Hugo Ramírez-Saad

Gut-Associated bacteria throughout the life cycle of the Bark Beetle "Dendroctonus rhizophagus" Thomas and Bright (Curculionidae: Scolytinae) and their cellulolytic activities

Dendroctonus rhizophagus Thomas and Bright (Curculionidae: Scolytinae) is an endemic economically important insect of the Sierra Madre Occidental in Mexico. This bark beetle has an atypical behavior within the genus because just one beetle couple colonizes and kills seedlings and young trees of 11 pine species. In this work, the bacteria associated with the Dendroctonus rhizophagus gut were analyzed by culture-dependent and culture-independent methods. Analysis of 16S rRNA sequences amplified directly from isolates of gut bacteria suggests that the bacterial community associated with Dendroctonus rhizophagus, like that of other Dendroctonus spp. and Ips pini, is limited in number. Nine bacterial genera of γ-Proteobacteria and Actinobacteria classes were detected in the gut of Dendroctonus rhizophagus. Stenotrophomonas and Rahnella genera were the most frequently found bacteria from Dendroctonus rhizophagus gut throughout their life cycle. Stenotrophomonas maltophilia, Ponticoccus gilvus, and Kocuria marina showed cellulolytic activity in vitro. Stenotrophomonas maltophilia, Rahnella aquatilis, Raoultella terrigena, Ponticoccus gilvus, and Kocuria marina associated with larvae or adults of Dendroctonus rhizophagus could be implicated in nitrogen fixation and cellulose breakdown, important roles associated to insect development and fitness, especially under the particularly difficult life conditions of this beetle.

Characterization of novel antibiotic resistance genes identified by functional metagenomics on soil samples.

The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (γ- and α-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (K(m) of 8.9 µM for NADPH and 3.7 µM for dihydrofolate and IC(50) of 20 µM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences.

Petroleum-influenced beach sediments of the Campeche Bank, Mexico: diversity and bacterial community structure assessment.

The bacterial diversity and community structure were surveyed in intertidal petroleum-influenced sediments of ≈ 100 km of a beach, in the southern Gulf of Mexico. The beach was divided in twenty sampling sites according to high, moderate and low petroleum influence. Densities of cultured heterotrophic (HAB) and hydrocarbon degrading bacteria (HDB) were highly variable in sediments, with little morphological assortment in colonies. PCR-RISA banding patterns differentiated distinct communities along the beach, and the bacterial diversity changed inversely to the degree of petroleum hydrocarbon influence: the higher TPH concentration, the lower genotype diversity. Seven DNA sequences (Genbank EF191394 -EF191396 and EF191398 -EF191401) were affiliated to uncultured members of Gemmatimonas, Acidobacterium, Desulfobacteraceae, Rubrobacterales, Actinobacterium and the Fibrobacteres/Acidobacteria group; all the above taxa are known for having members with active roles in biogeochemical transformations. The remaining sequences (EF191388 - EF191393 and EF191397) affiliated to Pseudoalteromonas, and to oil-degrading genera such as Pseudomonas, Vibrio and Marinobacter, being the last one an obligate oil-degrading bacterium. An exchange of bacteria between the beach and the oil seep environment, and the potential cleaning-up role of bacteria at the southern Gulf of Mexico are discussed.

Bacterial community in the rhizosphere of the cactus species Mammillaria carnea during dry and rainy seasons assessed by deep sequencing

Background and aimsThe Tehuacán-Cuitcatlán reserve is an area of unique plant biodiversity mostly in the form of xerophytes, with exceptionally high numbers of rare and endemic species. This endemism results partly from the characteristics of the climate of this area, with two distinct seasons: rainy and dry seasons. Although rhizosphere communities must be critical in the function of this ecosystem, understanding the structure of these communities is currently limited. This is the first molecular study of the microbial diversity present in the rhizosphere of Mamillaria carnea.MethodsTotal DNA was obtained from soil and rhizosphere samples at three locations in the Tehuacán Cuicatlán Reserve, during dry and rainy seasons. Temperature gradient gel electrophoresisis (TGGE) fingerprinting, 16S rRNA gene libraries and pyrosequencing were used to investigate bacterial diversity in the rhizosphere of Mammillaria carnea and changes in the microbial community between seasons.ResultsDeep sequencing data reveal a higher level of biodiversity in the dry season. Statistical analyses based on these data indicates that the composition of the bacterial community differed between both seasons affecting to members of the phyla Acidobacteria, Cyanobacteria, Gemmatimonadetes, Plantomycetes, Actinobacteria and Firmicutes. In addition, the depth of sequencing performed (>24,000 reads) enables detection of changes in the relative abundance of lower bacterial taxa (novel bacterial phylotypes) indicative of the increase of specific bacterial populations due to the season.ConclusionsThis study states the basis of the bacterial diversity in the rhizosphere of cacti in semi-arid environments and it is a sequence-based demonstration of community shifts in different seasons.

Rhizosphere-Bacterial Community in Eperua falcata (Caesalpiniaceae) a Putative Nitrogen-Fixing Tree from French Guiana Rainforest

The rainforest of French Guiana is still largely unaffected by human activity. Various pristine sites like the Paracou Research Station are devoted to study this tropical ecosystem. We used culture-independent techniques, like polymerase chain reaction-temperature gradient gel electrophoresis, and construction of clone libraries of partial 16S rRNA and nifH genes, to analyze the composition of the bacterial community in the rhizosphere of mature trees of Eperua falcata and Dicorynia guianensis, both species within the Caesalpiniaceae family. E. falcata is one of the more abundant pioneer tree species in this ecosystem and so far, no root nodules have ever been found. However, its nitrogen-fixing status is regarded as “uncertain”, whereas D. guianensis is clearly considered a non-nitrogen-fixing plant. The rhizospheres of these mature trees contain specific bacterial communities, including several currently found uncultured microorganisms. In these communities, there are putative nitrogen-fixing bacteria specifically associated to each tree: D. guianensis harbors several Rhizobium spp. and E. falcata members of the genera Burkholderia and Bradyrhizobium. In addition, nifH sequences in the rhizosphere of the latter tree were very diverse. Retrieved sequences were related to bacteria belonging to the α-, β-, and γ-Proteobacteria in the E. falcata rhizoplane, whereas only two sequences related to γ-Proteobacteria were found in D. guianensis. Differences in the bacterial communities and the abundance and diversity of nifH sequences in E. falcata rhizosphere suggest that this tree could obtain nitrogen through a nonnodulating bacterial interaction.

DNA extraction from actinorhizal nodules

The nitrogen-fixing actinomycete Frankia has its main ecological niche in the root nodules that are formed with a wide taxonomic range of host plants, called by this fact actinorhizal plants. This generic name comprises more than 200 species of vascular dicotyledonous plants, distributed in 20 genera and eight families [5].

Hydrocarbon Biodegradation Potential of Native and Exogenous Microbial Inocula in Mexican Tropical Soils

Soil and water contamination by oil is one of the central environmental problems in Mexico. In the southeastern part of the country, especially in Tabasco and Veracruz states there are several oil facilities involved in extraction, transportation, processing and storage of oil and oil products. Historically in this region there have been spills that affect large areas of soil and eventually rivers, streams and lagoons. Pollutants often persist for long periods in the soil, sediments and water due to different factors like the environmental conditions prevailing in the region (heavy rains and extensive wetlands), the nature of the soils (mostly clayed soils adjacent to water bodies), and the recalcitrance of oil components (Adams et al. 2011).

Evaluation of the simultaneous removal of recalcitrant drugs (bezafibrate, gemfibrozil, indomethacin and sulfamethoxazole) and biodegradable organic matter from synthetic wastewater by electro-oxidation coupled with a biological system

ABSTRACT Pharmaceutical degradation in conventional wastewater treatment plants (WWTP) represents a challenge since municipal wastewater and hospital effluents contain pharmaceuticals in low concentrations (recalcitrant and persistent in WWTP) and biodegradable organic matter (BOM) is the main pollutant. This work shows the feasibility of coupling electro-oxidation with a biological system for the simultaneous removal of recalcitrant drugs (bezafibrate, gemfibrozil, indomethacin and sulfamethoxazole (BGIS)) and BOM from wastewater. High removal efficiencies were attained without affecting the performance of activated sludge. BGIS degradation was performed by advanced electrochemical oxidation and the activated sludge process for BOM degradation in a continuous reactor. The selected electrochemical parameters from microelectrolysis tests (1.2 L s−1 and 1.56 mA cm−2) were maintained to operate a filter press laboratory reactor FM01-LC using boron-doped diamond as the anode. The low current density was chosen in order to remove drugs without decreasing BOM and chlorine concentration control, so as to avoid bulking formation in the biological process. The wastewater previously treated by FM01-LC was fed directly (without chemical modification) to the activated sludge reactor to remove 100% of BGIS and 83% of BOM; conversely, the BGIS contained in wastewater without electrochemical pre-treatment were persistent in the biological process and promoted bulking formation.

Polymorphism in the regulatory regions of genes CgYPS1 and CgYPS7 encoding yapsins in Candida glabrata is associated with changes in expression levels

ABSTRACT Candida glabrata is an opportunistic fungus infecting mainly immunocompromised people. Its adherence capacity and exoenzymes contribute to damaging host cells. In particular, the yapsins are a family of aspartyl proteases involved in maturation of proteins and cell wall function, and yapsins 1 and 7, respectively encoded by genes CgYPS1 and CgYPS7, are potential virulence factors. In this study, the polymorphism of regulatory regions and the expression profiles of both genes were compared in C. glabrata clinical strains. The sequence analysis of regulatory regions revealed that the distribution of transcription factor binding sites (TFBSs) was similar, although some TFBSs were not universally distributed. The quantita‐tive expression of CgYPS1 and CgYPS7 genes of different C. glabrata strains in rich and poor media was estimated by RT‐qPCR. The primary sequences of genes CgYPS1 and CgYPS7 of C. glabrata strains were highly conserved among different strains, but the regulatory regions were polymorphic, harboring different TFBS arrays, and showing differential expression profiles. &NA; Graphical Abstract Figure. The differential expression of CgYPS1 and CgYPS7 genes under the same physiological conditions is a consequence of polymorphism in the promotor regions in Candida glabrata clinical isolates.

DINÁMICA DE POBLACIONES BACTERIANAS Y ACTIVIDAD DESHIDROGENASA DURANTE LA BIORREMEDIACIÓN DE SUELO RECIÉN CONTAMINADO E INTEMPERIZADO CON HIDROCARBUROS

Se evaluo la actividad deshidrogenasa y la dinamica de poblaciones bacterianas nativas en suelo contaminado con hidrocarburos durante su biorremediacion por bioestimulacion y atenuacion natural. Se realizaron ensayos a escala mesocosmos, en la que se compararon dos condiciones: 1) contaminacion reciente (28 dias) y 2) intemperizacion (entre 28 a 56 dias), con suelo artificialmente contaminado (30 000 mg de hidrocarburos totales de petroleo /kg suelo). Se estimo la riqueza de las comunidades bacterianas con base en los perfiles genomicos generados mediante electroforesis en gel con gradiente de desnaturalizantes (DGGE, por sus siglas en ingles). Ademas, se identificaron las poblaciones por secuenciacion de las bandas dominantes en los perfiles de DGGE. La evolucion de la biodegradacion se estimo a traves de la actividad deshidrogenasa durante los ensayos. El analisis molecular de los extractos de ADN, revelo una riqueza de genotipos similar en ambos tratamientos recien contaminados y una disminucion importante de este parametro por efecto de intemperismo. Los niveles maximos de actividad de la enzima se presentaron en el suelo recien contaminado-bioestimulado (3146.84 mg de iodo-nitro-formazan (INF)/g) en comparacion con el suelo recien contaminado bajo atenuacion natural (1160.19 μg INF/g). Para los suelos intemperizados, la actividad deshidrogenasa disminuyo en comparacion con los demas tratamientos. Se determino una comunidad bacteriana estable a lo largo del tiempo en el suelo intemperizado, mientras que la adicion de nutrientes favorecio el incremento de la riqueza de genotipos tanto en los suelos recien contaminados como en los intemperizados. Se identifico la presencia de bacterias hidrocarbonoclastas ( Burkholderia, Pelobacter ) en todos los tiempos de muestreo en ambos tratamientos.