Soil Bacterial Communities From the Chilean Andean Highlands: Taxonomic Composition and Culturability

The Atacama Desert is a highly complex, extreme ecosystem which harbors microorganisms remarkable for their biotechnological potential. Here, a soil bacterial prospection was carried out in the high Altiplano region of the Atacama Desert (>3,800 m above sea level; m a.s.l.), where direct anthropogenic interference is minimal. We studied: (1) soil bacterial community composition using high-throughput sequencing of the 16S rRNA gene and (2) bacterial culturability, by using a soil extract medium (SEM) under a factorial design of three factors: temperature (15 and 30°C), nutrient content (high and low nutrient disposal) and oxygen availability (presence and absence). A total of 4,775 OTUs were identified and a total of 101 isolates were selected for 16S rRNA sequencing, 82 of them corresponded to unique or non-redundant sequences. To expand our view of the Altiplano landscape and to obtain a better representation of its microbiome, we complemented our Operational Taxonomic Units (OTUs) and isolate collection with data from other previous data from our group and obtained a merged set of OTUs and isolates that we used to perform our study. Taxonomic comparisons between culturable microbiota and metabarcoding data showed an overrepresentation of the phylum Firmicutes (44% of isolates vs. 2% of OTUs) and an underrepresentation of Proteobacteria (8% of isolates vs. 36% of OTUs). Within the Next Generation Sequencing (NGS) results, 33% of the OTUs were unknown up to genus, revealing an important proportion of putative new species in this environment. Biochemical characterization and analysis extracted from the literature indicated that an important number of our isolates had biotechnological potential. Also, by comparing our results with similar studies on other deserts, the Altiplano highland was most similar to a cold arid desert. In summary, our study contributes to expand the knowledge of soil bacterial communities in the Atacama Desert and complements the pipeline to isolate selective bacteria that could represent new potential biotechnological resources

The Role of Fur in the Transcriptional and Iron Homeostatic Response of Enterococcus faecalis

The ferric uptake regulator (Fur) plays a major role in controlling the expression of iron homeostasis genes in bacterial organisms. In this work, we fully characterized the capacity of Fur to reconfigure the global transcriptional network and influence iron homeostasis in Enterococcus faecalis. The characterization of the Fur regulon from E. faecalis indicated that this protein (Fur) regulated the expression of genes involved in iron uptake systems, conferring to the system a high level of efficiency and specificity to respond under different iron exposure conditions. An RNAseq assay coupled with a systems biology approach allowed us to identify the first global transcriptional network activated by different iron treatments (excess and limited), with and without the presence of Fur. The results showed that changes in iron availability activated a complex network of transcriptional factors in E. faecalis, among them global regulators such as LysR, ArgR, GalRS, and local regulators, LexA and CopY, which were also stimulated by copper and zinc treatments. The deletion of Fur impacted the expression of genes encoding for ABC transporters, energy production and [Fe-S] proteins, which optimized detoxification and iron uptake under iron excess and limitation, respectively. Finally, considering the close relationship between iron homeostasis and pathogenesis, our data showed that the absence of Fur increased the internal concentration of iron in the bacterium and also affected its ability to produce biofilm. These results open new alternatives in the field of infection mechanisms of E. faecalis

Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)

<p>Abstract</p> <p>Background</p> <p>Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury.</p> <p>Results</p> <p>The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest.</p> <p>Conclusions</p> <p>Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest.</p

Testing the stress gradient hypothesis in soil bacterial communities associated with vegetation belts in the Andean Atacama Desert

17 Pág.Soil microorganisms are in constant interaction with plants, and these interactions shape the composition of soil bacterial communities by modifying their environment. However, little is known about the relationship between microorganisms and native plants present in extreme environments that are not affected by human intervention. Using high-throughput sequencing in combination with random forest and co-occurrence network analyses, we compared soil bacterial communities inhabiting the rhizosphere surrounding soil (RSS) and the corresponding bulk soil (BS) of 21 native plant species organized into three vegetation belts along the altitudinal gradient (2400-4500 m a.s.l.) of the Talabre-Lejía transect (TLT) in the slopes of the Andes in the Atacama Desert. We assessed how each plant community influenced the taxa, potential functions, and ecological interactions of the soil bacterial communities in this extreme natural ecosystem. We tested the ability of the stress gradient hypothesis, which predicts that positive species interactions become increasingly important as stressful conditions increase, to explain the interactions among members of TLT soil microbial communities.This study was funded by ANID FONDECYT Grants 1201278 to MG, 11200319 to DM, 3190194 to JM and 1211893 to VC, and ANID-MILENIO-CN2021-044. LAC was supported by ANID FB21006 and ACT210038. AG was supported by ANID Ph.D. Fellowship 21210808. Research was supported by the "Severo Ochoa Program for Centers of Excellence in R&D" from the Agencia Estatal de Investigación of Spain (Grant SEV-2016-0672 (2017-2021)) to the CBGP. BG-J was supported by a Postdoctoral contract associated to the Severo Ochoa Program. In addition, this research was partially supported by the supercomputing infrastructure of the NLHPC (ECM-02) (Powered@NLHPC).Peer reviewe

Identification of woolliness response genes in peach fruit after post-harvest treatments

Woolliness is a physiological disorder of peaches and nectarines that becomes apparent when fruit are ripened after prolonged periods of cold storage. This disorder is of commercial importance since shipping of peaches to distant markets and storage before selling require low temperature. However, knowledge about the molecular basis of peach woolliness is still incomplete. To address this issue, a nylon macroarray containing 847 non-redundant expressed sequence tags (ESTs) from a ripe peach fruit cDNA library was developed and used. Gene expression changes of peach fruit (Prunus persica cv. O'Henry) ripened for 7 d at 21 °C (juicy fruit) were compared with those of fruit stored for 15 d at 4 °C and then ripened for 7 d at 21 °C (woolly fruit). A total of 106 genes were found to be differentially expressed between juicy and woolly fruit. Data analysis indicated that the activity of most of these genes (>90%) was repressed in the woolly fruit. In cold-stored peaches (cv. O'Henry), the expression level of selected genes (cobra, endopolygalacturonase, cinnamoyl-CoA-reductase, and rab11) was lower than in the juicy fruit, and it remained low in woolly peaches after ripening, a pattern that was conserved in woolly fruit from two other commercial cultivars (cv. Flamekist and cv. Elegant Lady). In addition, the results of this study indicate that molecular changes during fruit woolliness involve changes in the expression of genes associated with cell wall metabolism and endomembrane trafficking. Overall, the results reported here provide an initial characterization of the transcriptome activity of peach fruit under different post-harvest treatments

Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indic

DMAP‐85: A τ‐Like Protein from Drosophila melanogaster Larvae

Abstract: Microtubule‐associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol‐polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85‐kDa protein versus tubulin in the polymers was 1:5.3 ± 0.2 mol/mol. It was shown that the 85‐kDa protein bound specifically to an affinity column of Sepharose‐βII‐(422–434) tubulin peptide, which contains the sequence of the MAP binding domain on βII‐tubulin. Affinity‐purified 85‐kDa protein enhanced microtubule assembly in a concentration‐dependent manner. This effect was signifi

The β-isoform of heat shock protein hsp-90 is structurally related with human microtubule-interacting protein Mip-90

Through major research advances in the study of cytoskeletal organization, an integrated view of the complexity of this system has emerged. Recent findings on the microtubule-interacting protein Mip-90, which associates with microtubules and actin filaments in different cell domains, have shed light on its roles in cytoskeletal regulation. In order to study structural features of Mip-90, we sequenced several peptide fragments. A comparative sequence analysis revealed a high degree of similarity between the primary structure of this protein and the human heat shock protein of 90 kDa (hsp-90). Taken together, the present studies indicate the identity between Mip-90 and the the β-isoform of hsp-90 (hsp-90β). Western blot assays with an anti-hsp-90 monoclonal antibody showed cross-reactivity of hsp-90 and Mip-90 affinity purified from HeLa cells. Furthermore, the observed structural identity of Mip-90 with the hsp-90β was sustained by immunoblot assays using monoclonal antibodies that spe

Cop-like operon: Structure and organization in species of the Lactobacillale order

Copper is an essential and toxic trace metal for bacteria and, therefore, must be tightly regulated in the cell. Enterococcus hirae is a broadly studied model for copper homeostasis. The intracellular copper levels in E. hirae are regulated by the cop operon, which is formed by four genes: copA and copB that encode ATPases for influx and efflux of copper, respectively; copZ that encodes a copper chaperone; and copY, a copper responsive repressor. Since the complete genome sequence for E. hirae is not available, it is possible that other genes may encode proteins involved in copper homeostasis. Here, we identified a cop-like operon in nine species of Lactobacillale order with a known genome sequence. All of them always encoded a CopY-like repressor and a copper ATPase. The alignment of the cop-like operon promoter region revealed two CopY binding sites, one of which was conserved in all strains, and the second was only present in species of Streptococcus genus and L. johnsonii. Additional proteins associated to copper metabolism, CutC and Cupredoxin, also were detected. This study allowed for the description of the structure and organization of the cop operon and discussion of a phylogenetic hypothesis based on the differences observed in this operon's organization and its regulation in Lactobacillale order