days after planting than plants inoculated with mutant MAd3A or uninoculated plants. These results indicate that the transfer of fixed N from A. diazotrophicus to sugarcane appears to be the major mechanism for plant growth promotion in this association. When N was not limiting, growth enhancement sometimes was observed in plants inoculated with either wild type or Nif - mutants, suggesting the additional effect of a plant growth-promoting factor provided by A. diazotrophicus. An 15N2 incorporation experiment demonstrated that A. diazotrophicus wild type strain but not the Nif - mutants actively fixed N2 inside sugarcane plants. In addition, a major 35 kb cluster of nif and associated genes of Acetobacter diazotrophicus, a nitrogen-fixing endophyte of sugarcane, was sequenced and characterized. Northern blots and promoter sequence analysis indicated that 5 transcriptional units are present in the cluster. The overall arrangement of genes is most similar to that in Azospirillum brasilense while the individual gene products are more similar to those in species of Rhizobiaceae (sp) or in Rhodobacter capsulatus.

Impacts
(N/A)

Publications

  • Teixeira KRS, Wulling M, Morgan T, Galler R, Zellerman EM, Baldani JI, Kennedy C and Meletzus D. 1999. Molecular analysis of the chromosomal region encoding the nifA and nifBgenes of A. diazotrophicus. FEMS Micr. Lets 176:301-309.
  • Teixeira KRS, Morgan T, Meletzus D, Galler R, Baldani JI, Kennedy C. 1999. Identification, sequencing and structural analysis of a nifA-like gene of Acetobacter diazotrophicus. An Acad Bras Cien 71:521-530.


Progress 01/01/97 to 12/31/97

Outputs
Further analysis of the nif/fix gene cluster of Acetobacter diazotrophicus showed some additional open reading frames not placed on the map earlier. These include some which are similar to small Orf's in other diazotrophs encoding small iron sulfur proteins of unknown function. At the end of the fix genes which occur 3' down from the nif cluster is a gene identified as mcpA which encodes a methyl-acceptor transfer protein in other organisms. Mutations introduced into mcpA and put into the A. diazotrophicus genome are much less mobile or attracted to chemical signals than the parent strain. Experiments are now aimed at determinine whether these mutants are less able to colonize sugarcane than the wild type parental strain. Earlier results suggest that A. diazotrophicus has two mechanisms for plant- growth promotion, one occuring due to transfer of fixed nitrogen from bacteria to plant tissues under N-limiting conditions, and the other possibly involving the production of plant growth hormone-like compounds. This has been further studied using plants grown for a longer time and other strains and their non-nitrogen fixing mutant derivatives. The most recent results confirm what was observed previously, that a factor in addition to nitrogen fixation is important in this bacterial-plant interaction in allowing sugarcane to grow better than plants not harboring the bacteria. Probes carrying genes for indole acetic acid biosynthesis in other soil organisms were obtained and are being used to isolate similar genes from A. dizaotrophicus so that they can be mutated. Combinations of Nif-minus, IAA-minus, single and double mutant strains will be constructed to help determine the relative contribution of these two factors to the growth enhancement observed.

Impacts
(N/A)

Publications

  • Colnaghi R, Rudnick P, Green A, Kennedy C (1997) Strategies for increased ammonium production in free-living or plant associated nitrogen fixing bacteria. Plant and Soil 194:145-154
  • Sevilla M, Kennedy C (1997) Analysis of genes for nitrogen fixation in free-living and plant-associating bacteria. Life Sup Biospher Sci 3:169-173
  • Sevilla M, Meletzus D, Teixeira K, Lee S, Nutakki A, Baldani I, Kennedy C (1997) Analysis of nif and regulatory genes in Acetobacter diazotrophicus. Soil Biol Biochem 29:871-874