Influence of form IA RubisCO and environmental dissolved inorganic carbon on the delta13C of the clam-chemoautotroph symbiosis Solemya velum

Many nutritive symbioses between chemoautotrophic bacteria and invertebrates, such as Solemya velum, have delta(13)C values of approximately -30 to -35%, considerably more depleted than phytoplankton. Most of the chemoautotrophic symbionts fix carbon with a form IA ribulose 1,5-bisphosphate carboxylase (RubisCO). We hypothesized that this form of RubisCO discriminates against (13)CO(2) to a greater extent than other forms. Solemya velum symbiont RubisCO was cloned and expressed in Escherichia coli, purified and characterized. Enzyme from this recombinant system fixed carbon most rapidly at pH 7.5 and 20-25 degrees C. Surprisingly, this RubisCO had an epsilon-value (proportional to the degree to which the enzyme discriminates against (13)CO(2)) of 24.4 per thousand, similar to form IB RubisCOs, and higher than form II RubisCOs. Samples of interstitial water from S. velum's habitat were collected to determine whether the dissolved inorganic carbon (DIC) could contribute to the negative delta(13)C values. Solemya velum habitat DIC was present at high concentrations (up to approximately 5 mM) and isotopically depleted, with delta(13)C values as low as approximately -6%. Thus environmental DIC, coupled with a high degree of isotopic fractionation by symbiont RubisCO likely contribute to the isotopically depleted delta(13)C values of S. velum biomass, highlighting the necessity of considering factors at all levels (from environmental to enzymatic) in interpreting stable isotope ratios.     

Differential scanning calorimetric study of the binding of the primary water of hydration to nucleosides

Differential scanning calorimetry was used to study the water of hydration of all of the nucleosides at 59% relative humidity. Hydration was observed in pristine samples of guanosine, (rG), deoxyguanosine, (dG), and deoxyadenosine, (dA). Two inequivalent water sites were observed in dG and one site in rG. Re-hydration was observed in rG and dG, but not in dA. The activation energies for rG were 0.697 +/- 0.051 eV and 0.683 plus minus 0.090 eV for pristine and cycled samples respectively. dG had activation energies of 0.726 +/- 0.026 eV and 0.997 +/- 0.034 eV for the first and second peaks in the pristine samples while the activation energy for cycled dG was 0.671 +/- 0.133 eV.     

Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.     

Role of mass spectrometry in the purification of peptides and proteins

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.     

Bias in Template-to-Product Ratios in Multitemplate PCR

Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.     

Isotopic discrimination and kinetic parameters of RubisCO from the marine bloom‐forming diatom,Skeletonema costatum

The cosmopolitan, bloom‐forming diatom, Skeletonema costatum, is a prominent primary producer in coastal oceans, fixing CO₂ with ribulose 1,5‐bisphosphate carboxylase/oxygenase (RubisCO) that is phylogenetically distinct from terrestrial plant RubisCO. RubisCOs are subdivided into groups based on sequence similarity of their large subunits (IA–ID, II, and III). ID is present in several major oceanic primary producers, including diatoms such as S. costatum, coccolithophores, and some dinoflagellates, and differs substantially in amino acid sequence from the well‐studied IB enzymes present in most cyanobacteria and in green algae and plants. Despite this sequence divergence, and differences in isotopic discrimination apparent in other RubisCO enzymes, stable carbon isotope compositions of diatoms and other marine phytoplankton are generally interpreted assuming enzymatic isotopic discrimination similar to spinach RubisCO (IB). To interpret phytoplankton δ¹³C values, S. costatum RubisCO was characterized via sequence analysis, and measurement of its K_CO2 and V_max, and degree of isotopic discrimination. The sequence of this enzyme placed it among other diatom ID RubisCOs. Michaelis‐Menten parameters were similar to other ID enzymes (K_CO2 = 48.9 ± 2.8 μm ; V_max = 165.1 ± 6.3 nmol min^?1 mg^?1). However, isotopic discrimination (ε = [¹²k/¹³k ? 1] × 1000) was low (18.5‰; 17.0–19.9, 95% CI) when compared to IA and IB RubisCOs (22–29‰), though not as low as ID from coccolithophore, Emiliania huxleyi (11.1‰). Variability in ε‐values among RubisCOs from primary producers is likely reflected in δ¹³C values of oceanic biomass. Currently, δ¹³C variability is ascribed to physical or chemical factors (e.g. illumination, nutrient availability) and physiological responses to these factors (e.g. carbon‐concentrating mechanisms). Estimating the importance of these factors from δ¹³C measurements requires an accurate ε‐value, and a mass‐balance model using the ε‐value for S. costatum RubisCO is presented. Clearly, appropriate ε‐values must be included in interpreting δ¹³C values of environmental samples.     

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.     

Chemosynthetic endosymbioses: adaptations to oxic-anoxic interfaces

Chemosynthetic endosymbioses occur ubiquitously at oxic-anoxic interfaces in marine environments. In these mutualisms, bacteria living directly within the cell of a eukaryotic host oxidize reduced chemicals (sulfur or methane), fueling their own energetic and biosynthetic needs, in addition to those of their host. In habitats such as deep-sea hydrothermal vents, chemosynthetic symbioses dominate the biomass, contributing substantially to primary production. Although these symbionts have yet to be cultured, physiological, biochemical and molecular approaches have provided insights into symbiont genetics and metabolism, as well as into symbiont-host interactions, adaptations and ecology. Recent studies of endosymbiont biology are reviewed, with emphasis on a conceptual model of thioautotrophic metabolism and studies linking symbiont physiology with the geochemical environment. We also discuss current and future research directions, focusing on the use of genome analyses to reveal mechanisms that initiate and sustain the symbiont-host interaction.     

Mechanical properties of spinal nerve roots subjected to tension at different strain rates

An understanding of the biomechanical and physiological properties of spinal nerve roots, particularly in response to tension, is critical in understanding the pathomechanisms of pain and nerve root injury and subsequent management of related injuries. Biomechanical properties of dorsal nerve roots at the lumbar and sacral levels were evaluated at various strain rates. Nerve roots were stretched at two different rates, 0.01 mm/s (Group A, quasistatic) and 15 mm/s (Group B, dynamic). Load, displacement and digital video data were obtained as the nerve roots were stretched until failure. Maximum stress, strain at maximum stress and modulus of elasticity (E) were calculated from the load-displacement measurements. Comparison of mechanical properties and failure patterns of nerve roots at two different rates revealed significant differences. Maximum load, maximum stress and E values of 5.7+/-2.7 gm, 257.9+/-111.3 kPa and 1.3+/-0.8 MPa were observed for Group A and 13.9+/-7.5 gm, 624.9+/-306.8 kPa and 2.9+/-1.5 MPa were observed for Group B, respectively. Higher maximum load, maximum stress and E values occurred at the dynamic stretch rate as compared to the quasistatic stretch rate, illustrating the strain-rate dependency of spinal nerve roots. No differences were observed in the strain values. Differences in mechanical behavior of nerve roots were also observed among the four root levels (L4-S1). A significant interaction effect was observed between nerve root diameter and stretch rates. Overall, results from the present study demonstrate viscoelastic material properties of spinal nerve roots and provide better insight on the tensile properties of nerve roots at different strain rates.     

Off-axis symbiosis found: Characterization and biogeography of bacterial symbionts of Bathymodiolus mussels from Lost City hydrothermal vents

Organisms at hydrothermal vents inhabit discontinuous chemical 'islands' along mid-ocean ridges, a scenario that may promote genetic divergence among populations. The 2003 discovery of mussels at the Lost City Hydrothermal Field provided a means of evaluating factors that govern the biogeography of symbiotic bacteria in the deep sea. The unusual chemical composition of vent fluids, the remote location, and paucity of characteristic vent macrofauna at the site, raised the question of whether microbial symbioses existed at the extraordinary Lost City. If so, how did symbiotic bacteria therein relate to those hosted by invertebrates at the closest known hydrothermal vents along the Mid-Atlantic Ridge (MAR)? To answer these questions, we performed microscopic and molecular analyses on the bacteria found within the gill tissue of Bathymodiolus mussels (Mytilidae, Bathymodiolinae) that were discovered at the Lost City. Here we show that Lost City mussels harbour chemoautotrophic and methanotrophic endosymbionts simultaneously. Furthermore, populations of the chemoautotrophic symbionts from the Lost City and two sites along the MAR are genetically distinct from each other, which suggests spatial isolation of bacteria in the deep sea. These findings provide new insights into the processes that drive diversification of bacteria and evolution of symbioses at hydrothermal vents.