A transducing bacteriophage for Caulobacter crescentus uses the paracrystalline surface layer protein as a receptor.

The bacteriophage phi Cr30, a transducing phage for Caulobacter crescentus strains, required the paracrystalline surface (S) layer for infectivity. Wild-type strains were phage resistant when rsaA, the gene for the 130K S-layer protein, was interrupted with an antibiotic resistance cassette. Strains that had lost the S layer by mutation were phage resistant, as were mutants that produce an S layer but which do not attach the structure to the cell surface. Phage sensitivity was restored to 130K-protein-deficient strains by introducing rsaA on a plasmid. Spontaneous phage-resistant strains produced expected phenotypes as follows (in order of decreasing frequency): S-layer cell attachment defects, no S layer, or an S layer that was wild type in appearance.     

Three-dimensional structure of the regular tetragonal surface layer of Azotobacter vinelandii.

Fragments of the Azotobacter vinelandii tetragonal surface (S) layer, free of outer membrane material, were obtained by treating whole cells with 100 microM EDTA. The three-dimensional structure of the S layer was reconstructed from tilted-view electron micrographs of the S-layer fragments, after computer-assisted image processing by correlation averaging. At a resolution of 1.7 nm, the S layer exhibited funnel-shaped subunits situated at one fourfold-symmetry axis and interconnected at the other fourfold-symmetry axis to form prominent cruciform linking structures. These data, in conjunction with a relief reconstruction of the surface of freeze-etched whole cells, indicated that the apex of the funnel-shaped subunit was associated with the outer membrane, while the funnel "opening" faced the environment; the cruciform linking structures were formed at the outermost surface of the S layer. Electron microscopy and image enhancement were used to compare the structure of the outer membrane-associated S layer with that of fragments of the S layer dislodged from the outer membrane. This analysis revealed an increase in the lattice constant of the S layer from 12.5 to 13.6 nm and an alteration in the position of the cruciform linking structures in the z direction. These conformational changes resulted in a reduction in the thickness of the S layer (minimum estimate, 5 nm) and an apparent increase in the size of the gaps between the subunits. In terms of the porosity of the S layer, this gave the appearance of a transition from a closed to a more open structure.     

Three-dimensional structure of an open form of the surface layer from the fish pathogen Aeromonas salmonicida.

Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.     

Charge distribution on the S layer of Bacillus stearothermophilus NRS 1536/3c and importance of charged groups for morphogenesis and function.

The distribution and functional significance of charged groups on the outer and inner faces of the S layer from Bacillus stearothermophilus NRS 1536/3c was investigated. Chemical modification of the exposed amino or carboxyl groups was performed on whole cells, isolated S layers self-assembled in vitro, and cell wall fragments (S layer attached to the peptidoglycan-containing sacculus). Without chemical modification, S layer self-assembly products could be labeled with polycationic ferritin, while S layers on whole cells could not. Following treatment with glutaraldehyde, whole cells were uniformly labeled with polycationic ferritin. Whole cells treated with glutaraldehyde and glycine methyl ester in the presence of carbodiimide did not bind polycationic ferritin significantly above background. Treatment of cell wall fragments with amino-specific, homobifunctional cross-linkers or with carbodiimide alone rendered the S layer protein nonextractable with sodium dodecyl sulfate. After amidation of the accessible carboxyl groups, the modified, guanidine hydrochloride-extractable S layer protomers did not self-assemble into regularly structured lattices. N-Amidination with ethylacetimidate did not interfere with the self-assembly of the isolated protomers. N-Acetylation resulted in a considerable destabilization of the S layer lattice, as seen by the release of a large amount of modified protomers during the reaction. N-Succinylation led to a complete disintegration of the protein lattice. These results indicated that only the inner face of the S layer carried a net negative charge. On both faces, free amino and carboxyl groups of adjacent protomers were arranged in proximity so as to contribute by electrostatic interactions to the cohesion of the protomers in the two-dimensional array. The native charge of the protomers was required for both the in vitro self-assembly of the isolated subunits and the maintenance of the structural integrity of the S layer lattice. Among other functions, the biological significance of the S layers may be in masking the electronegative charge of the cell wall proper.     

Characterization of a dynamic S layer on Bacillus thuringiensis.

The surfaces of three Bacillus thuringiensis strains possess an S layer composed of linear arrays of small particles arranged with p2 symmetry and with a = 8.5 nm, b = 7.2 nm, and gamma = 73 degrees. Platinum shadows of whole cells and S-layer fragments revealed the outer surface of the array to be smooth and the inner surface to be corrugated. Treatment with 2 M guanidine hydrochloride at pH 2.5 to 4 best removed the S layer for chemical characterization; it was a relatively hydrophilic 91.4-kilodalton protein with a pI of 5, no detectable carbohydrate, cysteine, methionine or tryptophan, and 21.2% nonpolar residues. No N-terminal homology with other S-layer proteins was evident. Antibody labeling experiments confirmed that the amount of S layer was proportional to the growth phase in broth cultures. Late-exponential- and stationary-growth-phase cells typically sloughed off fragments of S layer, and this may be the result of wall turnover. Indigenous autolytic activity in isolated walls rapidly digested the wall fabric, liberating soluble S-layer protein. At the same time, proteases frequently reduced the molecular weight of the 91.4-kilodalton protein, but these polypeptides could still be identified as S-layer components by immunoblotting. As cultures were serially subcultured, the frequency of appearance of the S layer diminished, and it was eventually lost. The dynamic nature of this S layer makes it atypical of most previously identified S layers and made it unusually difficult to characterize.     

Development of haematopoietic colonies on the macrophage layer formed in the peritoneal cavity of S1/S1d mice

The colony-forming ability of haematopoietic cells was examined on the macrophage layer formed in the peritoneal cavity of S1/S1d mice. The bone marrow cells of the congenic +/+ mice formed many macroscopic colonies on the macrophage layer of the S1/S1d mice although they did not form macroscopic colonies in the spleens of the same S1/S1d recipients. The size and the differentiation pattern of colonies on the macrophage layer of the S1/S1d mice were comparable to those of the colonies on the macrophage layer of the +/+ mice. There are two possible explanations for these results: (a) The microenvironmental defect of the S1/S1d mice has a more prominent effect on the development of spleen colonies than that of macrophage-layer colonies because 'Steel' locus may not be expressed significantly in the peritoneal macrophages or (b) because the cells that make colonies on the macrophage layer may be more differentiated cells than the multipotential stem cells that make colonies in the spleen.     

DEVELOPMENT OF HAEMATOPOIETIC COLONIES ON THE MACROPHAGE LAYER FORMED IN THE PERITONEAL CAVITY OF S1/S1D MICE

The colony-forming ability of haematopoietic cells was examined on the macrophage layer formed in the peritoneal cavity of S1/S1^d mice. the bone marrow cells of the congenic +/+ mice formed many macroscopic colonies on the macrophage layer of the S1/S1^d mice although they did not form macroscopic colonies in the spleens of the same S1/S1^d recipients. the size and the differentiation pattern of colonies on the macrophage layer of the S1/S1^d mice were comparable to those of the colonies on the macrophage layer of the +/+ mice. There are two possible explanations for these results: (a) the microenvironmental defect of the S1/S1^d mice has a more prominent effect on the development of spleen colonies than that of macrophage-layer colonies because ‘Steel’ locus may not be expressed significantly in the peritoneal macrophages or (b) because the cells that make colonies on the macrophage layer may be more differentiated cells than the multipotential stem cells that make colonies in the spleen.     

Molecular sieving through S layers of Bacillus stearothermophilus strains.

The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selected strains of Bacillus stearothermophilus were investigated. Measurements were performed of passive solute uptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were prepared from whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preserved the integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found to consist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete S layer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi the peptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selected for the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The S layers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm.     

西藏色季拉山暗针叶林凋落物层化学性质研究

The storage and chemical properties of the forest litter in dark coniferous forest of Sejila Mountain were studied. The results showed that the existing storage was 5. 863t·hm^-2 and the annual litter fall was 0. 3205 t·hm^-2 It implied that the forest litter decomposed slowly and accumulated quickly, and the turnover of nutrient circles was slow. The contents of N, Ca, Na, and Mn nutrient elements in litter layer were in the order of un-decomposed layer (U layer) > semi-decomposed layer (S layer) > decomposed layer (D layer), those of K, Fe, and Mg were in the order of D layer > S layer > U layer, and P element content was in the order of U layer > D layer> S layer. The pool of elements was 78. 483 kg·hm^-2 N, 3. 843 kg·hm^-2P, 48. 205 kg·hm^-2 K, 23.115 kg·hm^-2 Ca, 13. 157 kg·hm^-2 Na, 30.554 kg·hm^-2 Fe, 2. 113 kg·hm^-2 Mn and 27. 513 kg·hm^-2 Mg. The turnover of forest litter was the total of nutrient release accumulation. K, Fe, and Mg were enriched, and N,Ca, Na, Mn, and P were released with the turnover rate in the order of N > Ca > Na > Mn >P.     

野生露珠杜鹃林不同分解层的土壤化感潜力

探明野生杜鹃群落不同层次土壤浸提物的化学成分及其含量差异,为阐明杜鹃群落天然更新障碍与化感作用之间的关系提供基础数据,从化学生态学角度解释群落天然更新障碍的原因。通过种子发芽试验,比较凋落物层(L层)、腐殖质层(H层)和土壤表层(S层)浸提液对自身种子萌发的化感效应,采用内标法结合气相色谱-质谱(GC/MS)联用等技术鉴定土壤浸提液所含的有机化合物。(1)种子发芽试验显示,露珠杜鹃不同土壤层浸提液的化感效应不同。L层的抑制作用最为强烈,其浸提液显著抑制自身种子的萌发。H层和S层对自身具有一定的抑制作用,与对照相比不显著;(2)鉴定了不同层次土壤浸提液中所含的有机化合物,L层、H层和S层均检测出31种组分,其中相对含量大于5%的组分分别有6、8和8种。L层浸提液主要化感成分为丙三醇和棕榈酸,分别达到总量的19.56%和19.17%;H层主要化感成分为2-羟基丙酸和棕榈酸,分别达到总量的14.05%和12.48%;S层土壤浸提物的主要化感成分为棕榈酸和2-羟基乙酸,分别达到总量的14.91%和12.79%。野生露珠杜鹃不同土壤浸提物的化感物质含量以L层最高,L层作为群落土壤化感物质的主要来源;从化感物质组分来分,长链脂肪酸和有机酸类是H层和S层主要的化感物质种类,长链脂肪酸类和醇类是L层主要的化感物质种类。杜鹃群落林下土壤中存在的化感物质可能是其天然更新障碍的重要原因之一。     

不同林型兴安落叶松林火烧迹地物种组成及多样性研究

以内蒙古大兴安岭兴安落叶松林火烧迹地为研究对象,采用分层取样的方法对群落中植被进行了调查,分析比较了3种不同林型兴安落叶松林群落恢复过程中物种组成及多样性的差异。结果表明:不同林型兴安落叶松林群落各层α多样性指数总体表现为草本层灌木层乔木层。不同林型兴安落叶松林火烧迹地群落恢复12年后,草本层的Simpson和Shannon-Wiener指数均小于对照样地并呈现出草类-兴安落叶松林杜香-兴安落叶松林杜鹃-兴安落叶松林的趋势,而在灌木层则各指数均大于对照样地并呈现出草类-兴安落叶松林杜鹃-兴安落叶松林杜香-兴安落叶松林的趋势。     

城市公园生境类型对鸟类群落的影响

2011年12月—2012年11月,在上海世纪公园和滨江森林公园对鸟类群落和植物群落进行调查,通过对12个植被变量进行主成分分析,将两个公园分为8种不同的生境类型。结果表明:2个公园生境构成存在显著差异,滨江森林公园灌木层植物发达的生境(Habitat with developed shrub layer,S型)以及灌木层和地被层植物都发达的生境(Habitat with developed tree layer and shrub layer,T+S型)数量显著多于世纪公园,世纪公园地被层发达的生境(Habitat with developed ground cover layer,G型)以及乔木层和地被层植物都发达的生境(Habitat with developed tree layer and ground cover layer,T+G型)数量显著多于滨江森林公园。世纪公园不同生境中鸟种数差异显著,而滨江森林公园中差异不显著。2个公园有24种共有鸟种,对共有鸟种生境利用率的配对t检验结果表明,滨江森林公园鸟类生境利用率显著高于世纪公园。对2个公园共有生境类型中鸟种数进行分析,发现滨江森林公园鹟科(Muscicapidae)鸟类种数显著大于世纪公园。根据以上结果,上海城市公园不同生境类型对鸟类群落结构存在显著影响。因此,建议在规划和建设大型城市公园时,应构建植被分层结构复杂的生境,多样化种植各类乔木,林下多样化搭配灌木。在保留供游客休憩草坪区域的同时种植各类草本植物,以此提高鸟类生境利用率,增加城市公园的鸟类多样性。     

Microfibril orientation across the secondary cell wall of Radiata pine tracheids

Polarised light microscopy and transmission electron microscopy were used to measure the variation in cellulose microfibril orientation across the secondary cell wall of Pinus radiata D. Don tracheids using oblique sections. Variations in orientation for the S1, S2 and S3 layers were determined for radial and tangential cell walls from the juvenile and mature earlywood at the base of the stem and at 5 m height. Microfibrils in the S1 layer are usually arranged in an S-helix (>90°) varying from 79° to 117° among tracheids. Within individual tracheids the microfibril orientation in the S1 region can be quite variable, sometimes changing from an S to Z-helix but without a well-defined crossed structure. Microfibrils in the S2 layer form a Z-helix (<90°) with average orientation varying from 1° to 59° among tracheids, while the S3 layer is also often a Z-helix varying from 50° to113° in orientation. The S2 layer shows significant variation among sample points within the tree related to age and height, while the S1 and S3 layers show small random variations within the tree. Microfibril orientation in the S2 layer is very uniform, although when variation does occur the trend is exclusively for an increase towards the S1 region. Orientation in the S3 layer often varies continuously from the S2 boundary to the lumen. The S3 layer becomes much thinner with increasing age and height, making measurement by light microscopy more difficult. The innermost lamellae of microfibrils at the lumen surface sometimes show a distinctive clustering, forming macrofibrils of varying orientation but this may be the result of delignification treatment.     

无瓣海桑、海桑、秋茄红树人工林群落动态及物种多样性研究

对红树植物无瓣海桑、海桑、秋茄3种人工林群落动态及物种多样性特征的系统研究结果表明,无瓣海桑群落和海桑群落的乔木层明显分为上层和中层两个亚层,上层木为无瓣海桑或海桑,中层木主要为秋茄和桐花树;秋茄群落的乔木层为单一层次,基本由秋茄组成．在无瓣海桑群落和海桑群落中,优势种群无瓣海桑或海桑仅有高龄级个体存在,未出现自然更新现象;秋茄和桐花树为旺盛增长种群,有可能成为优势种群,表明无瓣海桑和海桑为先锋造林树种,在裸滩种植可以促进其它红树植物的天然定居生长;在秋茄群落中,秋茄为旺盛增长种群,能够自然：更新演替,桐花树和海莲属初生增长种群．无瓣海桑群落和海桑群落的物种组成和物种多样性指标较接近,基本包含秋茄群落中的主要物种秋茄、桐花和海莲。表明无瓣海桑和海桑能与这些物种协调共生,同时种植无瓣海桑或海桑可以形成多样化的红树林群落;无瓣海桑群落和海桑群落在形成初期,种植密度较大时,物种多样性较高;密度相近时。形成初期随林龄的增加。其物种多样性略有提高．     

施入不同土层的秸秆腐殖化特征及对玉米产量的影响

耕作和秸秆还田是打破犁底层、改善黑土肥力的重要措施.本研究利用田间试验,分析了耕作和秸秆还田对秸秆腐殖化系数、总有机质含量(SOC)和玉米产量的影响.结果表明:深耕+秸秆施入20～35 cm(ST+S)能够打破犁底层,与浅耕(TT)、深耕(ST)和浅耕+秸秆还田(TT+S)相比,试验6年间土壤容重平均降低了5.7%、3.3%和5.7%,其中ST和ST+S试验第一年效果最好;试验6年后秸秆腐解率表现为0～20 cm土层(72.0%)>20～35 cm土层(59.2%);0～20和20～35 cm土层秸秆腐殖化系数在试验的第一年达到了最大值,分别为15.9%和12.7%;与初始土壤相比,TT、ST和ST+S处理0～20 cm土层SOC和轻组有机碳(LFOC)含量呈下降趋势,而TT+S处理分别增加了2.9%和12.4%,ST+S处理20～35 cm土层分别增加了9.2%和9.9%;对玉米产量的影响表现为ST+S>TT+S>ST>TT,耕作和秸秆还田的时间效应明显,其中ST处理玉米产量的影响可以持续3年,而ST+S处理可以持续6年.因此,通过耕作的方式将秸秆施入20～35 cm土层是一种有效的、可持续改善黑土质量的农业措施.     

S layer protein of Clostridium tetani: purification and properties.

S layer protein of Clostridium tetani strain AO 174, a nontoxigenic derivative of strain Harvard A 47, was prepared from the cell walls by 4 M urea extraction and purified by DEAE-Sepharose CL-6B chromatography followed by a combination of anion-exchange chromatography and reverse-phase chromatography using an HPLC system. The molecular weight of the S layer protein was estimated to be 140 kilodaltons (kDa) by SDS-PAGE. The amino acid composition of the 140 kDa protein was very similar to those of S layer proteins from the other bacterial species: it was rich in acidic amino acid and lacked cysteine. Also, the protein was unique in its extremely low content of proline (0.02 to 0.03 mol%). Multiple isoelectric forms ranging from pH 4.0 to 4.5 were observed in the purified preparation. Immunodiffusion analysis showed that the 140 kDa protein was a common antigen to the three strains of C. tetani tested.     

Peptidoglycan of free-living anaerobic spirochetes

Electron microscope examination of negatively stained or thin-sectioned cells of Spirochaeta stenostrepta treated with penicillin or lysozyme showed that the peptidoglycan was present as a thin, electron-dense layer adjacent and external to the cytoplasmic membrane. The peptidoglycan was isolated from cells of S. stenostrepta and Spirochaeta litoralis by a procedure including treatments with sodium lauryl sulfate and Pronase. Hydrolysates of the isolated S. stenostrepta and S. litoralis peptidoglycans contained glucosamine, muramic acid, glutamic acid, l-ornithine, and alanine in molar ratios of 0.90:0.85:1.00:1.00:1.40 and of 0.63:0.63:0.99:1.00:1.41, respectively. Determination of N-terminal residues suggested that nearly 50% of the ornithine in S. stenostrepta and S. litoralis peptidoglycans was involved in peptide cross-linkage. The peptidoglycan layer of S. stenostrepta was sensitive to lysozyme and myxobacter AL-1 protease.     

Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus.

We determined that paracrystalline protein surface arrays (S layers) protected gram-negative eubacteria from predation by Bdellovibrio bacteriovorus. Aquaspirillum serpens VHA and MW5 and Aquaspirillum sinuosum were resistant to predation by B. bacteriovorus 6-5-S when fully covered by their S layers. The S layer of Aeromonas salmonicida A449 protected the cells from predication by B. bacteriovorus 109J. A predacious, plaque-forming vibrio that lysed an S-layer- variant of Caulobacter crescentus but was not predacious on the parental strain which possessed an S layer was isolated from raw sewage. Since S layers are stable components of many bacterial surfaces in nature, they can provide this protective function in both aquatic and terrestrial habitats where Bdellovibrio spp. are found.     

S100 protein-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

S100-immunoreactivity (ir) was examined in tooth pulp primary neurons of the rat. An immunofluorescence method demonstrated that the molar tooth pulp contained S100-immunoreactive (ir) nerve fibers. In the root pulp, pulp horn and roof of the pulp chamber, S100-ir smooth and varicose fibers ramified and formed subodontoblastic nerve plexuses. All the fibers became varicose at the base of the odontoblastic layer and extended to the odontoblastic layer. Some varicose endings could be traced into the dentin. The trigeminal neurons retrogradely labeled with fluorogold (FG) from the first and second maxillary molar tooth pulps exhibited S100- and parvalbumin-ir. Approximately 60% and 24% of the labeled cells were ir for S100 and parvalbumin, respectively. Virtually all parvalbumin-ir FG-labeled cells showed S100-ir, while 40% of S100-ir ones coexpressed parvalbumin-ir. An immunoelectron microscopic method revealed that all myelinated axons and half of the unmyelinated axons in the root pulp contained S100-ir. In the odontoblastic layer, predentin and dentin, S100-ir neurites lost the Schwann cell ensheathment and made close contact with cell bodies and processes of odontoblasts. The odontoblastic layer also contained parvalbumin-ir neurites. These neurites were devoid of the Schwann cell ensheathment and in close apposition to cell bodies and processes of odontoblasts. S100-ir pulpal axons seemed to be insensitive to repeated neonatal capsaicin treatment. This study suggests that S100-ir tooth pulp primary neurons are mostly myelinated and that S100-ir unmyelinated axons in the root pulp are preterminal segments of myelinated stem axons.     

Comparison of metabolic systems required to activate pro-mutagens/carcinogens in vitro for sister-chromatid exchange studies

Irradiated Syrian hamster fetal cells (feeder layer) and rat-liver homogenate (S9 mix) were used to compare their capacity to metabolize 3 known promutagens/carcinogens; BaP, 3-MC and DMBA. DNA-damaging potential was determined by the induction of SCE in V79 target cells. The S9 mix (1/20th strength) was toxic to the target cells and reduced the mitotic index by half with an exposure time of 2.5 h. The feeder layer was not toxic to the target cells and, therefore, was included for the duration of the Expt. The test chemicals elicited a dose-response with both activating systems. At similar concentrations of the test chemicals, the cells grown on the feeder layer showed a greater number of SCEs as compared to those activated by the S9 mix.