Program Responsible: Jaime Mora-Celis, Ph.D.
The Program has contributed with several and important achievements in the following relevant projects.
- Carbon and nitrogen metabolism in Rhizobium
Participants: Dr. Jaime Mora (direction), Dr. Sergio Encarnación, Dr. Michael Dunn, Dra. Carmen Vargas, Q.B.P. Hermenegildo Taboada.
- Proteomic and transcriptomic analysis of Rhizobium.
Participants: Dr. Sergio Encarnacion (direction), Ing. Magdalena Hernandez, Q.F.B. Sandra Contreras-Martínez, Emmanuel Salazar, Dr. Luis Treviño-Quntanilla
- Improvement of Nitrogen Fixation capacity of Rhizobium
Participants: Dr. Jaime Mora (direction), Dr. Humberto Peralta Díaz, Quim. Yolanda Mora.
- Genomic analysis of orthologs in Rhizobiales
Participants: Dr. Jaime Mora (direction), Dr. Humberto Peralta Díaz, Ing. Gabriela Guerrero Ruíz, Ing. Alejandro Aguilar Vera, M. en C. Rafael Díaz Méndez, Dr. Miguel Angel Villalobos López.
We began our studies in the determination of the cycle of the glutamine synthetase in the fungus Neurospora crassa and the enzymes that participate in the glutamate-glutamine interconversion in N. crassa and in the new model organism Rhizobium etli .
R. etli does not present the glutamate dehydrogenase (GDH) gene. Together with the GS-GOGAT pathway, GDH constitutes the enter point of nitrogen (as ammonium) to all the cells. We expressed the GDH activity in R etli in nodule by means of specific promoters from the symbiotic stage, and it caused a strong inhibition of the nitrogen fixation, demonstrating that the ammonium assimilation and the fixation are incompatible processes.
We reported, for the first time for Rhizobium , the characterization of GOGAT enzyme. GOGAT synthesizes glutamate, and along with the Glutamine synthetase (GS), are responsible of the ammonium assimilation. The mutation of GOGAT in symbiosis simultaneously produced greater nitrogen contribution to the plant and nitrogen fixing capacity. The GS/GOGAT activity is fundamental in the nitrogen metabolism of the bacteria and it is closely related to the carbon metabolism.
We found that R. etli presents two GS´s that are specifically expressed according to the ammonium availability. Also, we reported that the GS-II sequence can serve as a phylogenetic marker in these organisms.
R. etli does not grow in subcultures because it enters into a "fermentative" metabolic state. Since the bacterium is an aerobic-strict organism, this finding had an extreme importance. The "fermentative" state represents profound metabolic changes, such as: alteration of some enzymatic activities of the Krebs (or Tricarboxilic Acid, TCA) cycle because two of their key enzymes have low activities; also it accumulates a reserve polymer named PHB (poly-beta-hydroxybutyrate), it excretes a great amount of glutamate and also precipitates in cell conglomerates. This situation is very similar to that within the nodules. The metabolic change is mainly due to the low synthesis and oxydation of two vitamins, thiamin and biotin, that are cofactors of the enzymes that disappear in the TCA cycle.
In free living and symbiotic conditions, R. etli produces the polymer PHB that represents a carbon and reducing power reserve. Apparently, the deviation of carbon and reducing power to the PHB synthesis allows that in microaerobiosis (as in the nodule) Rhizobium can maintain an active carbon metabolism. We obtained a mutant that does not accumulate the polymer and we found that it excreted great amounts of organic acids and that the proportion of NADH/NAD+ was increased. It is, the mutation produced a diminished oxydative capacity in the cell and it was filled of reducing power. In symbiosis, the mutation produced a better nitrogen fixation capacity possibly by deriving the excess of carbon to the energy-consuming process of the nitrogenase.
When we began the proteomic project with R. etli the genomic sequence was not available. Now, with the finished sequence, we also extended the analysis to the Sinorhizobium meliloti genome. To date, applying the 2D polyacrilamide gel electrophoresis technique (2D-PAGE) we have analyzed several physiological conditions of these organisms such as growth in rich medium, minimal medium (6, 12, 24 hours), symbiosis (early to late stages), biofilm formation and stress (heat shock and oxidative stress). Utilizing the mass spectrometer MALDI-TOF we have identified in the gels around 1200 proteins. We are constructing the metabolic charts of these conditions and soon will be available to the scientific communitiy on the web. On the other hand, we have constructed the first microarray in Mexico of a complete replicon, that of the symbiotic plasmid from R. etli CFN42, the type strain. Currently, we are assaying with the genomic microarray of that strain in the free living and symbiotic conditions.
Another of our projects is the annotation of the Sinorhizobium meliloti metabolome (SinoCyc) with the use of the PathwayTools software.
The biofertilizers have a great perspective of application in the agricultural fields in Mexico because they are environmentally friendly, have a low cost and they are easily transported and applied. On the other hand, the nitrogen chemical fertilization represents high economic costs for the farmers, but the most serious effects of the massive fertilization in the world are environmental: industrial production contaminates the air, soils are deteriorated by the salinity, as well as the water-bearing mantles. Studying the transcriptional regulation of the reiterated sequences of the nitrogenase in R. etli and the carbon flow modification, we obtained modified strains that overexpress the enzyme and have very important effects in their association with common bean (a main staple in Mexico): increases in seed production (up to 50%) and in its nutritious value (up to 100%). The increase in the nitrogen fixation ability of Rhizobium , obtained with modifications without transgenes, are the highest reported to the date.
Additionally, we done metabolic and genetic characterization of rhizobia strains from Costa Rica with high nitrogen fixation ability in order to define the key factors influencing their performance. After four year-tests in the field, we promoted the use of all these strains as biofertilizers. In regard to the genetic modification, a national (PA/2002/003920) and international (PCT MX 03/00033) patent was requested and the strains were transferred by means of an Agreement to the company Asesoría Integral Agropecuaria y Administrativa ( ASIA ). In order to use the biofertilizer in the field, the procedure is very simple: add a small amount of water to the seed, add an adherent and the content of a 400 gram-bag, mix thoroughly and it is ready to hand- or machine-sowing. The biofertilizer costs 10 times less than the chemical fertilizer.
In Northern Mexico , in the state of Durango , the biofertilizer was applied at commercial level and obtained satisfactory results in production increase. If farmers accept to use the biofertilizer, the whole population also will receive benefits such as lesser environmental contamination and greater nutritious quality of the seed.
With the appearance of genomic sequences of Rhizobium -related organisms, we initiated a study that tried to define whether exists a relationship between gene order conservation (or synteny) and function. Therefore, the chromosomal genes of the Rhizobiales Sinorhizobium meliloti , Mesorhizobium loti , Agrobacterium tumefaciens and Brucella melitensis were classified. These organisms display a great versatility in their living styles, since both first are legume symbionts, the third is a pathogen for plants and the last is an animal pathogen. Also, their genomes show variability and, in the same order, they present two megaplasmids, a linear chromosome, a large chromosome with almost 7000 genes and a circular "accessory" chromosome, respectively. When we analyzed them, we found they share a great amount of genes, approximately 2000, the majority have a conserved position in the chromosomes (it is, they are syntenic), they are organized in groups and operons, they encode house-keeping functions and also present a high level of sequence identity (in pairs, and compared between species). These genes, besides reflecting a relatedness among the species, and the remains of an ancient, common chromosome, also code the typical metabolic functions of the group. We extended our analysis to the Enterobacteriales (gamma proteobacteria) as Escherichia coli, Salmonella typhimurium and Erwinia carotovora and found similar tendencies in gene organization, functional linkage and essential role for syntenic genes. We consider that our synteny approach represents an interesting contribution to the analysis of the bacterial genomes, because the resulting evidences of phylogenetic, structural and functional relationships among chromosomal genes of related organisms.