Rosa Isela Santamaría

Rapid evolutionary change of common bean (Phaseolus vulgaris L) plastome, and the genomic diversification of legume chloroplasts

BackgroundFabaceae (legumes) is one of the largest families of flowering plants, and some members are important crops. In contrast to what we know about their great diversity or economic importance, our knowledge at the genomic level of chloroplast genomes (cpDNAs or plastomes) for these crops is limited.ResultsWe sequenced the complete genome of the common bean (Phaseolus vulgari s cv. Negro Jamapa) chloroplast. The plastome of P. vulgaris is a 150,285 bp circular molecule. It has gene content similar to that of other legume plastomes, but contains two pseudogenes, rpl 33 and rps 16. A distinct inversion occurred at the junction points of trn H-GUG/rpl 14 and rps 19/rps 8, as in adzuki bean [1]. These two pseudogenes and the inversion were confirmed in 10 varieties representing the two domestication centers of the bean. Genomic comparative analysis indicated that inversions generally occur in legume plastomes and the magnitude and localization of insertions/deletions (indels) also vary. The analysis of repeat sequences demonstrated that patterns and sequences of tandem repeats had an important impact on sequence diversification between legume plastomes and tandem repeats did not belong to dispersed repeats. Interestingly, P. vulgaris plastome had higher evolutionary rates of change on both genomic and gene levels than G. max, which could be the consequence of pressure from both mutation and natural selection.ConclusionLegume chloroplast genomes are widely diversified in gene content, gene order, indel structure, abundance and localization of repetitive sequences, intracellular sequence exchange and evolutionary rates. The P. vulgaris plastome is a rapidly evolving genome.

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.

The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain

BackgroundBean-nodulating Rhizobium etli originated in Mesoamerica, while soybean-nodulating Sinorhizobium fredii evolved in East Asia. S. fredii strains, such as GR64, have been isolated from bean nodules in Spain, suggesting the occurrence of conjugative transfer events between introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pRet42d) requires cointegration with the endogenous self-transmissible plasmid pRet42a. Aiming at further understanding the generation of diversity among bean nodulating strains, we analyzed the plasmids of S. fredii GR64: pSfr64a and pSfr64b (symbiotic plasmid).ResultsThe conjugative transfer of the plasmids of strain GR64 was analyzed. Plasmid pSfr64a was self-transmissible, and required for transfer of the symbiotic plasmid. We sequenced pSfr64a, finding 166 ORFs. pSfr64a showed three large segments of different evolutionary origins; the first one presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; the second one harbored 51 ORFs with highest similarity to genes from pRet42d, including the replication, but not the symbiosis genes. Accordingly, pSfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, but did not contribute to symbiosis. The third segment contained 36 ORFs with highest similarity to genes localized on pRet42a, 20 of them involved in conjugative transfer. Plasmid pRet42a was unable to substitute pSfr64a for induction of pSym transfer, and its own transfer was significantly diminished in GR64 background. The symbiotic plasmid pSfr64b was found to differ from typical R. etli symbiotic plasmids.ConclusionsS. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We infer that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid cointegrate from R. etli to a S. fredii strain, and at least two recombination events among the R. etli plasmids and the S. fredii genome. As in R. etli CFN42, the S. fredii GR64 transmissible plasmid is required for the conjugative transfer of the symbiotic plasmid. In spite of the similarity in the conjugation related genes, the transfer process of these plasmids shows a host-specific behaviour.

Evolutionary Dynamics of Insertion Sequences in Relation to the Evolutionary Histories of the Chromosome and Symbiotic Plasmid Genes of Rhizobium etli Populations

ABSTRACT Insertion sequences (IS) are mobile genetic elements that are distributed in many prokaryotes. In particular, in the genomes of the symbiotic nitrogen-fixing bacteria collectively known as rhizobia, IS are fairly abundant in plasmids or chromosomal islands that carry the genes needed for symbiosis. Here, we report an analysis of the distribution and genetic conservation of the IS found in the genome of Rhizobium etli CFN42 in a collection of 87 Rhizobium strains belonging to populations with different geographical origins. We used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 and evaluated whether the IS were located in consistent genomic contexts. We found that the IS from the symbiotic plasmid were frequently present in the analyzed strains, whereas the chromosomal IS were observed less frequently. We then examined the evolutionary dynamics of these strains based on a population genetic analysis of two chromosomal housekeeping genes (glyA and dnaB) and three symbiotic sequences (nodC and the two IS elements). Our results indicate that the IS contained within the symbiotic plasmid have a higher degree of genomic context conservation, lower nucleotide diversity and genetic differentiation, and fewer recombination events than the chromosomal housekeeping genes. These results suggest that the R. etli populations diverged recently in Mexico, that the symbiotic plasmid also had a recent origin, and that the IS elements have undergone a process of cyclic infection and expansion.

Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types

ABSTRACT In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.

Alcoholysis reactions from starch with α‐amylases

The ability of α‐amylases from different sources to carry out reactions of alcoholysis was studied using methanol as substrate. It was found that while the enzymes from Aspergillus niger and Aspergillus oryzae, two well‐studied saccharifying amylases, are capable of alcoholysis reactions, the classical bacterial liquefying α‐amylases from Bacillus licheniformis and Bacillus stearothermophilus are not. The effect of starch and methanol concentration, temperature and pH on the synthesis of glucosides with α‐amylase from A. niger was studied. Although methanol may inactivate α‐amylase, a 90% substrate relative conversion can be obtained in 20% methanol at a high starch concentration (15% w/v) due to a stabilizing effect of starch on the enzyme. As the products of alcoholysis are a series of methyl‐oligosaccharides, from methyl‐glucoside to methyl‐hexomaltoside, alcoholysis was indirectly quantified by high performance liquid chromatography analysis of the total methyl‐glucoside produced after the addition of glucoamylase to the α‐amylase reaction products. More alcoholysis was obtained from intact soluble starch than with maltodextrins or pre‐hydrolyzed starch. The biotechnological implications of using starch as substrate for the production of alkyl‐glucosides is analyzed in the context of these results.

Introducing transglycosylation activity in a liquefying α-amylase

By mutating Ala‐289 by Phe or Tyr in the Bacillus stearothermophilus α‐amylase, we induced this enzyme to perform alcoholytic reactions, a function not present in the wild‐type enzyme. This residue was selected from homology analysis with neopullulanase, where the residue has been implicated in the control of transglycosylation [Kuriki et al. (1996) J. Biol. Chem. 271, 17321–17329]. We made some inferences about the importance of electrostatic and geometrical modifications in the active site environment of the amylase to explain the behavior of the modified enzyme.

Genomic lineages of Rhizobium etli revealed by the extent of nucleotide polymorphisms and low recombination

BackgroundMost of the DNA variations found in bacterial species are in the form of single nucleotide polymorphisms (SNPs), but there is some debate regarding how much of this variation comes from mutation versus recombination. The nitrogen-fixing symbiotic bacteria Rhizobium etli is highly variable in both genomic structure and gene content. However, no previous report has provided a detailed genomic analysis of this variation at nucleotide level or the role of recombination in generating diversity in this bacterium. Here, we compared draft genomic sequences versus complete genomic sequences to obtain reliable measures of genetic diversity and then estimated the role of recombination in the generation of genomic diversity among Rhizobium etli.ResultsWe identified high levels of DNA polymorphism in R. etli, and found that there was an average divergence of 4% to 6% among the tested strain pairs. DNA recombination events were estimated to affect 3% to 10% of the genomic sample analyzed. In most instances, the nucleotide diversity (π) was greater in DNA segments with recombinant events than in non-recombinant segments. However, this degree of recombination was not sufficiently large to disrupt the congruence of the phylogenetic trees, and further evaluation of recombination in strains quartets indicated that the recombination levels in this species are proportionally low.ConclusionOur data suggest that R. etli is a species composed of separated lineages with low homologous recombination among the strains. Horizontal gene transfer, particularly via the symbiotic plasmid characteristic of this species, seems to play an important role in diversity but the lineages maintain their evolutionary cohesiveness.

Genomic basis of symbiovar mimosae in Rhizobium etli

BackgroundSymbiosis genes (nod and nif) involved in nodulation and nitrogen fixation in legumes are plasmid-borne in Rhizobium. Rhizobial symbiotic variants (symbiovars) with distinct host specificity would depend on the type of symbiosis plasmid. In Rhizobium etli or in Rhizobium phaseoli, symbiovar phaseoli strains have the capacity to form nodules in Phaseolus vulgaris while symbiovar mimosae confers a broad host range including different mimosa trees.ResultsWe report on the genome of R. etli symbiovar mimosae strain Mim1 and its comparison to that from R. etli symbiovar phaseoli strain CFN42. Differences were found in plasmids especially in the symbiosis plasmid, not only in nod gene sequences but in nod gene content. Differences in Nod factors deduced from the presence of nod genes, in secretion systems or ACC-deaminase could help explain the distinct host specificity. Genes involved in P. vulgaris exudate uptake were not found in symbiovar mimosae but hup genes (involved in hydrogen uptake) were found. Plasmid pRetCFN42a was partially contained in Mim1 and a plasmid (pRetMim1c) was found only in Mim1. Chromids were well conserved.ConclusionsThe genomic differences between the two symbiovars, mimosae and phaseoli may explain different host specificity. With the genomic analysis presented, the term symbiovar is validated. Furthermore, our data support that the generalist symbiovar mimosae may be older than the specialist symbiovar phaseoli.

Evolutionary Genomics of the Nitrogen-Fixing Symbiotic Bacteria

Soil contains the most complex communities of microorganisms (Tringe et al. 2005). The earth’s global ecology depends largely on the metabolic activities of different soil bacteria. One of these processes is biological nitrogen fixa-tion. Nitrogen from the atmosphere is made available to plants either by free-living bacteria or symbionts associated with leguminous plants. There are ample bacterial species that are able to establish nitrogen fixing symbiosis. Ordinarily known as rhizobia, they are grouped in different taxonomic families of the α-proteobacteria classified as Rhizobiaceae, Phylobacteriaceae and Bradyrhizobiaceae (Table 7.1) (Garrity et al. 2002). Recently, it was reported that some strains of