Lipid Membrane Structure and Dynamics Studied by All-Atom Molecular Dynamics Simulations of Hydrated Phospholipid Bilayers

Abstract

The structure and dynamics of phosphatidylcholine bilayers are examined by reviewing the results of several nanoseconds of molecular dynamics simulations on a number of bilayer and monolayer models. The lengths of these simulations, the longest single one of which was 2 nanoseconds, were sufficiently long to effectively sample many of the longer-scale motions governing the behaviour of biomembranes. These simulations reproduce many experimental observables well and provide a degree of resolution currently unavailable experimentally.     

Diversity and Metabolism of Marine Bacteria Cultivated on Dissolved DNA

Dissolved DNA (dDNA) is a potentially important source of energy and nutrients in aquatic ecosystems. However, little is known about the identity, metabolism, and interactions of the microorganisms capable of consuming dDNA. Bacteria from Eel Pond (Woods Hole, MA) were cultivated on low-molecular-weight (LMW) or high-molecular-weight (HMW) dDNA, which served as the primary source of carbon, nitrogen, and phosphorus. Cloning and sequencing of 16S rRNA genes revealed that distinct bacterial assemblages with comparable levels of taxon richness developed on LMW and HMW dDNA. Since the LMW and HMW dDNA used in this study were stoichiometrically identical, the results confirm that the size alone of dissolved organic matter can influence bacterial community composition. Variation in the growth and metabolism of isolates provided insight into mechanisms that may have generated differences in bacterial community composition. For example, bacteria from LMW dDNA enrichments generally grew better on LMW dDNA than on HMW dDNA. In contrast, bacteria isolated from HMW dDNA enrichments were more effective at degrading HMW dDNA than bacteria isolated from LMW dDNA enrichments. Thus, marine bacteria may experience a trade-off between their ability to compete for LMW dDNA and their ability to access HMW dDNA via extracellular nuclease production. Together, the results of this study suggest that dDNA turnover in marine ecosystems may involve a succession of microbial assemblages with specialized ecological strategies.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Laminin Fibrils in Newt Gastrulae Visualized by the Immunofluorescent Staining

An anastomosing network of extracellular fibrils on the inner surface of the ectoderm layer of amphibian gastrulae has been shown to provide an adequate substratum for attachment and migration by the mesodermal cells. These fibrils contain fibronectin as shown by immunostaining at the light and electron microscope levels. Now we report the presence of laminin, another cell adhesion glycoprotein, as a fibrillar network on the inner surface of the ectoderm layer in gastrulae of the Japanese newt ( Cynops pyrrhogaster ), but its absence on the blastula ectoderm layer, by the immunofluorescent staining using an antiserum specific for mouse laminin. The same antiserum was shown to stain basement membranes of adult newt organs as expected.     

盐酸酸化可改善核抗原免疫荧光染色

免疫荧光染色(immunofluorescent staining, IF)技术广泛用于细胞或组织内抗原定性、定量或定位检测。然而,依常规染色步骤操作,在有些胞核抗原的检测中很难得到令人满意的结果。有研究者采用盐酸酸化预处理用于细胞增殖标记物5-溴-2-脱氧尿嘧啶核苷(BrdU)的荧光染色并获得良好效果。但此方法是否也适用于其他类型的胞核抗原,尚不清楚。为系统全面分析盐酸酸化在胞核抗原免疫荧光染色中的作用,本文以成年C57BL小鼠主要嗅觉表皮(MOE)为材料,分别对Ki-67、5-甲基胞嘧啶(5mC)和5-羟甲基胞嘧啶(5hmC)三种不同类型的胞核抗原进行盐酸酸化处理免疫荧光染色。结果显示,当血清封闭和抗体浓度等条件一致时,室温下盐酸酸化2 h,Ki-67的染色效果最佳,而阴性对照与未酸化组均未出现阳性信号;同样经过盐酸处理2 h,5mC和5hmC染色也呈现较强的阳性信号。该研究表明,在一些胞核抗原免疫荧光染色中,使用盐酸酸化处理可显著提高染色效果。     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria

A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.     

Immunofluorescent Staining of Salmonella Species with Flagellar Sera

Salmonellae stained with flagellar sera by either the direct or indirect fluorescent-antibody technique fluoresced at the cell surface. Investigations showed that fimbriae, flagellar material, and capsule antigens were not the participating antigens in this reaction. Cell surface staining was inhibited by mannose but was unaffected by glucose, galactose, lactose, maltose, and sucrose. Specific absorption of the flagellar sera by intact cells and purified somatic antigen showed that O antigens were the site of the surface staining. The advantages of using flagellar sera in fluorescent-antibody screening of materials for the presence of salmonellae are discussed.     

Diversity of Thiosulfate-Oxidizing Bacteria from Marine Sediments and Hydrothermal Vents

Species diversity, phylogenetic affiliations, and environmental occurrence patterns of thiosulfate-oxidizing marine bacteria were investigated by using new isolates from serially diluted continental slope and deep-sea abyssal plain sediments collected off the coast of New England and strains cultured previously from Galapagos hydrothermal vent samples. The most frequently obtained new isolates, mostly from 10³- and 10⁴-fold dilutions of the continental slope sediment, oxidized thiosulfate to sulfate and fell into a distinct phylogenetic cluster of marine alpha-Proteobacteria. Phylogenetically and physiologically, these sediment strains resembled the sulfate-producing thiosulfate oxidizers from the Galapagos hydrothermal vents while showing habitat-related differences in growth temperature, rate and extent of thiosulfate utilization, and carbon substrate patterns. The abyssal deep-sea sediments yielded predominantly base-producing thiosulfate-oxidizing isolates related to Antarctic marine Psychroflexus species and other cold-water marine strains of the Cytophaga-Flavobacterium-Bacteroides phylum, in addition to gamma-proteobacterial isolates of the genera Pseudoalteromonas and Halomonas-Deleya. Bacterial thiosulfate oxidation is found in a wide phylogenetic spectrum of Flavobacteria and Proteobacteria.     

Immunofluorescent Staining of Leptospires in Pepsin Treated Histologic Sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Detection of Escherichia coli in Foods: Indole Staining Methods for Cellulosic and Polysulfone Membrane Filters

Optimized procedures for staining Escherichia coli colonies on cellulosic and polysulfone membrane filters are described. An explanation for the behavior of the Ehrlich reaction on membrane filters is suggested.     

Phytol Degradation by Marine Bacteria

Microbial degradation of phytol is often proposed to be the primary source of the acyclic isoprenoid acids observed in sediments, yet only a limited number of these acids have been found in bacterial cultures grown on phytol. This study reports detailed capillary gas chromatography and gas chromatography-mass spectrometry analyses of the products resulting from growth of marine bacteria on phytol as the sole carbon source. We examined two strains of bacteria which were able to oxidize phytol to phytenic acid but were unable to further degrade phytol. The third isolate studied converted phytol to a mixture of five saturated isoprenoid acids. The C₁₇ isoprenoid acid produced was of particular interest, since its genesis from phytol would have involved several unusual intermediates. It is suggested that this acid is produced by bacterial metabolism of the C₁₈ isoprenoid ketone (produced from phytol abiologically under oxic conditions) and that its abundance is thus a sensitive indicator of sedimentary depositional conditions.     

Concentration of Enteroviruses on Membrane Filters

Enteroviruses can be made to adsorb or to pass through membrane filters by manipulation of the suspending medium. Salts facilitate virus adsorption, but membrane-coating components (MCC) interfere. Because cells release MCC into the culture medium during viral growth, MCC must be removed before virus can be adsorbed to membranes. Adsorbed virus can be eluted with diluents containing MCC (cell extracts or serum) or agents that reduce surface tension (sodium lauryl sulfate). By membrane adsorption and elution, enteroviruses can be readily concentrated and quantitatively recovered from crude virus harvests.     

The Negative Staining of Bacteria

The author does not propose new methods but calls attention to the value of Burri's India ink technic and Fischer's use of nigrosin for the negative staining of bacteria. The latter is particularly useful. The technic is as follows: Boil 10 g. nigrosin in 100 cc. water about 30 minutes. Filter several times thru the same filter paper, adding 10 drops of formalin before the last filtration as a preservative. Place a small loopful of this solution on a clean slide and add the bacteria with a needle or loop. After mixing spread the mixture a little irregularly on the slide and dry either at room temperature or slowly over a low flame. The preparation may be examined in oil immersion without the use of a cover slip.     

Dynamics,Diversity and Function of Endophytic Siderophore-Producing Bacteria in Rice

Siderophore production confers to bacteria competitive advantages to colonize plant tissues and to exclude other microorganisms from the same ecological niche. This work shows that the community of endophytic siderophore-producing bacteria (SPB) associated to Oryza sativa cultivated in Uruguayan soils is dynamic and diverse. These bacteria were present in grains, roots, and leaves, and their density fluctuated between log₁₀ 3.44 and log₁₀ 5.52 cfu g⁻¹ fresh weight (fw) during the plant growth. Less than 10% of the heterotrophic bacteria produced siderophores in roots and leaves of young plants, but most of the heterotrophic bacteria were siderophore-producers in mature plants. According to their amplified restriction DNA ribosomal analysis (ARDRA) pattern, 54 of the 109 endophytic SPB isolated from different plant tissues or growth stages from replicate plots, were unique. Bacteria belonging to the genera Sphingomonas, Pseudomonas, Burkholderia, and Enterobacter alternated during plant growth, but the genus Pantoea was predominant in roots at tillering and in leaves at subsequent stages. Pantoea ananatis was the SPB permanently associated to any of the plant tissues, but the genetic diversity within this species—revealed by BOX-PCR fingerprinting- showed that different strains were randomly distributed along time and plant tissue, suggesting that a common trait of the species P. ananatis determined the interaction with the rice plant. Several isolates were stronger IAA producers than Azospirillum brasilense or Herbaspirillum seropedicae. In vitro inhibition assays showed that SPB of the genus Burkholderia were good antagonists of pathogenic fungi and that only one SPB isolate of the genus Pseudomonas was able to inhibit A. brasilense and H. seropedicae. These results denoted that SPB were selected into the rice plant. P. ananatis was the permanent and dominant associated species which was unable to inhibit two of the relevant plant growth-promoting bacteria.     

Diversity and Population Dynamics of Methanogenic Bacteria in a Granular Consortium

Upflow anaerobic sludge blanket bioreactor granules were used as an experimental model microbial consortium to study the dynamics and distribution of methanogens. Immunologic methods revealed a considerable diversity of methanogens that was greater in mesophilic granules than in the same granules 4 months after a temperature shift from 38 to 55°C. During this period, the sizes of the methanogenic subpopulations changed with distinctive profiles after the initial reduction caused by the shift. Methanogens antigenically related to Methanobrevibacter smithii PS and ALI, Methanobacterium hungatei JF1, and Methanosarcina thermophila TM1 increased rapidly, reached a short plateau, and then fell to lower concentrations that persisted for the duration of the experiment. A methanogen related to Methanogenium cariaci JR1 followed a similar profile at the beginning, but it soon diminished below detection levels. Methanothrix rods weakly related to the strain Opfikon increased rapidly, reaching a high-level, long-lasting plateau. Two methanogens related to Methanobrevibacter arboriphilus AZ and Methanobacterium thermoautotrophicum ΔH emerged from very low levels before the temperature shift and multiplied to attain their highest numbers 4 months after the shift. Histochemistry and immunohistochemistry revealed thick layers, globular clusters, and lawns of variable density which were distinctive of the methanogens related to M. thermoautotrophicum ΔH, M. thermophila TM1, and M. arboriphilus AZ and M. soehngenii Opfikon, respectively, in thin sections of granules grown at 55°C for 4 months. Mesophilic granules showed a different pattern of methanogenic subpopulations.     

Effects of Bacteriophages on the Population Dynamics of Four Strains of Pelagic Marine Bacteria

Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.