Jaime Mora

New Horizons in Nitrogen Fixation

this volume) to become associated with apodinitrogenase. The role of general chaperonins in promoting accumulation of nif gene products (Govezensky et al.1991) will be discussed in chapters of this section and may suggest roles for some of the nif-specific gene products to which no role has yet been ascribed. It is hoped that by the time of the next meeting, the enzymatic or transfer reactions carried out by the various nif genes involved in metal cluster assembly will be known in detail. Acknowledgements. The author wishes to acknowledge the contributions of his collaborators, GP Roberts, VK Shah, RM Allen, MJ Homer, R Chatterjee, J Allen and J Roll at the University of WI. Work from the laboratories of the author and his collaborators has been generously supported by the NIH and NRI/CGP.

Conserved symbiotic plasmid DNA sequences in the multireplicon pangenomic structure of Rhizobium etli.

ABSTRACT Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.

Taxonomy of rhizobia and agrobacteria from the Rhizobiaceae family in light of genomics

Phylogenomic analyses showed two major superclades within the family Rhizobiaceae that corresponded to the Rhizobium/Agrobacterium and Shinella/Ensifer groups. Within the Rhizobium/Agrobacterium group, four highly supported clades were evident that could correspond to distinct genera. The Shinella/Ensifer group encompassed not only the genera Shinella and Ensifer but also a separate clade containing the type strain of Rhizobium giardinii. Ensifer adhaerens (Casida A(T)) was an outlier within its group, separated from the rest of the Ensifer strains. The phylogenomic analysis presented provided support for the revival of Allorhizobium as a bona fide genus within the Rhizobiaceae, the distinctiveness of Agrobacterium and the recently proposed Neorhizobium genus, and suggested that R. giardinii may be transferred to a novel genus. Genomics has provided data for defining bacterial-species limits from estimates of average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH). ANI reference values are becoming the gold standard in rhizobial taxonomy and are being used to recognize novel rhizobial lineages and species that seem to be biologically coherent, as shown in this study.

AniA Regulates Reserve Polymer Accumulation and Global Protein Expression in Rhizobium etli

Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::OmegaSm(r)/Sp(r) mutant CAR1, which is unable to synthesize poly-beta-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes beta-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.

Regulation and function of glutamate synthase in Neurospora crassa

Abstract In Neurospora crassa two enzymes can provide glutamate: the NADPH dependent GDH and the NADH dependent GOGAT. An elevated GOGAT activity was found in Neurospora wild-type under ammonium limitation in contrast to a 4-fold lower activity on excess of a m monium. Glutamate and glutamine repress this enzyme. On excess of ammonium the GDH-NADPH deficient mutant am -1 grows poorly with an elevated GOGAT activity. A GOGAT less mutant was found. It presented a lag-phase to grow on ammonium. It is concluded that N. crassa glutamate synthase provides glutamate from low am-monium concentrations. The enzyme was purified to homogeneity and shown to be composed of a single type of monomer with a molecular weight above 200,000.

Characterization of the NifA-RpoN regulon in Rhizobium etli in free life and in symbiosis with Phaseolus vulgaris.

ABSTRACT The NifA-RpoN complex is a master regulator of the nitrogen fixation genes in alphaproteobacteria. Based on the complete Rhizobium etli genome sequence, we constructed an R. etli CFN42 oligonucleotide (70-mer) microarray and utilized this tool, reverse transcription (RT)-PCR analysis (transcriptomics), proteomics, and bioinformatics to decipher the NifA-RpoN regulon under microaerobic conditions (free life) and in symbiosis with bean plants. The R. etli NifA-RpoN regulon was determined to contain 78 genes, including the genes involved in nitrogen fixation, and the analyses revealed 42 new NifA-RpoN-dependent genes. More importantly, this study demonstrated that the NifA-RpoN regulon is composed of genes and proteins that have very diverse functions, that play fundamental and previously less appreciated roles in regulating the normal physiology of the cell, and that have important functions in providing adequate conditions for efficient nitrogen fixation in symbiosis. The R. etli NifA-RpoN regulon defined here has some components in common with other NifA-RpoN regulons described previously, but the vast majority of the components have been found only in the R. etli regulon, suggesting that they have a specific role in this bacterium and particular requirements during nitrogen fixation compared with other symbiotic bacterial models.

Rhizobium etli genetically engineered for the heterologous expression of Vitreoscilla sp. hemoglobin : Effects on free-living and symbiosis

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibi...

Evolutionary, structural and functional relationships revealed by comparative analysis of syntenic genes in Rhizobiales.

BackgroundComparative genomics has provided valuable insights into the nature of gene sequence variation and chromosomal organization of closely related bacterial species. However, questions about the biological significance of gene order conservation, or synteny, remain open. Moreover, few comprehensive studies have been reported for rhizobial genomes.ResultsWe analyzed the genomic sequences of four fast growing Rhizobiales (Sinorhizobium meliloti, Agrobacterium tumefaciens, Mesorhizobium loti and Brucella melitensis). We made a comprehensive gene classification to define chromosomal orthologs, genes with homologs in other replicons such as plasmids, and those which were species-specific. About two thousand genes were predicted to be orthologs in each chromosome and about 80% of these were syntenic. A striking gene colinearity was found in pairs of organisms and a large fraction of the microsyntenic regions and operons were similar. Syntenic products showed higher identity levels than non-syntenic ones, suggesting a resistance to sequence variation due to functional constraints; also, an unusually high fraction of syntenic products contained membranal segments. Syntenic genes encode a high proportion of essential cell functions, presented a high level of functional relationships and a very low horizontal gene transfer rate. The sequence variability of the proteins can be considered the species signature in response to specific niche adaptation. Comparatively, an analysis with genomes of Enterobacteriales showed a different gene organization but gave similar results in the synteny conservation, essential role of syntenic genes and higher functional linkage among the genes of the microsyntenic regions.ConclusionSyntenic bacterial genes represent a commonly evolved group. They not only reveal the core chromosomal segments present in the last common ancestor and determine the metabolic characteristics shared by these microorganisms, but also show resistance to sequence variation and rearrangement, possibly due to their essential character. In Rhizobiales and Enterobacteriales, syntenic genes encode a high proportion of essential cell functions and presented a high level of functional relationships.

Diversification of DNA Sequences in the Symbiotic Genome of Rhizobium etli

Bacteria of the genus Rhizobium and related genera establish nitrogen-fixing symbioses with the roots of leguminous plants. The genetic elements that participate in the symbiotic process are usually compartmentalized in the genome, either as independent replicons (symbiotic plasmids) or as symbiotic regions or islands in the chromosome. The complete nucleotide sequence of the symbiotic plasmid of Rhizobium etli model strain CFN42, symbiont of the common bean plant, has been reported. To better understand the basis of DNA sequence diversification of this symbiotic compartment, we analyzed the distribution of single-nucleotide polymorphisms in homologous regions from different Rhizobium etli strains. The distribution of polymorphisms is highly asymmetric in each of the different strains, alternating regions containing very few changes with regions harboring an elevated number of substitutions. The regions showing high polymorphism do not correspond with discrete genetic elements and are not the same in the different strains, indicating that they are not hypervariable regions of functional genes. Most interesting, some highly polymorphic regions share exactly the same nucleotide substitutions in more than one strain. Furthermore, in different regions of the symbiotic compartment, different sets of strains share the same substitutions. The data indicate that the majority of nucleotide substitutions are spread in the population by recombination and that the contribution of new mutations to polymorphism is relatively low. We propose that the horizontal transfer of homologous DNA segments among closely related organisms is a major source of genomic diversification.

Engineering the nifH Promoter Region and Abolishing Poly-β-Hydroxybutyrate Accumulation in Rhizobium etli Enhance Nitrogen Fixation in Symbiosis with Phaseolus vulgaris

ABSTRACT Rhizobium etli, as well as some other rhizobia, presents nitrogenase reductase (nifH) gene reiterations. Several R. etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c). Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant (Phaseolus vulgaris), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds. Additionally, expression of the chimeric nifHDK operon in a poly-β-hydroxybutyrate-negative R. etli strain produced an additive effect in enhancing symbiosis. To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in Rhizobium.