Looking inside the box: using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time

Stoichiometry of microbial biomass is a key determinant of nutrient recycling in a wide variety of ecosystems. However, little is known about the underlying causes of variance in microbial biomass stoichiometry. This is primarily because of technological constraints limiting the analysis of macromolecular composition to large quantities of microbial biomass. Here, we use Raman microspectroscopy (MS), to analyze the macromolecular composition of single cells of two species of bacteria grown on minimal media over a wide range of resource stoichiometry. We show that macromolecular composition, determined from a subset of identified peaks within the Raman spectra, was consistent with macromolecular composition determined using traditional analytical methods. In addition, macromolecular composition determined by Raman MS correlated with total biomass stoichiometry, indicating that analysis with Raman MS included a large proportion of a cell''s total macromolecular composition. Growth phase (logarithmic or stationary), resource stoichiometry and species identity each influenced each organism''s macromolecular composition and thus biomass stoichiometry. Interestingly, the least variable peaks in the Raman spectra were those responsible for differentiation between species, suggesting a phylogenetically specific cellular architecture. As Raman MS has been previously shown to be applicable to cells sampled directly from complex environments, our results suggest Raman MS is an extremely useful application for evaluating the biomass stoichiometry of environmental microorganisms. This includes the ability to partition microbial biomass into its constituent macromolecules and increase our understanding of how microorganisms in the environment respond to resource heterogeneity.     

Rate of follicle growth,change in follicle volume and stages of macromolecular synthesis during ovarian development in Musca domestica

At 21°C the first egg generation takes 6 days to develop. During this period the oöcyte volume increases by a factor of ×5000, and compared with an oögonium the growth factor amounts to ×100,000. The growth rate of the oöcyte increases with each stage of oögenesis, until at stage 5 it reaches 2.5 × 10⁶ μm³/hr. About 35% of the substance stored in the oöcyte originates from the nurse chamber. 30% of the volume is formed in the stage where the oöcyte synthesizes almost exclusively glycogen. Accordingly, about 30% of the volume is provided by the euplasmatic protein synthesis and by the yolk uptake.     

Packaging double-helical DNA into viral capsids

DNA packaging in bacteriophage P4 has been examined using a molecular mechanics model with a reduced representation containing one pseudoatom per turn of the double helix. The model is a discretized version of an elastic continuum model. The DNA is inserted piecewise into the model capsid, with the structure being reoptimized after each piece is inserted. Various optimization protocols were investigated, and it was found that molecular dynamics at a very low temperature (0.3 K) produces the optimal packaged structure. This structure is a concentric spool, rather than the coaxial spool that has been commonly accepted for so many years. This geometry, which was originally suggested by Hall and Schellman in 1982 (Biopolymers Vol. 21, pp. 2011-2031), produces a lower overall elastic energy than coaxial spooling.     

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis^1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)⁴ and secretory diarrhea⁵. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States⁶. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut⁷.Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo^8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP^20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)²⁰. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities²². This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells^16-18.Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay^16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR^16-19,28,29.     

The phloem, a miracle of ingenuity

This review deals with aspects of the cellular and molecular biology of the sieve element/companion cell complex, the functional unit of sieve tubes in angiosperms. It includes the following issues: (a) evolution of the sieve elements; (b) the specific structural outfit of sieve elements and its functional significance; (c) modes of cellular and molecular interaction between sieve element and companion cell; (d) plasmodesmal trafficking between sieve element and companion cell as the basis for macromolecular long‐distance signalling in the phloem; (e) diversity of sieve element/companion cell complexes in the respective phloem zones (collection phloem, transport phloem, release phloem); (f) deployment of carriers, pumps and channels on the plasma membrane of sieve element/companion cell complexes in various phloem zones; and (g) implications of the molecular‐cellular equipment of sieve element/companion cells complexes for mass flow of water and solutes in a whole‐plant frame.     

Structural studies of the retroviral proteinase from avian myeloblastosis associated virus.

The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has been determined and refined at 2.2 A resolution. This structure is compared with those of homologous proteinases from Rous sarcoma virus (RSV) and human immunodeficiency type 1 virus (HIV). Through comparison with the structure of a proteinase-inhibitor complex from HIV, a model of a complex between MAV proteinase and a peptide substrate has been generated. Examination of this model suggests structural basis for the diverse specifications of viral proteinases.     

Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs

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Macromolecular docking of a three-body system: the recognition of human growth hormone by its receptor.

Human growth hormone (hGH) binds to its receptor (hGHr) in a three-body interaction: one molecule of the hormone and two identical monomers of the receptor form a trimer. Curiously, the hormone-receptor interactions in the trimer are not equivalent and the formation of the complex occurs in a specific kinetic order (Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA, 1991, Science 254:821-825). In this paper, we model the recognition of hGH to the hGHr using shape complementarity of the three-dimensional structures and macromolecular docking to explore possible binding modes between the receptor and hormone. The method, reported previously (Hendrix DK, Kuntz ID, 1998, Pacific symposium on biocomputing 1998, pp 1234-1244), is based upon matching complementary-shaped strategic sites on the molecular surface. We modify the procedure to examine three-body systems. We find that the order of binding seen experimentally is also essential to our model. We explore the use of mutational data available for hGH to guide our model. In addition to docking hGH to the hGHr, we further test our methodology by successfully reproducing 16 macromolecular complexes from X-ray crystal structures, including enzyme-inhibitor, antibody-antigen, protein dimer, and protein-DNA complexes.     

Singular value decomposition as a tool for background corrections in time-resolved XFEL scattering data

The development of new X-ray light sources, XFELs, with unprecedented time and brilliance characteristics has led to the availability of very large datasets with high time resolution and superior signal strength. The chaotic nature of the emission processes in such sources as well as entirely novel detector demands has also led to significant challenges in terms of data analysis. This paper describes a heuristic approach to datasets where spurious background contributions of a magnitude similar to (or larger) than the signal of interest prevents conventional analysis approaches. The method relies on singular-value decomposition of no-signal subsets of acquired datasets in combination with model inputs and appears generally applicable to time-resolved X-ray diffuse scattering experiments.     

Impact of irradiance on the C allocation in the coastal marine diatom Skeletonema marinoi Sarno and Zingone

Elemental stoichiometry and organic composition were investigated in an Adriatic strain of Skeletonema marinoi, cultured at 25 [low light (LL)] and 250 [high light (HL)]µmol photon m^?2 s^?1. Inorganic carbon acquisition, fixation and allocation, and silicic acid and orthophosphate uptake were also studied. The C : P ratio was below the Redfield ratio, especially at LL. In HL cells, N quota was halved, C quota was similar, silica quota was lower, growth rate and long‐term net primary productivity were almost doubled, relative to LL cells. The HL : LL cell quota ratios were 6 for lipid, 0.5 for protein and 0.4 for carbohydrate. Phosphoenolpyruvate carboxylase (PEPc) and glutamine synthetase (GS) activities were unaffected by the growth irradiance; phosphoenolpyruvate carboxykinase (PEPck) was 2.5‐fold more active in LL cells. This suggests that in S. marinoi, C₄ photosynthesis is unlikely, PEPc is anaplerotic and PEPck may be involved in the conversion of lipid C to carbohydrates, especially in LL cells. Because about 50% of the cost for the production of an HL cell is caused by lipid biosynthesis, we propose that the preferential allocation of C to lipid at HL takes advantage of the relatively high volume‐based energy content of lipids, in an organism that reduces its size at each vegetative cell division.