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At least 559 records · Page 31

Complete disintegration of the microtubular cytoskeleton precedes its auxin-mediated reconstruction in postmitotic maize root cells

The inhibitory action of 0.1 microM auxin (IAA) on maize root growth was closely associated with a rapid and complete disintegration of the microtubular (MT) cytoskeleton, as visualized by indirect immunofluorescence of tubulin, throughout the growth region. After 30 min of this treatment, only fluorescent spots were present in root cells, accumulating either around nuclei or along cell walls. Six h later, in addition to some background fluorescence, dense but partially oriented oblique or longitudinal arrays of cortical MTs (CMTs) were found in most growing cells of the root apex. After 24 h of treatment, maize roots had adapted to the auxin, as inferred from the slowly recovering elongation rate and from the reassembly of a dense and well-ordered MT cytoskeleton which showed only slight deviations from that of the control root cells. Taxol pretreatment (100 microM, 24 h) prevented not only the rapid auxin-mediated disintegration of the MT cytoskeleton but also a reorientation of the CMT arrays, from transversal to longitudinal. The only tissue to show MTs in their cells throughout the auxin treatment was the epidermis. Significant resistance of transverse CMT arrays in these cells towards auxin was confirmed using a higher auxin concentration (100 microM, 24 h). The latter auxin dose also revealed inter-tissue-specific responses to auxin: outer cortical cell files reoriented their CMTs from the transversal to longitudinal orientation, whereas inner cortical cell files lost their MTs. This high auxin-mediated response, associated with the swelling of root apices, was abolished with the pretreatment of maize root with taxol.

Zea mays/cytology/drug effects/growth & developmen↗

The mechanics of plant morphogenesis

Understanding the mechanism by which patterned gene activity leads to mechanical deformation of cells and tissues to create complex forms is a major challenge for developmental biology. Plants offer advantages for addressing this problem because their cells do not migrate or rearrange during morphogenesis, which simplifies analysis. We synthesize results from experimental analysis and computational modeling to show how mechanical interactions between cellulose fibers translate through wall, cell, and tissue levels to generate complex plant tissue shapes. Genes can modify mechanical properties and stresses at each level, though the values and pattern of stresses differ from one level to the next. Here, the dynamic cellulose network provides elastic resistance to deformation while allowing growth through fiber sliding, which enables morphogenesis while maintaining mechanical strength.

59 BASIC BIOLOGICAL SCIENCES↗

High-rate/high-temperature capability of a single-layer zicar-separator nickel-hydrogen cell

A 50 Ampere-hour nickel-hydrogen cell with a single-layer Zircar separator stack design was fully charged and then discharged at a 2C current rate to an end voltage of 1 volt. This extreme test resulted in high temperatures which were recorded at three locations on the cell, i.e., the cell wall, the boss (barrel of the compression seal), and a terminal. The results provide new information about the high-temperature and high-discharge-rate capabilities of nickel-hydrogen cells. This information also adds to the growing data base for single-layer zirconium-oxide-cloth (Zircar) separator cell designs.

Wheeler, James R.↗

Gold-Nanocluster-Mediated Delivery of siRNA to Intact Plant Cells for Efficient Gene Knockdown

RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a "green" alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium -mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗

Phytochrome induces changes in the immunodetectable level of a wall peroxidase that precede growth changes in maize seedlings

The regulatory pigment phytochrome induces rapid and opposite growth changes in different regions of etiolated maize seedlings: it stimulates the elongation rate of coleoptiles and inhibits that of mesocotyls. As measured by a quantitative immunoassay, phytochrome also promotes rapid and opposite changes in the extractable content of a Mr 98,000 anionic isoperoxidase in the cell walls of these same organs: it induces a decrease of this peroxidase in coleoptiles and an increase in mesocotyls. The peroxidase changes precede the growth changes. As measured by video stereomicroscopy or a position transducer, red light (R), which photoactivates phytochrome, stimulates coleoptile elongation with a lag of about 15-20 min and suppresses mesocotyl growth with a lag of 45-50 min. R also induces a 50% reduction in the extractable level of the anionic peroxidase in coleoptile walls in less than 10 min and a 40% increase in the level of this peroxidase in mesocotyl walls within 30 min. Ascorbic acid, an inhibitor of peroxidase activity, blocks the effects of R on mesocotyl section growth. These results are relevant to hypotheses that postulate that certain wall peroxidases can participate in light-induced changes in growth rate by their effects on wall extensibility.

Non-NASA Center↗

Impact of Fatty-Acid Labeling of Bacillus subtilis Membranes on the Cellular Lipidome and Proteome

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.

59 BASIC BIOLOGICAL SCIENCES↗

A Structural and Molecular Approach for the Study Biomarkers

Investigation of the nucleation and growth of crystals in both abiotic and biotic systems is critical to seemingly diverse disciplines of geology, biology, environmental science, and astrobiology. While there are abundant studies devoted to the determination of the structure and composition of inorganic crystals, as well as to the development of thermodynamic and kinetic models, it is only recently that research efforts have been directed towards understanding mineralization in biological systems (i.e., biomineralization). Biomineralization refers to the processes by which living organisms form inorganic solids. Studies of the processes of biomineralization under low temperature aqueous conditions have focused primarily on magnetite forming bacteria and shell forming marine organisms. Many of the biological building materials consist of inorganic minerals (calcium carbonate, calcium phosphate, silica or iron oxide) intricately combined with organic polymers (like proteins). More recently, efforts have been undertaken to explore the nature of biological activities in ancient rocks. In the absence of well-preserved microorganisms or genetic material required for the polmerase chain reaction (PCR) method in molecular phylogenetic studies, using biominerals as biomarkers offers an alternative approach for the recognition of biogenic activity in both terrestrial and extraterrestrial environments. The primary driving force in biomineralization is the interaction between organic and inorganic phases. Thus, the investigation of the ultrastructure and the nature of reactions at the molecular level occurring at the interface between inorganic and organic phases is essential to understanding the processes leading to the nucleation and growth of crystals. It is recognized that crystal surfaces can serve as the substrate for the organization of organic molecules that lead to the formation of polymers and other complex organic molecules, and in discussions of the origins of life, is referred to as organic synthesis on mineral surfaces. Furthermore, it is suggested that the interaction between mineral surfaces and simple organic molecules resulted in the formation of amino acids, RNA, and perhaps other more complex molecules such as proteins. On the other hand, in natural systems, it is recognized that functional groups on cell walls or membranes of microorganisms serve as sites of nucleation and crystallization. The precise replication of biominerals with controlled structure, morphology, size and texture is not confined to higher organisms as it also occurs in primitive prokaryotic cells such as magnetotactic bacteria and cyanobacteria. This suggests that the principal strategies of biomineralization were established early on in the evolutionary history of organisms. It is critical, therefore, to search for common mechanisms within diverse biological systems. One such common factor is the capability for organization and self-assembly. Organic macromolecules such as proteins and lipids can aggregate and polymerize forming membranes or extracellular matrix. At the organic-inorganic interface, several factors such as lattice geometry, polarity, stereochemistry and topography may act in concert to control nucleation and growth of crystals. Although several models have been proposed that discuss the significance of these factors for biomineralization, no comprehensive experimental data are available. In contrast to crystallization in exclusively inorganic systems, the kinetics of reaction and structural relationships between organic and inorganic phases in biominerals or biomimetic material is poorly understood. For example, it is not clear if the concept of epitactic growth (geometrical matching of unit cells at the interface of a secondary crystal growing on a primary crystal) applies to organic-inorganic systems. In contrast to inorganic templates that often have a smooth and rigid surface that promotes epitactic growth, biological substrates are usually rough and result in a large degree of mismatch. It is apparent that factors controlling the reaction at the crystal-matrix interface are strongly dependent upon the nature of the substrate. Therefore, characterization of the assembled organic surface and surface structure of the inorganic phase is crucial to understanding the processes of biomineralization. The focus of our research is the investigation of the processes leading to the nucleation and growth of crystals on both natural and synthetic systems through an interdisciplinary approach that integrates molecular biology, morphology and mineralogy using advanced preparation and analytical techniques. We have studied run-products, particularly magnetite, siderite and other carbonates, that resulted from extracellular biomineralization by extremophiles isolated from a variety of extreme environments ranging from permafrost to hydrothermal vent systems. The results of this study are critical to recognizing biomarkers in terrestrial and extraterrestrial environments.

Thomas-Keprta, Kathie↗

A Micromechanics Finite Element Model for Studying the Mechanical Behavior of Spray-On Foam Insulation (SOFI)

A micromechanics model has been constructed to study the mechanical behavior of spray-on foam insulation (SOFI) for the external tank. The model was constructed using finite elements representing the fundamental repeating unit of the SOFI microstructure. The details of the micromechanics model were based on cell observations and measured average cell dimensions discerned from photomicrographs. The unit cell model is an elongated Kelvin model (fourteen-sided polyhedron with 8 hexagonal and six quadrilateral faces), which will pack to a 100% density. The cell faces and cell edges are modeled using three-dimensional 20-node brick elements. Only one-eighth of the cell is modeled due to symmetry. By exercising the model and correlating the results with the macro-mechanical foam behavior obtained through material characterization testing, the intrinsic stiffness and Poisson s Ratio of the polymeric cell walls and edges are determined as a function of temperature. The model is then exercised to study the unique and complex temperature-dependent mechanical behavior as well as the fracture initiation and propagation at the microscopic unit cell level.

Ghosn, Louis J.↗

Localized growth and remodelling drives spongy mesophyll morphogenesis

The spongy mesophyll is a complex, porous tissue found in plant leaves that enables carbon capture and provides mechanical stability. Unlike many other biological tissues, which remain confluent throughout development, the spongy mesophyll must develop from an initially confluent tissue into a tortuous network of cells with a large proportion of intercellular airspace. How the airspace in the spongy mesophyll develops while the tissue remains mechanically stable is unknown. Here, we use computer simulations of deformable polygons to develop a purely mechanical model for the development of the spongy mesophyll tissue. By stipulating that cell wall growth and remodelling occurs only near void space, our computational model is able to recapitulate spongy mesophyll development observed in Arabidopsis thaliana leaves. We find that robust generation of pore space in the spongy mesophyll requires a balance of cell growth, adhesion, stiffness and tissue pressure to ensure cell networks become porous yet maintain mechanical stability. The success of this mechanical model of morphogenesis suggests that simple physical principles can coordinate and drive the development of complex plant tissues like the spongy mesophyll.

Science & Technology - Other Topics↗

Point-implicit relaxation strategies for viscous, hypersonic flows

An upwind-biased, point-implicit relaxation algorithm for obtaining the numerical solution to the governing equations for 3D, viscous, hypersonic flows in chemical and thermal nonequilibrium is described. The algorithm is derived using a finite-volume formulation in which the inviscid components of flux across cell walls are described with a modified Roe's averaging and Harten's entropy fix with second-order corrections based on Yee's symmetric total variation diminishing scheme. Newton relaxation of the fully coupled equation set is employed on a cell-to-cell basis. Under-relaxation of the inviscid and over-relaxation of the viscous contributions to the residual are implemented. Computational work is easily partitioned among many processors in an asynchronous, dynamic mode for convergence acceleration. An overview of the physical models employed herein for thermochemical nonequilibrium is included. Several test cases and comparisons with experimental data are presented involving hypersonic flow over blunt bodies which illustrate the qualitative and quantitative capabilities of this approach.

Gnoffo, P. A.↗

Ionic signaling in plant responses to gravity and touch

Touch and gravity are two of the many stimuli that plants must integrate to generate an appropriate growth response. Due to the mechanical nature of both of these signals, shared signal transduction elements could well form the basis of the cross-talk between these two sensory systems. However, touch stimulation must elicit signaling events across the plasma membrane whereas gravity sensing is thought to represent transformation of an internal force, amyloplast sedimentation, to signal transduction events. In addition, factors such as turgor pressure and presence of the cell wall may also place unique constraints on these plant mechanosensory systems. Even so, the candidate signal transduction elements in both plant touch and gravity sensing, changes in Ca2+, pH and membrane potential, do mirror the known ionic basis of signaling in animal mechanosensory cells. Distinct spatial and temporal signatures of Ca2+ ions may encode information about the different mechanosignaling stimuli. Signals such as Ca2+ waves or action potentials may also rapidly transfer information perceived in one cell throughout a tissue or organ leading to the systemic reactions characteristic of plant touch and gravity responses. Longer-term growth responses are likely sustained via changes in gene expression and asymmetries in compounds such as inositol-1,4,5-triphosphate (IP3) and calmodulin. Thus, it seems likely that plant mechanoperception involves both spatial and temporal encoding of information at all levels, from the cell to the whole plant. Defining this patterning will be a critical step towards understanding how plants integrate information from multiple mechanical stimuli to an appropriate growth response.

Review↗

Rugged, Portable, Real-Time Optical Gaseous Analyzer for Hydrogen Fluoride

Hydrogen fluoride (HF) is a primary evolved combustion product of fluorinated and perfluorinated hydrocarbons. HF is produced during combustion by the presence of impurities and hydrogen- containing polymers including polyimides. This effect is especially dangerous in closed occupied volumes like spacecraft and submarines. In these systems, combinations of perfluorinated hydrocarbons and polyimides are used for insulating wiring. HF is both highly toxic and short-lived in closed environments due to its reactivity. The high reactivity also makes HF sampling problematic. An infrared optical sensor can detect promptly evolving HF with minimal sampling requirements, while providing both high sensitivity and high specificity. A rugged optical path length enhancement architecture enables both high HF sensitivity and rapid environmental sampling with minimal gaseous contact with the low-reactivity sensor surfaces. The inert optical sample cell, combined with infrared semiconductor lasers, is joined with an analog and digital electronic control architecture that allows for ruggedness and compactness. The combination provides both portability and battery operation on a simple camcorder battery for up to eight hours. Optical detection of gaseous HF is confounded by the need for rapid sampling with minimal contact between the sensor and the environmental sample. A sensor is required that must simultaneously provide the required sub-parts-permillion detection limits, but with the high specificity and selectivity expected of optical absorption techniques. It should also be rugged and compact for compatibility with operation onboard spacecraft and submarines. A new optical cell has been developed for which environmental sampling is accomplished by simply traversing the few mm-thick cell walls into an open volume where the measurement is made. A small, low-power fan or vacuum pump may be used to push or pull the gaseous sample into the sample volume for a response time of a few seconds. The optical cell simultaneously provides for an enhanced optical interaction path length between the environmental sample and the infrared laser. Further, the optical cell itself is comprised of inert materials that render it immune to attack by HF. In some cases, the sensor may be configured so that the optoelectronic devices themselves are protected and isolated from HF by the optical cell. The optical sample cell is combined with custom-developed analog and digital control electronics that provide rugged, compact operation on a platform that can run on a camcorder battery. The sensor is inert with respect to acidic gases like HF, while providing the required sensitivity, selectivity, and response time. Certain types of combustion events evolve copious amounts of HF, very little of other gases typically associated with combustion (e.g., carbon monoxide), and very low levels of aerosols and particulates (which confound traditional smoke detectors). The new sensor platform could warn occupants early enough to take the necessary countermeasures.

Pilgrim, Jeffrey↗

Xylella fastidiosa causes transcriptional shifts that precede tylose formation and starch depletion in xylem

Abstract Pierce's disease (PD) in grapevine ( Vitis vinifera ) is caused by the bacterial pathogen Xylella fastidiosa . X. fastidiosa is limited to the xylem tissue and following infection induces extensive plant‐derived xylem blockages, primarily in the form of tyloses. Tylose‐mediated vessel occlusions are a hallmark of PD, particularly in susceptible V. vinifera . We temporally monitored tylose development over the course of the disease to link symptom severity to the level of tylose occlusion and the presence/absence of the bacterial pathogen at fine‐scale resolution. The majority of vessels containing tyloses were devoid of bacterial cells, indicating that direct, localized perception of X. fastidiosa was not a primary cause of tylose formation. In addition, we used X‐ray computed microtomography and machine‐learning to determine that X. fastidiosa induces significant starch depletion in xylem ray parenchyma cells. This suggests that a signalling mechanism emanating from the vessels colonized by bacteria enables a systemic response to X. fastidiosa infection. To understand the transcriptional changes underlying these phenotypes, we integrated global transcriptomics into the phenotypes we tracked over the disease spectrum. Differential gene expression analysis revealed that considerable transcriptomic reprogramming occurred during early PD before symptom appearance. Specifically, we determined that many genes associated with tylose formation (ethylene signalling and cell wall biogenesis) and drought stress were up‐regulated during both Phase I and Phase II of PD. On the contrary, several genes related to photosynthesis and carbon fixation were down‐regulated during both phases. These responses correlate with significant starch depletion observed in ray cells and tylose synthesis in vessels.

59 BASIC BIOLOGICAL SCIENCES↗

Diploid-dominant life cycles characterize the early evolution of Fungi

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane– and cell-wall–associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

59 BASIC BIOLOGICAL SCIENCES↗

Mineralized Remains of Morphotypes of Filamentous Cyanobacteria in Carbonaceous Meteorites

The quest for conclusive evidence of microfossils in meteorites has been elusive. One difficulty arises from the fact than many abiotic or inorganic microstructures, mineral grains, and coating artifacts can mimic the smaller representatives of the microbial world that possess very simple morphologies (unicellular cocci or bacilli). However, there exist a wide variety of large, filamentous trichomic prokaryotic microorganisms (cyanobacteria and sulfur bacteria) with sufficiently well known and complex morphologies that they can be recognized and are known to be of unquestionable biogenic origin. The taphonomic modes of fossilization and their of their life habits and processes frequently result in distinctive chemical biosignatures associated with carbonization, silicification, calcification, phosphatization and metal-binding properties of their cell-walls, trichomes, sheaths and extracellular polymeric substances (EPS). Strong differences of mineral concentrations in closely associated and visibly differentiated cellular microstructures provide strong evidence of biogenicity. This evidence is further enhanced by the detection of recognizable and distinct microstructures (e.g., uniseriate or multiseriate filaments, trichomes, sheaths, cells of proper sizes and size distributions) and growth characteristics (e.g., basal or apical cells, true or false branching of trichomes, tapered or uniform filaments, robust or thin sheaths) and reproductive and nitrogen fixation habits (e.g., baeocytes, hormogonia, akinetes and heterocysts), Microfossils of cyanobacteria and cyanobacterial mats and stromatolites have been recognized a described from many of the most ancient rocks on Earth. The crucial problem lies in developing valid protocols and methodologies for establishing that the putative microfossils are truly indigenous and not merely recent microbial contaminants. During the past several years, we have conducted Field Emission Scanning Electron Microscopy (FESEM) investigations of freshly fractured interior surfaces of carbonaceous meteorites, terrestrial rocks, and recent microbial extremophiles and filamentous cyanobacteria. These studies have resulted in the detection in a several carbonaceous meteorites of the mineralized remains of a wide variety of complex filamentous trichomic microorganisms. These embedded forms are consistent in size and microstructure with well-preserved morphotypes of mat- forming filamentous trichomic cyanobacteria and the degraded remains of microfibrils of cyanobacterial sheaths. We present the results of comparative imaging studies and EDAX elemental analyses of recent cyanobacteria (e.g. Calothrix, Oscillatoria, and Lyngbya) that are similar in size, morphology and microstructure to morphotypes found embedded in meteorites. EDAX elemental studies reveal that forms found in carbonaceous meteorites often have highly carbonized sheaths in close association with permineralized filaments, trichomes and microbial cells. Ratios of critical bioelements (C:O, C:N, C:P, and C:S) reveal dramatic differences between microfossils in Earth rocks and meteorites and in filaments, trichomes, hormogonia, and cells of recent cyanobacteria.

Hoover, Richard B.↗

Quantum dots as strain- and metabolism-specific microbiological labels

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.

NASA Center JPL↗

Glycosyl transferase GT2 genes mediate the biosynthesis of an unusual (1,3;1,4)‐β‐glucan exopolysaccharide in the bacterium Sarcina ventriculi

Abstract Linear, unbranched (1,3;1,4)‐β‐glucans (mixed‐linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram‐positive bacteriumSarcina ventriculiare capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic ofSarcinaspecies. Heterologous expression of these two genes shows that they are capable of producing MLGsin planta, including an MLG that is chemically identical to the MLG secreted fromS. ventriculicells but which has regularly spaced (1,3)‐β‐linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designatedSvBmlgs1andSvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)‐β‐ and (1,4)‐β‐glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)‐β‐glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large‐scale production of MLG for medical, food and beverage applications.

Biochemistry & Molecular Biology↗

Tubulinlike protein from Spirochaeta bajacaliforniensis

Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures. Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes. These proteins have been the subject of intense biochemical and biophyiscal interest since the early 1970s and are of evolutionary interest as well. If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable. Tubulin proteins are widely thought to be limited to eukaryotes. Yet both azotobacters and spirochetes have shown immunological cross-reactivity with antitubulin antibodies. In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity. Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter. This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules. Spirochetes are helically shaped gram-negative motile prokaryotes. They differ from all other bacterial in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall. Some of the largest spirochetes have longitudinally aligned 240 angstrom microtubules. Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable. However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers displays antitubulin immunoreactivity in whole-cell preparations. Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken. Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold. After several cycles of sedimentation and redissolution the microtubule fraction is comprised of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs). In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation. Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

Non-NASA Center↗