Degradation of outer membrane proteins of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis>

To examine the possibility that outer membrane proteins (OMP) of Synechococcus sp. remain in seawater, we investigated the stability of OMPs in vitro and in situ. Some fractions prepared from Synechococcus sp. CSIRO-94 were treated with trypsin and proteinase K. Four tightly bound OMPs were separated from Synechococcus. We designated the two major OMPs of 52 kDa and 48 kDa as Omp52Sy and Omp48Sy, respectively. Degradation of the OMP in natural seawater was monitored in microcosms to which intact Synechococcus cells and outer membrane (OM) were added. Omp52Sy and Omp48Sy were the most stable against trypsin and proteinase K among the OMPs when they were embedded in the OM. However, in the microcosm experiment using intact cells, Omp52Sy and Omp48Sy were detected in the particulate fraction only during the first 4 days, after which they could not longer be detected. Omp52Sy and Omp48Sy were the most stable proteins among the Synechococcus OMPs in vitro, but they might be degraded in situ. This indicates that stability of Synechococcus porin differs depending on complex formation with other membrane molecules, which might cause different preservation of microbial membrane proteins in the dissolved protein pool in the ocean. This study suggests that Gram negative bacterial OM with thin peptidoglycan forms a lipid bilayer that proptects OMP, but Synechococcus OM with thick peptidoglycan cannot form a lipid bilayer. The incomplete bilayer might not be able to protect from protease attack in the natural environment.     

Detection of Vibrio (Listonella) Anguillarum Porin Homologue Proteins and Their Source Bacteria from Coastal Seawater

The molecular distribution of dissolved proteins in seawater from coastal marine environments in Uranouchi Bay, Kochi Prefecture, is first reported in this article. Occurrence of bacteria-derived dissolved proteins and their source bacteria were examined using a probe of the antibody (anti-Omp35La) against a porin outer membrane protein (Omp35La) of the fish pathogenic bacterium Vibrio (Listonella) anguillarum. The electrophoretograms of dissolved proteins from coastal seawater showed a large number of discrete and individual proteins overlapped each other over a wide range of molecular masses indicating active processes in coastal environments in transferring proteins from organisms to the inanimate dissolved protein pool. Among the dissolved proteins, 37 kDa- and 18 kDa-proteins reacted with the Omp35La. In order to isolate the source bacteria of such dissolved proteins, bacteria from seawater and diseased fish were screened by colony Western blotting with anti-Omp35La. The reactive strains were further examined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting to verify the presence of Omp35La homologues among the outer membrane proteins of such strains. Outer membrane proteins reacting with anti-Omp35La were detected in only 4 strains of the 129 strains that were positive in the colony Western blotting. The level of possible source bacteria of 37 kDa- and 18 kDa-dissolved proteins was suggested to be 5–6 orders of magnitude lower than the total bacterial count. The present study leads us to hypothesize that a minor portion of the bacterial assemblage is responsible for the dissolved proteins in the coastal waters.     

Similarity of electrophoretic dissolved protein spectra from coastal to pelagic seawaters

Dissolved proteins in seawater samples collected from a coastal area of Tokyo Bay, Sagami Bay and a location off the Kuroshio Current were investigated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE). Four to nine protein bands were detected in SDS-PAGE in the apparent molecular weight (MW) range from 12 kilo Dalton (kDa) to 49 kDa. The 2-DE technique distinguished 10 to 46 protein spots exhibiting isoelectric point (pI)/MW ranging 4.3–9.2/12–63 kDa. The elecrophoretic patterns were similar between the coastal and pelagic samples, as well as previously reported patterns from various pelagic areas. The close similarity of electrophoretic mobility on both SDS-PAGE and 2-DE gels indicates the compositional homogeneity of dissolved proteins in seawater throughout a broad range of marine environments. Proteinaceous dissolved organic matter (DOM) that was unresolved and smeared staining characteristics on both SDS-PAGE and 2-DE gels was first observed in Tokyo Bay waters in the present study and its possible sources are discussed. Although the two protein species, 48 kDa and 39 kDa proteins, have been identified as homologues of Porin P and low molecular weight-alkaline phosphatase of Pseudomonas aeruginosa PAO1, respectively, four strains of P. aeruginosa and two species of Pseudomonas spp. have been newly identified as the source organisms of these proteins using the N-terminal amino acid sequence data determined in previous studies.     

The inventory and chemical characterization of dissolved proteins in oceanic waters

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE) were applied to separate protein molecules in dissolved organic matter (DOM) from oceanic waters. Results were: (1) The 2-DE distinguished a total of 412 protein spots in 10 samples from five water columns over the Pacific, although fewer than 30 proteins were resolved as bands from the identical samples by SDS-PAGE. (2) Major and ubiquitous protein bands (34 and 39 kDa proteins) on the SDS-PAGE gel were resolved into horizontally spread arrays (trains) of spots on the 2-DE gels, indicating that these bands were a mixture of protein species that have the same molecular weight (MW) but different isoelectric points (pIs). (3) Proteins that exhibited such electrophoretic patterns on the 2-DE gels were glycosylated with variable linkages between the sugar and polypeptide chains. (4) N-terminal amino-acid sequencing demonstrated that individual spots within each train of spots had identical N-terminal amino-acid sequences.The N-terminal amino-acid sequences of the 39 and 34 kDa glycoprotein spots in samples collected at different sites were also identical. Protein isoforms with the same amino-acid sequence but different glycosylation profiles, termed glycoforms, were often observed on the 2-DE gel. Thirty-one and 24 spots on the 2-DE gels were glycoforms of two glycoproteins with MWs of 39 and 34 kDa, respectively; they were one protein species. The glycoforms of the 39 kDa protein were identified as a low molecular weight alkaline phosphatase (L-AP) of Pseudomonas aeruginosa PAO1 by a homology search through five amino-acid sequence databases. The present and earlier work indicates that all identified source organisms of dissolved proteins belong to the Pseudomonas group. We propose the hypothesis that proteins associated with membrane vesicles liberated from a minor member of the bacterioplankton assemblage, the marine Pseudomonas group, are one of the important sources of dissolved proteins in oceanic waters. This hypothesis may apply to the source pathway and survival not only of proteins and also to the universally occurring bacterial peptidoglycan and lipopolysaccharide components in DOM.     

海湾区海水中的溶存甲烷 Ⅰ.分布特征及成因

本文基于日本东京湾、伊势湾和浜名湖的调查资料，详细地讨论了海湾区海水中溶存甲烷的分布特征、成因及其来源。溶存甲烷在海湾中的分布主要受物理混合过程所控制；由河流携入和沉积物中产生是海湾区海水中溶存甲烷的两个主要来源。     

Metaproteomic characterization of dissolved organic matter in coastal seawater

We performed a comprehensive metaproteomic analysis of the dissolved organic matter (DOM) in Japanese coastal waters using liquid chromatography–tandem mass spectrometry and demonstrated that these proteomes were characterized by proteins with various functions, including metabolic enzymes, membranes, and photosynthetic proteins. The protein sources included cyanobacteria, heterotrophic bacteria, and eukaryotic phytoplankton. Most of the components were similar among samples and also similar to pelagic components. We also observed differences in the compositions of the microbial communities of origin among the different dissolved protein samples and differences in the relative abundance of specific dissolved protein types (e.g., cytoskeletal proteins), possibly indicating potential dynamics in the coastal DOM pool.     

动态式抽取、气相色谱法测定海水中的甲烷

本文报道了用动态（ｄｙｎａｍｉｃ）技术，经抽取、气相色谱法测定海水中溶存甲烷浓度的方法．详细论述了溶存甲烷经起泡、收集（吸附）、解吸和检测四步完成，并逐步讨论了起泡时间、起泡气体流速以及收集温度等有关因子对测定的影响。结果表明：该方法分析速度快、精密度好、灵敏度高，可广泛应用于海上现场测定海水中溶存的甲烷。     

小角刺藻生长过程中溶解游离氨基酸含量在海水中的变化

于1986年12月-1987年2月在温度为25±1℃下培养小角刺藻，以HPLC法测定该藻在培养过程中海水的溶解游离氨基酸（DFAA）含量变化。结果表明，小角刺藻不仅是海水中DFAA的主要生产者，而且也是DFAA的消耗者。小角刺藻不仅在缺乏硝酸盐的时候要吸收DFAA，即使在硝酸盐充足的情况下也会吸收。在小角刺藻的生长初期，水体中DFAA的含量迅速降低，而在后期却又大大增高。在小角刺藻的不同生长阶段，水体中DFAA的组成也不同。这些都可以说明海洋中DFAA的含量、组成和地区分布是密切地同浮游植物群落的繁殖消亡过程相关联的。     

海湾区海水中的溶存甲烷 Ⅱ.浓度、海气交换通量

本文基于日本东京湾、伊势湾和浜名湖的调查资料，详细地比较了这3个海湾海水中的溶存甲烷浓度、饱和度。表层海水中，溶存甲烷浓度以供名湖最高，达95nmol／dm3，伊势湾和东京湾相近，分别为41nmol／dm3和37nmol／dm3；其表层测定浓度是其平衡浓度的13～78倍，并分别计算了这3个海湾中甲烷的海气交换通量；用平均值外推法得出全球海洋环境中总的甲烷海气交换通量为6．3Tg／a。     

The diffusion coefficient of dissolved silica revisited

The diffusion coefficient of dissolved silica was determined for two different salinities, 36 and 0, at temperatures ranging from 2 °C to 30 °C and at an average pH value of 8.1. Our results show limited influence of salinity and a variation by a factor of 2 to 3 of the silica diffusion coefficient within the temperature range considered in this study. The values obtained at 25 °C are in agreement with previous work carried out at room temperature for seawater and freshwater. The dependency on temperature and viscosity of the diffusion coefficient agrees well with the Einstein–Stokes equation. The composition of the solvent appears to be an important factor because it modifies the viscosity and allows for the complexation of the dissolved silica with less mobile ions, while its pH controls the dissolved silica speciation. In seawater, the higher viscosity and the presence of dissociated and polymeric species result in a decrease of the diffusion coefficient compared to freshwater systems.     

Investigations on dissolved copper—organic substances in Baltic waters

A technique for the pH-controlled isolation and separation of dissolved copper—organic substances from large amounts of seawater is described. The method has been applied in two cruises in the Bothnian and Gotland Seas of the Baltic. In both areas the correlation between the amount of isolated dissolved organic material (DOM) and the concentration of organic copper in seawater has been found to be highly significant. Under the conditions established in our experiments, organic copper fractions detected in Baltic waters were in the range of about 5% of total copper. HPLC chromatograms and UV irradiation experiments from DOM extracts strongly indicate the existence of dissolved organic-copper compounds in Baltic waters.     

Use of an optical oxygen sensor to measure dissolved oxygen in seawater

The use of an optical oxygen sensor to measure dissolved oxygen in seawater was investigated. The sensor is based on the dynamic quenching of an oxygen-sensitive fluorochrome embedded in the tip. Dissolved oxygen in seawater samples collected from eight stations at depths ranging from 3000 to 6000 m was analyzed both with the optical sensor and by the Winkler titration method. The two sets of data did not differ significantly. The stability and simplicity of the method and the good agreement of the results with those of the titration method indicate that the sensor would be useful for fieldwork.     

溶解有机碳在混合水中的行为研究

研究溶解有机碳(DOC)在海水和黄河水所组成的混合水中的迁移转化行为,结果表明,海水和已过滤黄河水混合时,DOC呈近似的保守性;海水与未过滤的黄河水混合时,DOC呈明显的非保守性。通过研究沉积物对DOC的解吸过程,发现沉积物在盐水中能解吸出DOC,随着盐度的增大,其解吸量也迅速增大。因此可以认为,DOC在河口区的非保守行为是DOC的絮凝和颗粒物的解吸共同作用的结果。     

Advanced characterization of marine dissolved organic matter by combining reversed-phase liquid chromatography and FT-ICR-MS

Marine dissolved organic matter (DOM) was separated by reversed-phase (RP) liquid chromatography method and analyzed with fluorescence/absorption detection and Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS). The three key characteristics of the RP method are: (a) The C18-RP column chosen provides enhanced separation when the aqueous phase is 100% buffer-free water, and it does not degrade over time; (b) the water eluent adjusted to pH 7 significantly improves the resolution of water soluble compounds; (c) the initial flow maintained at low levels improves the separation of polar compounds. In samples, containing “fresh” DOM, specific peaks were detected, which were absent in “old” DOM samples. The combination with size exclusion chromatography (SEC) also demonstrated the relation between polarity and molecular size of DOM. FT-ICR-MS was applied to evaluate the quality of separation on a molecular scale demonstrating that physico-chemical characteristics of DOM can be related to molecular formulas. Sample extracts were separated into 4 preparative fractions, and a large suite of the identified molecular formulas only occurred in specific fractions. This is an important basis for the application of further analytical techniques in order to perform a more target-oriented analysis aiming at the determination of source and process biomarkers for DOM.     

Measurement of Noble Gas Solubility in Seawater Using a Quadrupole Mass Spectrometer

A quadrupole-based mass spectrometric system for determining noble gas (He, Ne, Ar, Kr and Xe) abundances in a liquid sample has been developed and applied to seawater equilibrated with the atmosphere at several temperatures. After known amounts of isotopic spikes (²²Ne, ³⁶Ar, ⁸⁶Kr and ¹²⁴Xe) were introduced into the preparation vacuum line, dissolved gases were extracted from seawater and well mixed with the spikes by ultrasonic vibration. Noble gases were purified using three-stage hot Ti getters and separated by two activated charcoal traps held at low temperature (liquid nitrogen and dry ice—ethanol). Noble gas abundances were measured by a relatively inexpensive quadrupole mass spectrometer based on the isotope dilution technique. Precisions obtained by repeated measurements for He, Ne, Ar, Kr and Xe are 1.5%, 0.4%, 0.4%, 0.4% and 0.6%, respectively. Noble gas abundances in air-saturated seawater at eight temperature steps (1.0°C, 5.5°C, 10.0°C, 14.4°C, 17.0°C, 21.3°C, 24.6°C, and 29.6°C) were measured using the analytical system. The overall variation of the abundances with temperature agrees well with those of NaCl brines reported in the literature. We propose here new constants to fit observed data to empirical equation of noble gas solubility in seawater with its temperature variation.     

长牡蛎nacrein 基因的克隆、结构及进化分析

首次在长牡蛎(Crassostrea gigas)中克隆得到一种新的贝壳基质蛋白nacrein-like protein F3的全长c DNA序列。nacrein-like protein F3基因c DNA全长1499bp,其中编码区长度为1242bp,编码一条含413个氨基酸残基的多肽链。氨基酸序列比对和结构域分析均表明其为合浦珠母贝(Pinctada fucata)nacrein的同源蛋白,含有1个保守的α-碳酸酐酶结构域,但由于重复结构域的插入,α-碳酸酐酶结构域被间隔成2个亚结构域。系统进化分析显示nacrein-like protein F3与贻贝(Mytilus californianus)nacrein-like protein进化关系最近。此外,在软体动物中,双壳纲nacrein-like proteins进化速度相对较快,推测与寒武纪时期剧烈的环境变化有关,如影响贝壳形成的海水化学变化。     

Chemical studies of dissolved carbohydrates in seawater

Charcoal column chromatography and a dialysis system using a Visking tube were used for the concentration and separation of dissolved carbohydrates in seawater. Recoveries of mono-, oligo-, and polysaccharides were 46–74, 61–89 and 70%, respectively, relative to the total amount of sugars initially submitted to analysis. The combined charcoal column chromatography and dialysis system was used to separate dissolved carbohydrates in seawater samples from Mikawa Bay into mono-, oligo- and polysaccharide fractions. The polysaccharides are separated into two fractions with molecular weights of <4,000 and >4,000 using Sephadex G-25 column chromatography. Monosaccharides, oligosaccharides, polysaccharides with M.W.<4,000 and polysaccharides with M.W. >4,000 accounted for 7–9, 15–26, 7–13 and 20–33% of total dissolved carbohydrates, respectively. However, 32–38% of dissolved carbohydrates were lost during the analytical procedures. Glucose, galactose, mannose, xylose, arabinose, ribose, fucose, and rhamnose were found upon acid hydrolysis of the oligo- and polysaccharide fractions, while fucose and rhamnose were not present in the monosaccharide fraction. Possible sources of the carbohydrates in these fractions are discussed with reference to the monosaccharide composition of cellular carbohydrate in marine algae.     

A discussion on vertical profiles of dissolved Cu,Cd and Ni in the northwestern Pacific, south of Japan

Some of the results about vertical profile of heavy metals of seawater to the south of Japan in Oct. 1990 are presented and discussed in relation to the concentration of dissolved Cu, Cd and Ni to biogeochemical environments. It points out that the distribution of dissolved Cu is higher in surface water than that in/upper 500 m layer, and maxima value attains the 8. 2 n mol/dm3 in depth of 4 000 m. The concentrations of dissolved Ni ranges from 3. 4 n mol/dm3 in surface seawater to 8. 5 n mol/dm3 in the deep to the south of Japan. The highest values are observed in the colder waters. This paper shows also that the vertical profile of dissolved Cd is perfect nutrient-type distribution. And dissolved Cd and Phosphate are linearly correlated by the regression equation. The ·Cd : ·N : ·P atomic ratio is 3. 5 × 10-4 : 14 : 1.     

The free amino acid composition of seawater obtained without desalting and preconcentration

A conventional amino acid analyzer was modified to allow detection of dissolved free amino acids in a direct injection of 0.5 to 2.0 ml of seawater. The amino acids were monitored fluorimetrically by reaction with o-phthaldialdehyde after column chromatography. The accuracies of two commonly employed desalting methods, cation exchange resin and copper Chelex, were determined by comparison with the direct injection method. After correcting the results of the desalting methods for the procedural blank and recovery rate for each individual amino acid, the amino acid compositions on a mole percent basis as determined by these methods resembled approximately the true composition determined by direct injection. However, on an absolute basis the cation resin method overestimates and the copper Chelex method underestimates the total amount of dissolved free amino acids. The reasons for these discrepancies are discussed. Using the direct injection method, the spectra of dissolved free amino acids in surface seawater samples from different locations were determined. As opposed to the relatively uniform patterns reported in most past investigations, the amino acid spectra found were highly variable. In a number of samples, the acidic amino acids dominated the spectra. Valine was also high in many samples. In a depth profile the deep samples were dominated by arginine. Application of the method to real-time shipboard analyses of dissolved free amino acids is discussed.     

pH对SPE-HPLC-ESI-Q-TOF-MS检测海水中小分子活性物质的影响研究

海水溶解有机物(dissolvedorganicmatter,DOM)中含有的生物活性物质在海洋生态系统中作用巨大,但因缺乏适合的分离提取方法而严重阻碍了对其不同组分在生态系统中作用的探索。固相萃取法对富集提取海水DOM十分有效,在用其提取海水DOM时,海水pH对活性物质提取效果的影响很大,但目前针对海水的这种影响尚存在很大争议。本文以天然近海海水作为基质,探究不同pH条件下用亲水-疏水平衡(hydrophilic-lipophilicbalanced,HLB)固相萃取小柱萃取海水中活性分子的提取效率,并使用高效液相色谱-四极杆飞行时间质谱(HPLC-Q-TOF-MS)在负离子(ESI-)模式下检测解析提取物的组成。研究结果表明,当海水样品pH为中性和强酸性时都能获得较好的提取效率,随着pH的降低,提取物质谱的整体响应值降低,但可识别的谱峰数目增加,提取出有机物的分子量和性质差异都更广泛。分析提取物分子在范克雷维伦(van Krevelen)图和质荷比-氢碳比(m/z-H/C)图上的分布发现,中性条件适合提取饱和度较高的小分子化合物,而具有生物活性的带有不饱和基团的化合物及蛋白质、糖类等生物大分子在强酸性提取条件时提取效果和分辨率更好。综合提取效率、有效峰数目和分子组成特征考虑,用HLB固相萃取小柱提取近海海水中的小分子活性物质时,将海水样品pH调节为2较为适宜。