Lorenzo Segovia

Rhizobium tropici, a Novel Species Nodulating Phaseolus vulgaris L. Beans and Leucaena sp. Trees

A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.

Reclassification of American Rhizobium leguminosarum Biovar Phaseoli Type I Strains as Rhizobium etli sp. nov.

A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16S ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nodA and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16S ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16S rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal.

Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.

BackgroundExpansins and expansin-like proteins loosen cellulose microfibrils, possibly through the rupture of intramolecular hydrogen bonds. Together with the use of lignocellulolytic enzymes, these proteins are potential molecular tools to treat plant biomass to improve saccharification yields.ResultsHere we describe a new type of expansin-related fungal protein that we have called loosenin. Its corresponding gene, loos1, from the basidiomycete Bjerkandera adusta, was cloned and heterologously expressed in Saccharomyces cerevisiae. LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent. LOOS1 binds tightly to cellulose and chitin, and we demonstrate that cotton fibers become susceptible to the action of a commercial cellulase following treatment with LOOS1. Natural fibers of Agave tequilana also become susceptible to hydrolysis by cellulases after loosenin treatment.ConclusionsLOOS1 is a new type of protein with disrupting activity on cellulose. LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose.

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop α-8 of domain II

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop α‐8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop α‐8, β‐4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop α‐8 of Cry11A in receptor interaction was demonstrated by phage display and site‐directed mutagenesis. We isolated a peptide‐displaying phage (P5.tox), that recognizes loop α‐8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop α‐8 mutants affected in toxicity and receptor binding were characterized.

γN‐crystallin and the evolution of the βγ‐crystallin superfamily in vertebrates

The β and γ crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.

A network perspective on the evolution of metabolism by gene duplication.

BackgroundGene duplication followed by divergence is one of the main sources of metabolic versatility. The patchwork and stepwise models of metabolic evolution help us to understand these processes, but their assumptions are relatively simplistic. We used a network-based approach to determine the influence of metabolic constraints on the retention of duplicated genes.ResultsWe detected duplicated genes by looking for enzymes sharing homologous domains and uncovered an increased retention of duplicates for enzymes catalyzing consecutive reactions, as illustrated by the ligases acting in the biosynthesis of peptidoglycan. As a consequence, metabolic networks show a high retention of duplicates within functional modules, and we found a preferential biochemical coupling of reactions that partially explains this bias. A similar situation was found in enzyme-enzyme interaction networks, but not in interaction networks of non-enzymatic proteins or gene transcriptional regulatory networks, suggesting that the retention of duplicates results from the biochemical rules governing substrate-enzyme-product relationships. We confirmed a high retention of duplicates between chemically similar reactions, as illustrated by fatty-acid metabolism. The retention of duplicates between chemically dissimilar reactions is, however, also greater than expected by chance. Finally, we detected a significant retention of duplicates as groups, instead of single pairs.ConclusionOur results indicate that in silico modeling of the origin and evolution of metabolism is improved by the inclusion of specific functional constraints, such as the preferential biochemical coupling of reactions. We suggest that the stepwise and patchwork models are not independent of each other: in fact, the network perspective enables us to reconcile and combine these models.

gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates.

The β and γ crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.

Comparative Metagenomics of Two Microbial Mats at Cuatro Ciénegas Basin I: Ancient Lessons on How to Cope with an Environment Under Severe Nutrient Stress

The Cuatro Ciénegas Basin (CCB) is an oasis in the desert of Mexico characterized by low phosphorus availability and by its great diversity of microbial mats. We compared the metagenomes of two aquatic microbial mats from the CCB with different nutrient limitations. We observed that the red mat was P-limited and dominated by Pseudomonas, while the green mat was N-limited and had higher species richness, with Proteobacteria and Cyanobacteria as the most abundant phyla. From their gene content, we deduced that both mats were very metabolically diverse despite their use of different strategies to cope with their respective environments. The red mat was found to be mostly heterotrophic, while the green mat was more autotrophic. The red mat had a higher number of transporters in general, including transporters of cellobiose and osmoprotectants. We suggest that generalists with plastic genomes dominate the red mat, while specialists with minimal genomes dominate the green mat. Nutrient limitation was a common scenario on the early planet; despite this, biogeochemical cycles were performed, and as a result the planet changed. The metagenomes of microbial mats from the CCB show the different strategies a community can use to cope with oligotrophy and persist.

RNA silencing of rotavirus gene expression.

Abstract RNA interference (RNAi) is a double-stranded RNA (dsRNA)-triggered mechanism for suppressing gene expression, which is conserved in evolution and has emerged as a powerful tool to study gene function. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, and a genome composed of 11 segments of dsRNA. Here, we show that the RNAi machinery can be triggered to silence rotavirus gene expression by sequence-specific short interfering RNAs (siRNAs). RNAi is also useful for the study of the virus-cell interactions, through the silencing of cellular genes that are potentially important for the replication of the virus. Interestingly, while the translation of mRNAs is readily stopped by the RNAi machinery, the viral transcripts involved in virus genome replication do not seem to be susceptible to RNAi. Since gene silencing by RNAi is very efficient and specific, this system could become a novel therapeutic approach for rotavirus and other virus infections, once efficient methods for in vivo delivery of siRNAs are developed. Although the use of RNAi as an antiviral therapeutic tool remains to be demonstrated, there is no doubt that this technology will influence drastically the way postgenomic virus research is conducted.

The DNA-binding domain as a functional indicator: the case of the AraC/XylS family of transcription factors

The AraC/XylS family of transcription factors, which include proteins that are involved in the regulation of diverse biological processes, has been of considerable interest recently and has been constantly expanding by means of in silico predictions and experimental analysis. In this work, using a HMM based on the DNA binding domain of 58 experimentally characterized proteins from the AraC/XylS (A/X), 1974 A/X proteins were found in 149 out of 212 bacterial genomes. This domain was used as a template to generate a phylogenetic tree and as a tool to predict the putative regulatory role of the new members of this family based on their proximity to a particular functional cluster in the tree. Based on this approach we assigned a functional regulatory role for 75% of the TFs dataset. Of these, 33.7% regulate genes involved in carbon-source catabolism, 9.6% global metabolism, 8.3% nitrogen metabolism, 2.9% adaptation responses, 8.9% stress responses, and 11.7% virulence. The abundance of TFs involved in the regulation of metabolic processes indicates that bacteria have optimized their regulatory systems to control energy uptake. In contrast, the lower percentage of TFs required for stress, adaptation and virulence regulation reflects the specialization acquired by each subset of TFs associated with those processes. This approach would be useful in assigning regulatory roles to uncharacterized members of other transcriptional factor families and it might facilitate their experimental analysis.