Isolation of broad-host-range replicons from marine sediment bacteria

Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888-895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the alpha and gamma subclasses of the class Proteobacteria. Three of the marine sediment isolates are related to the gamma-3 subclass organisms Vibrio splendidus and Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication in Escherichia coli DH5alpha and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera Achromobacter, Pseudomonas, Serratia, and Vibrio. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.     

In situ morphologies of deep-sea and sediment bacteria

Deep-sea and sediment bacteria at the bottom of an approximately 1200-m water column were sampled by means of pressure vessels attached to a remote underwater manipulator. Cells were immediately fixed in situ with glutaraldehyde, and after processing in the laboratory their morphologies were observed with the scanning electron microscope. Most bacteria were coccoid or rod-lide and less than 0.4 mum in diameter or width. Few filamentous bacteria were observed. Bacteria were in aggregates or free-living. It is concluded that morphologies of deep-sea bacteria collected and fixed at the hydrostatic pressure of their environment are, in general, similar to the observed morphologies of deep-sea bacteria determined at 1 atm pressure after collection and decompression during ascent through the water column.     

Isolation of Aeromonas salmonicida in association with purple-pigmented bacteria in sediment from a Scottish loch

Aeromonas salmonicida was recovered in close association with an unidentified purple-pigmented organism, which was isolated from sediment in a Scottish loch during November (1997) and February (1998). However, there has not been any evidence of A. salmonicida infections, specifically furunculosis, associated with the fish in this loch.     

Hydrogen-bond interactions of the primary donor of the photosynthetic purple sulfur bacterium Chromatium tepidum

We have used near-infrared Fourier transform (pre)resonance Raman spectroscopy to determine the protein interactions with the bacteriochlorophyll (BChl) dimer constituting the primary electron donor, P, in the reaction center (RC) from the thermophilic purple sulfur bacterium Chromatium tepidum. In addition, we report the alignment of partial sequences of the L and M protein subunits of C. tepidum RCs in the vicinity of the primary donor with those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. Taken together, these results enable us to propose the hydrogen-bonding pattern and the H-bond donors to the conjugated carbonyl groups of P. Selective excitation (1064-nm laser radiation) of the FT (pre)-resonance Raman spectra of P in its neutral (P degree) and oxidized (P degree +) states were obtained via their electronic absorption bands at 876 and 1240 nm, respectively. The P degree spectrum exhibits vibrational frequencies at 1608, 1616, 1633, and 1697 cm-1 which bleach upon P oxidation. The P degree + spectrum exhibits new bands at 1600, 1639, and 1719 cm-1. The 1608-cm-1 band, which downshifts to 1600 cm-1 upon oxidation, is assigned to a CaCm methine bridge stretching mode of the P dimer, indicating that each BChl molecule possesses a single axial ligand (His L181 and His M201, from the sequence alignment). The 1616- and 1633-cm-1 bands correspond to two H-bonded pi-conjugated acetyl carbonyl groups of each BChl molecule. with different H-bond strengths: the 1616-cm-1 band is assigned to the PL C2 acetyl group which is H-bonded to a histidine residue (His L176), while the 1633-cm-1 band is assigned to the PM C2 acetyl carbonyl, H-bonded to a tyrosine residue (Tyr M196). Both PL and PM C9 keto carbonyls are free from interactions and vibrate at the same frequency (1697 cm-1). Thus, the H-bond pattern of the primary donor of C. tepidum differs from that of Rb. sphaeroides in the extra H-bond to the PM C2 acetyl carbonyl group; that of PL is H-bonded to a histidine residue in both primary donors (His L168 in Rb. sphaeroides and His L176 in C. tepidum). The P degree/P degree + redox midpoint potentials were measured to be +497 and +526 mV for isolated C. tepidum RCs with and without the associated tetraheme cytochrome c subunit, respectively, and +502 mV for intracytoplasmic membranes. The positive charge localization was estimated to be 69% in favor of PL, indicating a more delocalized situation over the primary donor of C. tepidum than that of Rb. sphaeroides (estimated to be 80% on PL). These differences in physicochemical properties are discussed with respect to the proposed structural model for the microenvironment of the primary donor of C. tepidum.     

Phylogenetic and physiological diversity of sulphate-reducing bacteria isolated from a salt marsh sediment

Xenopus blastula cells activate different mesodermal genes as a concentration-dependent response to activin, which behaves like a morphogen. To understand how cells recognize morphogen concentration, we have bound naturally labeled activin to cells and related this to choice of gene activation. We find that the increasing occupancy of a single receptor type can cause cells to switch gene expression. Cells sense ligand concentration by the absolute number of occupied receptors per cell (100 and 300 molecules of bound activin induce Xbra and Xgsc, respectively, i.e., 2% and 6% of the total receptors) and not by a ratio of occupied to unoccupied receptors. The long duration of occupancy explains a previously described ratchet effect. Our results suggest a new concept of morphogen gradient formation and interpretation that is particularly well suited to the needs of early development.     

Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components

The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria. To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects. Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype. Each of these mutants was defective in chemotactic response. Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response. In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface. When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity. Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant. These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade.     

Widespread use of the glu-tRNAGln transamidation pathway among bacteria. A member of the alpha purple bacteria lacks glutaminyl-trna synthetase

The expression of the Rhizobium meliloti glutamyl-tRNA synthetase gene in Escherichia coli under the control of a trc promoter results in a toxic effect upon isopropyl-beta-D-thiogalactopyranoside induction, which is probably caused by a misacylation activity. To further investigate this unexpected result, we looked at the pathway of Gln-tRNAGln formation in R. meliloti. No glutaminyl-tRNA synthetase activity has been found in R. meliloti crude extract, but we detected a specific aminotransferase activity that changes Glu-tRNAGln to Gln-tRNAGln. Our results show that R. meliloti, a member of the alpha-subdivision of the purple bacteria, is the first Gram-negative bacteria reported to use a transamidation pathway for Gln-tRNAGln synthesis. A phylogenetic analysis of the contemporary glutamyl-tRNA synthetase and glutaminyl-tRNA synthetase amino acid sequences reveals that a close evolutionary relationship exists between R. meliloti and yeast mitochondrial glutamyl-tRNA synthetases, which is consistent with an origin of mitochondria in the alpha-subdivision of Gram-negative purple bacteria. A 256-amino acid open reading frame closely related to bacterial glutamyl-tRNA synthetases, which probably originates from a glutamyl-tRNA synthetase gene duplication, was found in the 4-min region of the E. coli chromosome. We suggest that this open reading frame is a relic of an ancient transamidation pathway that occurred in an E. coli ancestor before the horizontal transfer of a eukaryotic glutaminyl-tRNA synthetase (Lamour, V., Quevillon, S., Diriong, S., N'Guyen, V. C., Lipinski, M., and Mirande, M.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8670-8674) and that it favored its stable acquisition. From these observations, a revisited model for the evolution of the contemporary glutamyl-tRNA synthetases and glutaminyl-tRNA synthetases that differs from the generally accepted model for the evolution of aminoacyl-tRNA synthetases is proposed.     

LF-RH工艺生产极低硫钢硫含量控制分析

研究了LF炉渣脱氧剂加入量、LF动力学条件及RH后期钙处理对脱硫效果的影响.结果表明:向LF精炼渣中加入300 kg以上的铝粒时可以将渣中w(FeO+ MnO)控制在0.5％以下,从而将钢液中w(S)由原来的30×10-6降至6×10-6;当底吹搅拌氩气流量为500 L/min,搅拌10 min后,钢液中w(S)可以降至6×10-6,过高或过低的吹氩流量都会影响脱硫效率;RH后期钙处理没有脱硫能力,但是钙线的加入有利于抑制回硫,并将硫含量保持在较低水平.     

Biotransformation of 2,4,6-trinitrotoluene (TNT) by a Methanococcus sp. (strain B) isolated from a lake sediment

The newly developed antihypertensive drugs, the long-acting beta-blocker propranolol and the sustained release calcium antagonist verapamil, are compared in their antihypertensive, platelet function, rheological properties and metabolic effects. The trial was a double-blind, randomised, placebo-controlled cross-over study. Thirty patients with mild to moderate hypertension received propranolol (40-120 mg) or verapamil (80-200 mg) once daily in two separate ten week courses. After ten weeks treatment both drugs had significantly reduced both SBP and DBP. Beta-thromboglobulin (beta-TG) concentration, reflecting the status of platelet activation in vivo, was significantly decreased after propranolol (129.6 +/- 13.5 vs. 77.9 +/- 8.6 ng/ml) and verapamil (129.6 +/- 13.5 vs. 90.7 +/- 10.1 ng/ml) treatments while platelet aggregation induced by ADP, collagen, arachidonic acid or adrenaline and the production of thromboxane B2 (TXB2), 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) and platelet cyclic 3'-5' adenosine monophosphate (C-AMP) concentration were not affected. Significant alterations in rheological parameters such as plasma and whole blood viscosity, fibrinogen level and red cell deformability were not found. Higher cholesterol and low-density lipoprotein-cholesterol (LDL-C) levels were observed after propranolol treatment but not in verapamil treatment. Side-effects were mild, tolerated and no patient had to be withdrawn from the study. In conclusion, propranolol and verapamil are generally effective antihypertensive as well as rheologically safe drugs. Compared with the metabolic effect on serum lipid, verapamil may be a better choice. Both drugs possess the tendency to inhibit platelet properties which is desirable in hypertension treatment.     

Origin attribution of polychlorinated dibenzo-p-dioxins and dibenzofurans in sediment and soil from a Japanese freshwater lake area through congener-specific data analysis

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDDs/PCDFs) in sediment and soil from a freshwater lake area were congener-specifically determined. The obtained data were examined to estimate the major origins of these compounds, with the aid of principal component analysis (PCA). Four major principal components (PCs) were obtained, and three of them were attributed to PCDDs/PCDFs in the atmosphere, in a diphenyl ether herbicide and in pentachlorophenol, based on congener-specific comparisons with references. One PC remained unattributed. These four were interpreted as the major origins of PCDDs/PCDFs in the area. The relative influence of the origins was also investigated.     

Leaching of marine manganese nodules by acidophilic bacteria growing on elemental sulfur

This article describes the bioleaching of manganese nodules by thermophilic and mesophilic sulfuroxidizing bacteria, in which oxidized sulfur compounds are biologically produced from elemental sulfur added to liquid medium and are simultaneously used to leach nodules. The thermophile Acidianus brierleyi solubilized the manganese nodules faster at 65 °C than did the mesophiles Thiobacillus ferrooxidans and Thiobacillus thiooxidans at 30 °C. Leaching experiments with A. brierleyi growing on elemental sulfur were used to optimize various process parameters, such as medium pH, initial sulfur-liquid loading ratio, and initial cell concentration. The observed dependencies of the leaching rates at a pH optimum on the initial amounts of elemental sulfur and A. brierleyi cells were qualitatively consistent with model simulations for microbial sulfur oxidation. Under the conditions determined as optimum, the leaching of nodule particles (−330+500 mesh) by A. brierleyi yielded 100 pct extraction of both copper and zinc within 4 days and high extractions of nickel (85 pct), cobalt (70 pct), and manganese (55 pct) for 10 days. However, the iron leaching was practically negligible.     

Application of antisera raised against sulfate-reducing bacteria for indirect immunofluorescent detection of immunoreactive bacteria in sediment from the German Baltic Sea

Polyclonal rabbit antisera raised against sulfate-reducing bacteria (SRB) could detect several distinct populations of bacteria in sediment from the German Baltic Sea. The depth distribution of immunoreactive bacteria was determined by an indirect immunofluorescence filter method. Anti-Desulfovibrio desulfuricans DSM 1926 serum showed maximum bacterial numbers at a depth of 18 cm, with a concentration of 60 x 10(6) cells cm-3. With anti-Desulfovibrio baculatus DSM 2555 serum, counts were highest at the same depth, approaching 0.7 x 10(6) cells cm-3. Other significantly smaller populations were observed. Anti-SRBStrain 1 (lactate,vibrio) maxima were at 0 to 4 cm and at 17 to 18 cm. Anti-SRBStrain 2 (lactate,vibrio) serum showed several local maxima. Anti-SRBStrain 3 (lactate,oval) serum detected one single peak at a depth of 10 to 12 cm. Also determined were rates of sulfate reduction, total bacterial counts by acridine orange staining, and the viable counts by dilution series on anaerobic lactate medium. The total bacterial counts were highest (180 x 10(6) cells cm-3) at 3 to 4 cm and dropped to 24 x 10(6) cells cm-3 at 10 to 11 cm but showed additional local maxima reaching 140 x 10(6) cells cm-3 at a depth of 17 to 18 cm. Viable counts probable number) were above 10(5) CFU cm-3 at 0 to 3.6 cm but remained below 10(3) CFU at 7.2 to 18 cm. The sulfate reduction rate was maximal (107 nmol cm-3 day-1) at a depth of 1 to 2 cm, dropped to 10 nmol cm-3 day-1 at 12 to 13 cm, and reached 38 nmol cm-3 day-1 at 17 to 18 cm.     

Temporal changes in concentration of radiocaesium in lake sediment and fish of southern Finland as related to environmental factors

Strain Henrichsen S 735 (NCTC 10234) of Streptococcus suis serotype 2 reference and three other such strains (strains S 4005, S 3921 and T 141) were tested for virulence by inoculating pigs intranasally and intravenously. The taxonomical properties of each strain were determined. Phenotypes were determined by Western blotting based on MRP and EF protein expression and genotypes were determined by Southern hybridization analysis of the mrp and epf genes. Reference strain S 735 and strain S 3921 produced the 136 kDa MRPh and a 180 kDa form of EF, and hence these strains belong to the MRP + EF phenotype. In accordance with previous experiments with this phenotype, strains S 735 and S 3921 appeared to be only weakly virulent for newborn gnotobiotic pigs. Strain S 4005 produced the 136 kDa MRP and the 110 kDa form of EF, hence it belongs to the MRP + EF + phenotype. This strain was highly virulent for pigs. Strain T 141 did not produce MRP or EF, and hence belongs to the MRP-EF- phenotype. It was nonvirulent for pigs. The route of inoculation did not influence the frequency or severity of clinical signs of disease or lesions, which demonstrated that the 110 kDa EF is not essential during invasion. Southern blot analysis showed that all four S. suis type 2 strains contain sequences that are homologous to the epf and mrp genes. For studies on pathogenesis of S. suis type 2 infections in pigs, we recommend the use of strains that have been tested in a standardized pig model and that belong to the MRP + EF + phenotype, such as strain S 4005.     

焦炭硫分影响因素分析

简要归纳了国内目前焦炭硫分预测方法,指出了不足。通过32个单种煤硫分形态与焦炭硫分关系研究,分析了硫分形态对焦炭硫分的影响;通过分析炼焦过程炼焦煤硫分变化,提出传统室式炼焦过程脱硫反应和固硫反应并存,煤气中H2具有脱硫功能,灰中CaO和MgO是固硫剂;60个样品试验数据的回归分析结果表明,煤的全硫和灰中(CaO+MgO)含量是影响焦炭硫分的主要因素。     

粉末压片制样-波长色散X射线荧光光谱法测定土壤和水系沉积物中硫和氯

土壤和水系沉积物中S、Cl含量采用高频燃烧-红外吸收光谱法、离子色谱法等方法测定时,耗时相对较长;采用粉末压片法制样,波长色散X射线荧光光谱法(WD-XRF)测定时,同一样片Cl元素只能测定1次,造成标准物质大量浪费,且S元素因受粒度效应影响测试难度较大。实验选用粒度48 μm样品,在压力30 MPa、保压30 s条件下制备的样片能有效改善粒度效应对X射线荧光强度的影响;将水系沉积物和土壤标准物质研磨至粒度48 μm建立校准曲线;同时采用经验系数法进行基体效应校正,通过校正MoLα对SKα的谱线干扰,MoLγ1对ClKα的谱线干扰,以及减少样片放置时间、优先测量Cl元素等操作,可保证WD-XRF测量S、Cl的准确度。试验发现测试完毕后的标准样品重新研磨压片或超过半年的长期放置后,Cl首次测量值基本恢复认定值,解决了Cl元素只能测定1次,标准物质大量浪费的问题。试验比对了WD-XRF、红外碳硫分析仪和元素分析仪的准确度,结果表明3种方法测定值基本相符;WD-XRF中S、Cl检出限分别为30 μg/g和18 μg/g,对数偏差(ΔlgC)为0.003～0.054,相对标准偏差(RSD,n=12)为0.7％～8.7％,符合DZ/T 0258—2014《多目标区域地球化学调查规范(1∶250 000)》要求。方法适用于日常大批量分析测试。     

粉末压片制样-波长色散X射线荧光光谱法测定土壤和水系沉积物中硫和氯

土壤和水系沉积物中S、Cl含量采用高频燃烧-红外吸收光谱法、离子色谱法等方法测定时,耗时相对较长;采用粉末压片法制样,波长色散X射线荧光光谱法(WD-XRF)测定时,同一样片Cl元素只能测定1次,造成标准物质大量浪费,且S元素因受粒度效应影响测试难度较大。实验选用粒度48 μm样品,在压力30 MPa、保压30 s条件下制备的样片能有效改善粒度效应对X射线荧光强度的影响;将水系沉积物和土壤标准物质研磨至粒度48 μm建立校准曲线;同时采用经验系数法进行基体效应校正,通过校正MoLα对SKα的谱线干扰,MoLγ1对ClKα的谱线干扰,以及减少样片放置时间、优先测量Cl元素等操作,可保证WD-XRF测量S、Cl的准确度。试验发现测试完毕后的标准样品重新研磨压片或超过半年的长期放置后,Cl首次测量值基本恢复认定值,解决了Cl元素只能测定1次,标准物质大量浪费的问题。试验比对了WD-XRF、红外碳硫分析仪和元素分析仪的准确度,结果表明3种方法测定值基本相符;WD-XRF中S、Cl检出限分别为30 μg/g和18 μg/g,对数偏差(ΔlgC)为0.003～0.054,相对标准偏差(RSD,n=12)为0.7％～8.7％,符合DZ/T 0258—2014《多目标区域地球化学调查规范(1∶250 000)》要求。方法适用于日常大批量分析测试。     

KR脱硫渣矿相及硫在渣中分布

 为探讨KR脱硫渣的脱硫机理,利用现场取脱硫渣,通过炉渣淬火实验,对渣中矿相组成和硫在渣中分布进行研究与分析。研究结果表明：KR渣主要位于CaO-SiO2-CaF2-CaS四元系,渣中含有单一的CaS相、以CaO为主的CaO-CaF2-CaS相和以CaO、SiO2为主的CaO-SiO2-CaF2-CaS相,且CaS相中的硫含量明显高于其他2种矿相。通过统计渣相中CaS相的面积分数,并结合炉渣总的硫含量,得出渣相中的硫主要以单一的CaS形式存在。因此,通过提高渣相中CaS相的面积分数,可提高炉渣硫含量。     

Diagnostic value of a miracidium in urinary sediment

The Medical School of the Ljubljana University has a long lasting tradition in teaching the history of medicine. Since 1934, all students of medicine and dentistry in Slovenia have attended the courses in history of medicine which were and still are mandatory. The first years of lectures on the history of medicine at the Ljubljana University Medical School coincided with the struggle for the establishment and recognition of a full medical curriculum in Slovenia. Dr Ivan Pintar (1888-1963), the first lecturer of the history of medicine (1934-1963), was a man of great ethical and national values and an ardent advocate for the promotion of Slovene contribution to medical culture in Slovenia, former Yugoslavia, and Europe. Pintar's concept of history of medicine included general historical, sociological, ethical, cultural, psychological, biotechnical, and other aspects. During 30 years of his endeavors, he did a pioneer work in the research on the history of medicine in Slovenia. For many years he was the editor of the Medical Chamber Bulletin, chairman of the Slovene Medical Society, and the founder of the Section for the History of Medicine in 1951. He wrote the first Slovene textbook on the history of medicine and published many articles on prominent physicians from Slovenia. He paved the way for the new generation of medical historians in Slovenia. Owing to him, contemporary Slovene physicians and dentists are aware of the importance of professional tradition. They know how to use the past experiences for new achievements.