Pre-Columbian Floristic Legacies in Modern Homegardens of Central Amazonia

Historical ecologists have demonstrated legacy effects in apparently wild landscapes in Europe, North America, Mesoamerica, Amazonia, Africa and Oceania. People live and farm in archaeological sites today in many parts of the world, but nobody has looked for the legacies of past human occupations in the most dynamic areas in these sites: homegardens. Here we show that the useful flora of modern homegardens is partially a legacy of pre-Columbian occupations in Central Amazonia: the more complex the archaeological context, the more variable the floristic composition of useful native plants in homegardens cultivated there today. Species diversity was 10% higher in homegardens situated in multi-occupational archaeological contexts compared with homegardens situated in single-occupational ones. Species heterogeneity (β-diversity) among archaeological contexts was similar for the whole set of species, but markedly different when only native Amazonian species were included, suggesting the influence of pre-conquest indigenous occupations on current homegarden species composition. Our findings show that the legacy of pre-Columbian occupations is visible in the most dynamic of all agroecosystems, adding another dimension to the human footprint in the Amazonian landscape.     

Moderately Thermophilic Magnetotactic Bacteria from Hot Springs in Nevada

Populations of a moderately thermophilic magnetotactic bacterium were discovered in Great Boiling Springs, Nevada, ranging from 32 to 63°C. Cells were small, Gram-negative, vibrioid to helicoid in morphology, and biomineralized a chain of bullet-shaped magnetite magnetosomes. Phylogenetically, based on 16S rRNA gene sequencing, the organism belongs to the phylum Nitrospirae.Magnetotactic bacteria are a metabolically, morphologically, and phylogenetically heterogeneous group of prokaryotes that passively align and actively swim along magnetic field lines (3). This behavior, called magnetotaxis, is due to the presence of intracellular, membrane-bounded, single-magnetic-domain crystals of magnetite (Fe₃O₄) and/or greigite (Fe₃S₄) (3).Most known cultured magnetotactic bacteria are mesophilic and do not grow much above 30°C (e.g., Magnetospirillum species and Desulfovibrio magneticus strains MV-1 and MC-1 [D. A. Bazylinski, unpublished data]). Uncultured magnetotactic bacteria have been observed in numerous habitats that were mostly at 30°C and below. There is only one report describing thermophilic magnetotactic bacteria despite a number of efforts to look for them (e.g., in hydrothermal vents [D. A. Bazylinski, unpublished data]). Nash (12) reported the presence of thermophilic magnetotactic bacteria in microbial mats at about 45 to 55°C adjacent to the main flow in Little Hot Creek (but not in other springs in the same area at 40 to 80°C) and in microbial mats of other springs in central California at up to 58°C, all on the east side of the Sierra Nevada mountains. Cells biomineralized bullet-shaped crystals of magnetite and were phylogenetically affiliated with the phylum Nitrospirae (12). Few additional details were provided regarding the organisms and their habitat.In this study, water and surface sediment samples were taken from the Great Boiling Springs (GBS) geothermal field in Gerlach, NV. GBS is a series of hot springs that range from ambient temperature to ∼96°C (2, 5). The geology, chemistry, and microbial ecology of the springs have been described in some detail (2, 5). The pHs of the samples ranged from 6.4 to 7.5, while the salinities were about 4 to 5 ppt, as determined with a handheld Palm Abbe PA203 digital refractometer (MISCO Refractometer, Cleveland, OH). Samples were examined for the presence of magnetotactic bacteria using the hanging drop technique on-site and in the laboratory at room temperature with and without magnetic enrichment of the sample (15). Some samples taken back to the laboratory were kept at an elevated temperature (∼62°C), while others were kept at ambient temperature. There did not appear to be a significant difference in the number of magnetotactic cells in samples taken back to the laboratory and kept at these two temperatures. Only one morphotype of magnetotactic bacteria was found in samples from nine springs whose temperatures ranged from 32 to 63°C, and we estimate their numbers to be between 10³ to 10⁵ cells ml⁻¹ in surface sediments in sample bottles. We did not observe magnetotactic cells of this type in a large number of springs or pools that were at <32°C. Only one spring positive for the presence of these magnetotactic bacteria had sediment that was partially covered with a microbial mat, while sediment at most of the springs was dark gray in color. Cells were small (1.8 ± 0.4 by 0.4 ± 0.1 μm; n = 59), Gram negative, vibrioid to helicoid in morphology, and possessed a single polar flagellum (Fig. ​(Fig.1A).1A). Magnetotactic bacteria were not observed in springs that were at 67°C and above, suggesting the maximum survival and perhaps growth temperature for the organism is about 63°C. In the lab, cells remained viable and motile in samples kept at 25 to 62°C for several months. We refer to this organism as strain HSMV-1.Open in a separate windowFIG. 1.Transmission electron microscope (TEM) images of cells and magnetosomes of strain HSMV-1. (A) TEM image of unstained cell of HSMV-1 showing a single polar flagellum and a single chain of bullet-shaped magnetosomes. The electron-dense structures at the poles were found to be phosphorus-rich based on energy-dispersive X-ray analysis (data not shown) and therefore likely represent polyphosphate granules. (B) Higher-magnification TEM image of the magnetosome chain. (C) High-magnification TEM image of magnetosomes from which a selected area electron diffraction (SAED) pattern was obtained (inset of B). The SAED pattern corresponds to the [1 0−1] zone of magnetite, Fe₃O₄: reflection o, (0 0 0); reflection a, (1 −1 1) (0.48 nm); reflection b, (1 1 1) (0.48 nm); reflection c, (2 0 2) (0.30 nm); angle a-o-b, 70.5°. (D) Iron, sulfur, and oxygen elemental maps, derived from energy-filtering transmission electron microscopy (EFTEM), showing that the positions of the magnetosome crystals correlate with increased concentrations of Fe and O, but not S, consistent with the iron oxide magnetite (Fe₃O₄).Cells of HSMV-1 biomineralized a single chain of magnetosomes that traversed the cells along their long axis (Fig. 1A to C). Selected area electron diffraction (SAED) and energy-filtering transmission electron microscopy (EFTEM) elemental maps were determined on magnetosome crystals using a Tecnai model G2 F30 Super-Twin transmission electron microscope (FEI Company, Hillsboro OR). SAED patterns of HSMV-1 magnetosome crystals (Fig. ​(Fig.1B,1B, inset) indicated that they consisted of magnetite, while EFTEM elemental maps (Fe, O and S) (Fig. ​(Fig.1D)1D) clearly showed that the crystals consisted of an iron oxide and not an iron sulfide, again consistent with the mineral magnetite. Cells contained an average of 12 ± 6 magnetosome crystals per cell (n = 15 cells) that averaged 113 ± 34 by 40 ± 5 nm in size (n = 179). A plot of the length of the crystals as a function of the shape factor (width/length ratio) is provided in Figure S1 in the supplemental material and shows that the crystals fit in the theoretical single-magnetic-domain size range (4), along with all known mature magnetosome magnetite crystals from magnetotactic bacteria (3).Whole-cell PCR amplification of the 16S rRNA gene was performed by first magnetically purifying cells of HSMV-1 using the “capillary racetrack” described by Wolfe et al. (18). Purity of the collected cells was determined by microscopic examination, and contaminating cells were never observed. The 16S rRNA gene was amplified using bacteria-specific primers 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGHTACCTTGTTACGACTT-3′ (11). PCR products were cloned into pGEM-T Easy vector (Promega Corporation, Madison, WI) and sequenced (Functional Biosciences, Inc., Madison, WI). Six of eight clones sequenced had identical inserts.Alignment of 16S rRNA gene sequences was performed using the CLUSTAL W multiple alignment accessory application in the BioEdit sequence alignment editor (7). Phylogenetic trees were constructed using MEGA version 4 (17) by applying the neighbor-joining method (14). Bootstrap values were calculated with 1,000 replicates. The 16S rRNA gene sequence of strain HSMV-1 places the organism in the phylum Nitrospirae (Fig. ​(Fig.2),2), with its closest relative in culture being Thermodesulfovibrio hydrogeniphilus (87.2% identity) (8). Two other uncultured magnetotactic bacteria are phylogenetically affiliated with the phylum Nitrospirae, including the unnamed rod-shaped bacterium strain MHB-1 (86.5% identity) (6) and the very large Candidatus Magnetobacterium bavaricum (86.4% identity) (16). Interestingly, all the magnetotactic bacteria associated with the phylum Nitrospirae thus far (e.g., Candidatus Magnetobacterium bavaricum) contain bullet-shaped magnetite crystals in their magnetosomes.Open in a separate windowFIG. 2.Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain HSMV-1 in the phylum Nitrospirae. Bootstrap values at nodes are percentages of 1,000 replicates. The magnetotactic bacteria Desulfovibrio magneticus and Candidatus Magnetoglobus multicellularis (outgroup; deltaproteobacteria) were used to root the tree. GenBank accession numbers are given in parentheses. Bar represents 2% sequence divergence.Fluorescent in situ hybridization (FISH) was used to authenticate the 16S rRNA gene sequence. A specific Alexa594-labeled probe for HSMV-1 was designed (HSMVp, 5′-CCTTCGCCACAGGCCTTCTA-3′, complementary to nucleotides 690 to 709 of the 16S rRNA molecule) based on the alignment of 10 of the most similar 16S rRNA gene sequences found in GenBank after BLAST analysis (1) and on cultivated members of the phylum Nitrospirae. FISH with the Alexa594-labeled probe was carried out after fixation of magnetically concentrated cells directly on the wells of gelatin-coated hydrophobic microscope slides with 4% paraformaldehyde. FISH was performed according to the work of Pernthaler et al. (13). The hybridization solution contained 10 ng/ml of the probe, 20% formamide, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM Na₂EDTA, and 0.01% sodium dodecyl sulfate (SDS). Cells of HSMV-1 hybridized to the HSMVp probe, while other cells in the sample did not (Fig. ​(Fig.3),3), indicating that the 16S rRNA gene sequence we obtained is from the magnetotactic bacterium under study. Strain HSMV-1 clearly represents a new genus (Fig. ​(Fig.2),2), and based on the phylogeny and what we currently know phenotypically about strain HSMV-1, we propose the name Candidatus Thermomagnetovibrio paiutensis (the GBS site was originally occupied by the Paiute Indian Tribe).Open in a separate windowFIG. 3.Fluorescent in situ hybridization (FISH) of cells of strain HSMV-1 using an HSMV-1-specific oligonucleotide rRNA probe (HSMVp). Cells used for FISH were magnetically concentrated by placing a magnet next to the side of the sample bottle for 30 min and then removed with a Pasteur pipette. This technique was used rather than the magnetic racetrack method in order to have many HSMV-1 cells as well as some other cells that could be used as a negative control. (A) Differential interference contrast (DIC) image of HSMV-1 cells (filled arrows) and other cells (negative control; empty arrows) from hot spring samples; (B) cells stained with 4′,6-diamidino-2-phenylindole (DAPI); (C) cells hybridized with the specific probe HSMVp.Nash (12) first reported thermophilic magnetotactic bacteria phylogenetically affiliated with the Nitrospirae phylum in hot springs, and it would be interesting and important to compare these organisms and their habitats. However, little can be compared at this time due to lack of information. Nash (12) reported that the one spring at Little Hot Creek was freshwater and that microbial mats were present at all springs where thermophilic magnetotactic bacteria were found. The water at our sampling sites was brackish, not freshwater, and microbial mats were not an important feature of our springs. Thus, it is difficult to determine without knowing the relationship between the organisms found by Nash (12) and strain HSMV-1 what environmental parameters are important to the growth and survival of these bacteria.It is also difficult to determine the temperature ranges for the survival and growth for strain HSMV-1 without having a pure culture. Data presented here suggest that the temperature range for both is quite wide, and this would be important for the continued presence of HSMV-1 at GBS, as temperatures in the hot springs are known to fluctuate greatly (2). Even if the maximum growth temperature of HSMV-1 is slightly lower than the maximum survival temperature (a conservative estimate) that we know of (63°C), it would still be considered a moderately thermophilic bacterium.The results presented here clearly show that some magnetotactic bacteria can be considered at least moderately thermophilic. They extend the upper temperature limit for environments where magnetotactic bacteria exist and likely grow (∼63°C) and where magnetosome magnetite is deposited, a finding that may prove significant in the study and interpretation of magnetofossils (9, 10).     

Computer simulation of the rough lipopolysaccharide membrane of Pseudomonas aeruginosa.

Lipopolysaccharides (LPSs) form the major constituent of the outer membrane of Gram-negative bacteria, and are believed to play a key role in processes that govern microbial metal binding, microbial adsorption to mineral surfaces, and microbe-mediated oxidation/reduction reactions at the bacterial exterior surface. A computational modeling capability is being developed for the study of geochemical reactions at the outer bacterial envelope of Gram-negative bacteria. A molecular model for the rough LPS of Pseudomonas aeruginosa has been designed based on experimentally determined structural information. An electrostatic model was developed based on Hartree-Fock SCF calculations of the complete LPS molecule to obtain partial atomic charges. The exterior of the bacterial membrane was assembled by replication of a single LPS molecule and a single phospholipid molecule. Molecular dynamics simulations of the rough LPS membrane of P. aeruginosa were carried out and trajectories were analyzed for the energetic and structural factors that determine the role of LPS in processes at the cell surface.     

Study of Thermomyces lanuginosa Lipase in the Presence of Tributyrylglycerol and Water

The Thermomyces lanuginosa lipase has been extensively studied in industrial and biotechnological research because of its potential for triacylglycerol transformation. This protein is known to catalyze both hydrolysis at high water contents and transesterification in quasi-anhydrous conditions. Here, we investigated the Thermomyces lanuginosa lipase structure in solution in the presence of a tributyrin aggregate using 30 ns molecular-dynamics simulations. The water content of the active-site groove was modified between the runs to focus on the protein-water molecule interactions and their implications for protein structure and protein-lipid interactions. The simulations confirmed the high plasticity of the lid fragment and showed that lipid molecules also bind to a secondary pocket beside the lid. Together, these results strongly suggest that the lid plays a role in the anchoring of the protein to the aggregate. The simulations also revealed the existence of a polar channel that connects the active-site groove to the outside solvent. At the inner extremity of this channel, a tyrosine makes hydrogen bonds with residues interacting with the catalytic triad. This system could function as a pipe (polar channel) controlled by a valve (the tyrosine) that could regulate the water content of the active site.     

Signal 3 tolerant CD8 T cells degranulate in response to antigen but lack granzyme B to mediate cytolysis

Naive CD8 T cells that respond in vivo to Ag and costimulation in the absence of a third signal, such as IL-12, fail to develop cytolytic function and become tolerized. We show in this study that CD8 T cells purified from TCR transgenic mice and stimulated in vitro in the presence or absence of IL-12 form conjugates with specific target cells, increase intracellular Ca2+, and undergo degranulation to comparable extents. Perforin is also expressed at comparable levels in the absence or presence of a third signal, but expression of granzyme B depends upon IL-12. Levels of granzyme B also correlate strongly with the cytolytic activity of cells responding in vivo. In contrast, an increase in CD107a (lysosomal-associated membrane protein 1) expression resulting from degranulation cannot distinguish in vivo generated lytic effector cells from tolerized, noncytolytic cells. Thus, it appears that cells rendered tolerant as a result of stimulation in the absence of a third signal fail to lyse target cells because they are "shooting blanks."     

Magnetosome chain arrangement and stability in magnetotactic cocci

We have studied the disposition of chains of magnetosomes inside magnetotactic cocci with light and electron microscopy. Light microscopy of isolated cocci indicated that the chains of magnetosomes are disposed on opposite sides of the cell. Electron spectroscopic imaging of whole unprocessed bacteria, showed the magnetosome chains in the cells. Freeze-etching of the cell surface allowed the observation of the close association of the chain with the cell surface. During the replication process of the freeze-etching, the magnetosome chains remained attached to the replicas, which indicates that chains were very close to the cell surface before freezing. We provide evidence that the large area of the contact faces between magnetosomes in a chain may provide an extra mechanical stability that helps keep the magnetosomes in chains even after isolation from the bacteria. Comparison with pointed magnetosomes from different cocci present in the same samples showed that the maintenance of linear chains is more difficult to be achieved because of the geometry of the crystals.     

Protein under pressure: molecular dynamics simulation of the arc repressor

Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogen-bonding pattern of the intermolecular beta-sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the beta-sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.     

Polysaccharide isolated from Passiflora edulis: Characterization and antitumor properties

A hot water-extracted polysaccharide fraction (PFCM) of Passiflora edulis was characterized by microanalysis, infrared spectroscopy, NMR and high performance size-exclusion chromatography. The major component in PFCM is (1 → 4) linked galacturonic acid (esterified and unesterified). Neutral sugars such as arabinose, glucose, rhamnose, mannose, and fucose were also present. Traces of xylose and ribose were detected. The PFCM sample had a similar molar mass to that of pectin extracted from P. edulis under acidic conditions. Sarcoma 180 tumors treated with PFCM showed a growth inhibition ratio ranging from 40.59% to 48.73% depending on the dosage and type of PFCM administration (oral or intraperitoneal). Toxicological analysis shows that PFCM increases the cell types involved in primary defense mechanisms and no significant changes in the biochemical parameters and organs (e.g., kidney and liver) were observed. However, the use of PFCM treatment increased the spleen weight when compared with the use of 5-fluorouracil.     

Effects of different nitrogen sources on the biogas production - a lab-scale investigation

For anaerobic digestion processes nitrogen sources are poorly investigated although they are known as possible process limiting factors (in the hydrolysis phase) but also as a source for fermentations for subsequent methane production by methanogenic archaea. In the present study different complex and defined nitrogen sources were investigated in a lab-scale experiment in order to study their potential to build up methane. The outcome of the study can be summarised as follows: from complex nitrogen sources yeast extract and casamino acids showed the highest methane production with approximately 600ml methane per mole of nitrogen, whereas by the use of skim milk no methane production could be observed. From defined nitrogen sources l-arginine showed the highest methane production with almost 1400ml methane per mole of nitrogen. Moreover it could be demonstrated that the carbon content and therefore C/N-ratio has only minor influence for the methane production from the used substrates.     

Fixed Differences in the paralytic Gene Define Two Lineages within the Lutzomyia longipalpis Complex Producing Different Types of Courtship Songs

The sand fly Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), the most important vector of American visceral leishmaniasis, is widely distributed in Latin America. There is currently a consensus that it represents a species complex, however, the number and distribution of the different siblings is still uncertain. Previous analyses have indicated that Brazilian populations of this vector can be divided into two main groups according to the type of courtship song (Burst vs. Pulse) males produce during copulation. Nevertheless, no diagnostic differences have been observed between these two groups with most molecular markers used to date. We analyzed the molecular divergence in a fragment of the paralytic (para) gene, a locus involved in the control of courtship songs in Drosophila, among a number of Lu. longipalpis populations from Brazil producing Burst and Pulse-type songs. Our results revealed a very high level of divergence and fixed differences between populations producing the two types of songs. We also compared Lu. longipalpis with a very closely related species, Lutzomyia cruzi, which produces Burst-type songs. The results indicated a higher number of fixed differences between Lu. cruzi and the Pulse-type populations of Lu. longipalpis than with those producing Burst-type songs. The data confirmed our previous assumptions that the presence of different sibling species of the Lu. longipalpis complex in Brazil can be divided into two main groups, one representing a single species and a second more heterogeneous group that probably represents a number of incipient species. We hypothesize that para might be one of the genes directly involved in the control of the courtship song differences between these two groups or that it is linked to other loci associated with reproductive isolation of the Brazilian species.