Chemical analysis of the surface of microorganisms by X-ray photoelectron spectroscopy

Abstract A collection of microorganisms, including a microfungus and various yeasts and bacteria has been analyzed by X-ray photoelectron spectroscopy (XPS). A correlation is observed between the N/P atomic concentration ratio of the cell surface and the cell electrophoretic mobility measured at pH 4, indicating that the dehydrated surface analyzed by XPS is representative of the cell surface in contact with water.
Deprotonation of phosphate groups plays a predominant role in the development of the cell negative charge, and carboxylic groups are not involved appreciably; a partial neutralization is allowed by protonation of free amino groups of proteins.
These results advocate a broader use of XPS in order to understand physicochemical properties (electrostatic charge, hydrophobicity, ion binding) of the surface of cells, which are of prime importance in various processes occurring in nature and technology.     

微生物大片段DNA染色体整合技术研究进展

现代生物发酵工业聚焦于设计和创制高效的微生物细胞工厂,以实现原料向目标产品的定向转化。评判微生物细胞工厂性能优劣的主要标准是其合成能力及稳定性。由于质粒系统存在拷贝数不稳定、易于丢失等局限性,在菌株改造中将基因或产物合成途径整合至染色体上实现稳定表达通常是更优的选择。因此,染色体的基因整合技术作为实现这一目标的重要手段已受到广泛关注,并得到快速发展。本综述梳理了近年来微生物染色体上的大片段DNA整合方法的研究进展,归纳了各种技术的原理和特点,尤其是新兴的CRISPR相关转座系统,同时对未来的发展重点和方向进行了展望。     

Preliminary X-ray diffraction analysis of crystals of turnip yellow mosaic virus (TYMV)

Turnip yellow mosaic virus (TYMV) was purified from Chinese cabbage and crystallized in a form that permits high resolution structural analysis using X-ray diffraction. The crystals have a hexagonal bipyramidal morphology and often achieve dimensions of 1.0 × 1.0 × 0.5 mm. The crystals appear to be of hexagonal space group P6₂22 with a = b = 525 Å, c=315 Å, but we cannot strictly rule out the possibility that the space group is P622. They appear different than any crystals of TYMV previously reported. There are three T = 3 virus particles in the unit cell, which implies that one quarter of the particle, or 45 protein subunits, comprises the asymmetric unit of the crystal. Native data have been collected using synchrotron radiation to a resolution of 3.2 Å. © 1995 Wiley-Liss, Inc.     

AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells

The mechanical stiffness of individual cells is important in tissue homeostasis, cell growth, division and motility, and the epithelial-mesenchymal transition in the initiation of cancer. In this work, a normal squamous cell line (EPC2) and metaplastic (CP-A) as well as dysplastic (CP-D) Barrett's Esophagus columnar cell lines are studied as a model of pre-neoplastic progression in the human esophagus. We used the combination of an atomic force microscope (AFM) with a scanning confocal fluorescence lifetime imaging microscope to study the mechanical properties of single adherent cells. Sixty four force indentation curves were taken over the nucleus of each cell in an 8 x 8 grid pattern. Analyzing the force indentation curves, indentation depth-dependent Young's moduli were found for all cell lines. Stiffness tomograms demonstrate distinct differences between the mechanical properties of the studied cell lines. Comparing the stiffness for indentation forces of 1 nN, most probable Young's moduli were calculated to 4.7 kPa for EPC2 (n = 18 cells), 3.1 kPa for CP-A (n = 10) and 2.6 kPa for CP-D (n = 19). We also tested the influence of nuclei and nucleoli staining organic dyes on the mechanical properties of the cells. For stained EPC2 cells (n = 5), significant stiffening was found (9.9 kPa), while CP-A cells (n = 5) showed no clear trend (2.9 kPa) and a slight softening was observed (2.1 kPa) in the case of CP-D cells (n = 16). Some force-indentation curves show non-monotonic discontinuities with segments of negative slope, resembling a sawtooth pattern. We found the incidence of these 'breakthrough events' to be highest in the dysplastic CP-D cells, intermediate in the metaplastic CP-A cells and lowest in the normal EPC2 cells. This observation suggests that the microscopic explanation for the increased compliance of cancerous and pre-cancerous cells may lie in their susceptibility to 'crumble and yield' rather than their ability to 'bend and flex'.     

Preliminary X-ray fiber diffraction studies of cucumber green mottle mosaic virus, watermelon strain

Fiber diffraction patterns have been obtained for cucumber green mottle mosaic virus, watermelon strain (a distant relative of tobacco mosaic virus), and two heavy-atom derivatives. These patterns and the similarity between the cucumber and the tobacco virus offer the potential of a full structure determination of the cucumber virus.     

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5°C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5°C on a series of solutions prepared by low-temperature quenching after incubation at 20°C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.     

大型浅水湖泊鄱阳湖湿地微生物的研究现状

鄱阳湖是我国第一大淡水湖泊,同时也是一个典型的季节性通江浅水湖泊,独特的水文特征和多样的湿地景观类型形成了复杂多样的微生物群落。本文综述了鄱阳湖水文节律、营养盐及重金属含量对湖泊微生物群落组成的影响,以及水位高程和湿地围垦对鄱阳湖湿地土壤微生物分布特征的影响,同时还探讨了未来湖泊湿地微生物的研究方向和鄱阳湖所拥有的独特研究条件,以期为未来湖泊微生物研究提供重要参考。     

Solution X-ray scattering study of reconstitution process of tobacco mosaic virus particle using low-temperature quenching

The reconstitution process of tobacco mosaic virus (TMV) was investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. TMV assembly in an aqueous solution is completely stopped below 5 degrees C. The TMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation either at 15, 20 or 25 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. In order to account the time course of SAXS profiles in terms of the elongation of TMV assembly, a model calculation was performed to simulate the Guinier plot, the Kratky plot and the distance distribution function by applying Glatter's multibody method using models that were constituted of the spheres representing a column of piled two-layer disks of TMV-protein. The three simulated functions thus obtained support the conclusion derived from the three functions calculated from the experimental results that the incubation of the RNA and protein of TMV began to reconstitute TMV instantly after mixing, proceeded steeply to a long rod, and then extended asymptotic to the full length of the TMV particle. This process is in good agreement with that obtained from electron microscopic studies.     

Small angle X-ray scattering studies on local structure of tobacco mosaic virus RNA in solution

Effects of temperature and ionic strength (S) on the local structure of tobacco mosaic virus RNA in phosphate buffer solution are studied by analyzing the small-angle X-ray scattering (SAXS) curves. The root-mean-square radius of a cross-section of RNA chain was kept at 0.845+/-0.005 nm over a wide range of S from 0.2 to 0.003 at 20 degrees C, whereas it gradually diminished from 0.85 to 0.61 nm when the temperature is raised from 20 to 50 degrees C at S = 0.2. Nevertheless, all of SAXS curves reflecting the backbone structures were equally mimicked by theoretical ones of freely hinged rod (FHR) models, i.e. several straight rods joined with freely hinged joints in the form of a combination of the letter Y, if the constituent rod lengths in the models are adjusted. From these facts, it is suggested that the local structure of the RNA chain in aqueous solution is characterized by an essential feature that unpaired bases in the partially double-stranded helix are constantly far isolated from each other along the helix and the rod-like structure of the helix is preserved over a range of helical contents. Such a characteristic local structure of the chain is entirely collapsed in the formamide solution at 50 degrees C.     

Self-assembly of brome mosaic virus capsids: Kinetic study using neutron and X-ray solution scattering

The self-assembly of brome mosaic virus capsid has been studied kinetically by means of X-ray and neutron scattering. It appears to be a very fast process: for the concentrations used (5 to 8 mg/ml) the forward scattering reaches 50% of its maximal value in less than one second. Further, the assembly seems to proceed through intermediate states whose nature is still speculative.     

Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.     

Detection of microorganisms in the environment and the preliminary appraisal of their physiological state by X-ray microanalysis

The paper deals with the X-ray microanalysis of the elemental composition of bacteriomorphic particles in 170,000-year old Antarctic permafrost sediments and in indoor dust. A comparative analysis of the phosphorus, sulfur, calcium, and potassium contents and the Ca/K and P/S ratios in these particles and in reference microbial cells occurring in different physiological states showed that the absence of P and/or S peaks in the X-ray spectrum of an object may indicate that it is abiotic. Resting microbial forms can be revealed on the basis of the following characteristic features: an increased content of Ca, a high Ca/K ratio, and a low P/S ratio. Model experiments with nonviable bacterial and yeast micromummies with alterations in the structural and barrier functions of the cytoplasmic membrane showed that micromummies can be recognized by a super-high content of a marker element (e.g., P, K, or Si), accumulated due to facilitated diffusion along the deliberately created concentration gradient. Such an analysis of the permafrost sediment and dust made it possible to suggest the presence of mummified cells in these objects. The possibility of using X-ray microanalysis for the detection of microbial cells in natural habitats in order to enhance the efficiency of ecological monitoring of the environment is discussed.