Bacteriology of Manganese Nodules: III. Reduction of MnO2 by Two Strains of Nodule Bacteria1

MnO₂ reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolated from ferromanganese nodules, was assessed for 7 days. A 1-day lag was observed before the onset of MnO₂ reduction by either culture. Addition of HgCl₂ to a final concentration of about 10^-3 M caused a rapid cessation of MnO₂ reduction by the growing cultures. Neither culture reduced MnO₂ when grown under continued anaerobiosis from the start of an experiment. However, if conditions were made anaerobic after MnO₂ reduction was initiated, reduction continued at a rate only slightly lower than that under aerobic conditions. Resting-cell cultures reduced MnO₂ equally well aerobically and anaerobically, provided that ferricyanide was present to serve as electron carrier. These findings showed that oxygen is needed for culture adaptation to MnO₂ reduction, and that oxygen does not interfere with microbial MnO₂ reduction itself. Both cultures caused sharp drops in the pH of the medium during MnO₂ reduction: with coccus 32, during the entire incubation time; with Bacillus 29, for the first 3 days. The E_h of the medium fluctuated with either culture and never fell below 469 mv with Bacillus 29 and below 394 mv with coccus 32. The rates of glucose consumption and Mn²⁺ release by Bacillus 29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate production were not. Although acid production undoubtedly helped in the reduction of pyrolusite (MnO₂) by the bacteria, it did not appear to be important in the reduction of manganese oxide in ferromanganese nodules, as shown by the results with a nodule enrichment.     

Bacteriology of Manganese Nodules: I. Bacterial Action on Manganese in Nodule Enrichments

Bacteria, found in manganese nodules from the Atlantic Ocean, enhance the adsorption of Mn from sea water by crushed manganese nodules in the presence of peptone. When bacterial outgrowth from crushed manganese nodules was experimentally delayed, peptone did not enhance Mn adsorption by nodular substance, but hindered it in some cases. A mechanism to explain the role of bacteria in enhancing Mn adsorption by manganese nodules is presented. Oyster shells were shown to adsorb Mn in the absence of bacteria. Peptone did not enhance the rate of Mn adsorption. Adsorbed Mn was not visibly oxidized during experimental observation. These results suggest one way whereby nodule formation may be initiated in the oceans. Some bacteria in the nodules were found to release manganese from them in the presence of glucose and peptone. Bacteria may, therefore, play a role not only in nodule buildup but also in nodule breakdown.     

Bacteriology of Manganese Nodules: II. Manganese Oxidation by Cell-free Extract from a Manganese Nodule Bacterium

A cell-free extract from Arthrobacter 37, isolated from a manganese nodule from the Atlantic Ocean, exhibited enzymatic activity which accelerated manganese accretion to synthetic Mn-Fe oxide as well as to crushed manganese nodule. The reaction required oxygen and was inhibited by HgCl₂ and p-chloromercuribenzoate but not by Atebrine dihydrochloride. The rate of enzymatic action depended on the concentration of cell-free extract used. The enzymatic activity had a temperature optimum around 17.5 C and was destroyed by heating at 100 C. The amount of heat required for inactivation depended on the amount of nucleic acid in the preparation. In the cell-free extract, unlike the whole-cell preparation, peptone could not substitute for NaHCO₃ in the reaction mixture. An enzyme-containing protein fraction and a nucleic acid fraction could be separated from cell extract by gel filtration, when prepared in 3% NaCl but not in seawater. The nucleic acid fraction was not required for enzymatic activity.     

Bacteriology of Manganese Nodules: IV. Induction of an MnO2-Reductase System in a Marine Bacillus1

Bacillus 29, isolated from a ferromanganese nodule from the Atlantic Ocean, was shown to possess an MnO(2)-reductase system which is induced in the presence of manganous ion. Maximal activity of the enzyme system was induced in about 5 hr in the presence of 4.35 mm MnSO(4) and was minimally dependent on the presence of either glucose or peptone and oxygen. Induction of optimal activity required the simultaneous presence of glucose and peptone. At least 30% of maximal activity was induced in 5 hr in the presence of 0.4 mum MnSO(4). Actinomycin D (5 mug/ml) or chloramphenicol (35 mug/ml), when added to the induction medium, inhibited approximately 90% of MnO(2)-reductase synthesis and incorporation of uracil-2-(14)C or leucine-1-(14)C. Cell-free extracts having MnO(2)-reductase activity were prepared by sonic disruption of cell suspensions of induced Bacillus 29. Such extracts used glucose metabolism as a source of electrons. They had an average specific activity of 1.15 nmoles of Mn(II) produced per mg of protein per hr at 25 C. They had a temperature optimum of 18 C for reductase activity and retained 50% of their activity at 4 C, the approximate temperature of the natural habitat of the organism. Extracts were stable for several days at 4 C but rapidly lost over 50% of their activity on freezing and thawing. Over 90% of the activity of the extract could be destroyed by heating in a boiling-water bath for 5 min. At a concentration of 1 mm, HgCl(2) and atebrine dihydrochloride inhibited MnO(2)-reductase activity by at least 50%, but sodium azide was ineffective. The MnO(2)-reductase activity of induced cells and extracts from them was no greater in the absence of oxygen than in its presence, confirming an earlier observation that MnO(2) and O(2) do not compete as terminal electron acceptors in the respiratory activity of this organism.     

Bacteriology of manganese nodules: III. Reduction of MnO(2) by two strains of nodule bacteria

MnO₂ reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolated from ferromanganese nodules, was assessed for 7 days. A 1-day lag was observed before the onset of MnO₂ reduction by either culture. Addition of HgCl₂ to a final concentration of about 10^-3 M caused a rapid cessation of MnO₂ reduction by the growing cultures. Neither culture reduced MnO₂ when grown under continued anaerobiosis from the start of an experiment. However, if conditions were made anaerobic after MnO₂ reduction was initiated, reduction continued at a rate only slightly lower than that under aerobic conditions. Resting-cell cultures reduced MnO₂ equally well aerobically and anaerobically, provided that ferricyanide was present to serve as electron carrier. These findings showed that oxygen is needed for culture adaptation to MnO₂ reduction, and that oxygen does not interfere with microbial MnO₂ reduction itself. Both cultures caused sharp drops in the pH of the medium during MnO₂ reduction: with coccus 32, during the entire incubation time; with Bacillus 29, for the first 3 days. The E_h of the medium fluctuated with either culture and never fell below 469 mv with Bacillus 29 and below 394 mv with coccus 32. The rates of glucose consumption and Mn²⁺ release by Bacillus 29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate production were not. Although acid production undoubtedly helped in the reduction of pyrolusite (MnO₂) by the bacteria, it did not appear to be important in the reduction of manganese oxide in ferromanganese nodules, as shown by the results with a nodule enrichment.     

Effects of High Hydrostatic Pressure on Coastal Bacterial Community Abundance and Diversity

Hydrostatic pressure is an important parameter influencing the distribution of microbial life in the ocean. In this study, the response of marine bacterial populations from surface waters to pressures representative of those under deep-sea conditions was examined. Southern California coastal seawater collected 5 m below the sea surface was incubated in microcosms, using a range of temperatures (16 to 3°C) and hydrostatic pressure conditions (0.1 to 80 MPa). Cell abundance decreased in response to pressure, while diversity increased. The morphology of the community also changed with pressurization to a predominant morphotype of small cocci. The pressure-induced community changes included an increase in the relative abundance of Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Flavobacteria largely at the expense of Epsilonproteobacteria. Culturable high-pressure-surviving bacteria were obtained and found to be phylogenetically similar to isolates from cold and/or deep-sea environments. These results provide novel insights into the response of surface water bacteria to changes in hydrostatic pressure.     

The Release of Extrinsic Polypeptides and Manganese Cluster from Photosystem 2 Membranes under High Hydrostatic Pressure

Three extrinsic polypeptides and manganese cluster were sequentially released from the membrane when photosystem 2 (PS2) membranes were kept under high hydrostatic pressure. The 17 kDa polypeptide was the most sensitive, while the 33 kDa polypeptide was the most reluctant to the treatment with high pressure. The release of manganese was not simply correlated with the loss of 33 kDa polypeptide. The losing of oxygen-evolving activity of PS2 was synchronised with the releasing of extrinsic polypeptides and manganese.     

Effect of Anions on the Binding and Oxidation of Divalent Manganese and Iron in Modified Bacterial Reaction Centers

The influence of different anions on the binding and oxidation of manganous and ferrous cations was studied in four mutants of bacterial reaction centers that can bind and oxidize these metal ions. Light-minus-dark difference optical and electron paramagnetic resonance spectroscopies were applied to monitor electron transfer from bound divalent metal ions to the photo-oxidized bacteriochlorophyll dimer in the presence of five different anions. At pH 7, bicarbonate was found to be the most effective for both manganese and iron binding, with dissociation constants around 1 μM in three of the mutants. The pH dependence of the dissociation constants for manganese revealed that only bicarbonate and acetate were able to facilitate the binding and oxidation of the metal ion between pH 6 and 8 where the tight binding in their absence could not otherwise be established. The data are consistent with two molecules of bicarbonate or one molecule of acetate binding to the metal binding site. For ferrous ion, the binding and oxidation was facilitated not only by bicarbonate and acetate, but also by citrate. Electron paramagnetic resonance spectra suggest differences in the arrangement of the iron ligands in the presence of the various anions.     

高静水压培养对内皮酯酶表达的影响

探讨高静水压培养对内皮脂酶表达的影响及其机制．分大气压(0 mmHg)和高于1个标准大气压的递增压力(120、150、180 mmHg)培养人脐静脉内皮细胞,用蛋白酶体抑制剂MG132进行干预．RT-PCR检测内皮脂酶 mRNA的表达,间接免疫荧光技术和流式细胞术检测内皮脂酶蛋白的表达．结果显示,高静水压培养明显促进内皮脂酶mRNA和蛋白质的表达,180 mmHg培养24 h内皮脂酶mRNA的表达较对照组上调2.2倍(P < 0.001),内皮脂酶蛋白表达较对照组上调2.54倍(P < 0.001)．蛋白酶体抑制剂MG132干预明显抑制180 mmHg培养诱导的内皮脂酶 mRNA的表达,MG132干预组内皮脂酶mRNA表达约为180 mmHg培养组的50% (P < 0.05)．结果说明,高静水压上调内皮脂酶 mRNA和蛋白质的表达,其机制可能与核因子-κB活化有关．     

Effect of Hydrostatic Pressure on Growth and Viability of Vibrio parahaemolyticus

Six strains of marine bacteria, including three strains of Vibrio parahaemolyticus, two Vibrio spp isolated from coastal regions, and the deep ocean isolate Pseudomonas bathycetes, were examined for ability to survive and grow at deep ocean hydrostatic pressures. V. parahaemolyticus and the coastal Vibrio spp. were unable to survive or grow at 200, 400, 600, 800, or 1,000 atm of pressure. In contrast, the deep ocean isolate P. bathycetes was capable of survival and growth at these pressures. The evidence strongly supports the neritic or estuarine origin and habitat for V. parahaemolyticus.     

高压力对限制性内切酶活性的影响

以质粒ｐＳＰＯＲＴ１为底物研究了高压力对Ⅱ型限制性内切酶ＨｉｎｄⅢ和ＸｂａⅠ反应的影响。高压力未引起两酶底物碱基特异性的改变,但二者反应活力均随压力升高而逐渐下降,ＸｂａⅠ对高压力更敏感。引起ＨｉｎｄⅢ和ＸｂａⅠ限制性内切酶活性完全丧失的压力分别为２００和１８０ＭＰａ,半失活压力分别为１３０和７５ＭＰａ。     

The Effects of Hydrostatic Pressure on Living Aquatic Organisms V. Eurybiotic Environmental Capacity as a Factor in High Pressure Tolerance

Study on the total spectrum of organisms (72 species) subjected to hydrostatic pressure as of this date allows one to established categories of pressure tolerance (resistance): Extremely high – eurybiotic forms (1000–1200 atm), High – marine littoral, planktonic, freshwater (600–1000 atm), Moderate – marine littoral, planktonic, freshwater (400–600 atm), Low – planktonic, freshwater (100–300 atm), Extremely low – planktonic, freshwater (0–100 atm). The average pressure tolerance of marine littoral species is higher than that of planktonic species but not significantly different from freshwater species. Eurybiotic species which are not marine seem to show the highest pressure tolerance.     

Calorimetric Study of Cowpea Protein Isolates. Effect of Calcium and High Hydrostatic Pressure

The thermal properties of cowpea protein isolates (CPI) were studied by differential scanning calorimetry under the influence of various conditions. An increase in the pH of protein extraction, from 8.0 to 10.0, during CPI preparation promoted a partial denaturation of cowpea proteins. Increases in enthalpy change of denaturation (ΔH) and temperature of denaturation (Td) were detected with increasing protein concentration from 7.5 to 10.5% (w/w). This behavior suggests that denaturation involves a first step of dissociation of protein aggregates. Calcium induced thermal stabilization in cowpea proteins, the increase in Td was ca. 0.3 °C/mM for protein dispersions of 7.5% (w/w) for 0 to 40 mM CaCl₂. High hydrostatic pressure (HHP) induced denaturation in CPI in a pressure level dependent manner. The presence of calcium protected cowpea proteins towards HHP-induced denaturation when pressure level was 400 MPa, but not when it was 600 MPa. Thermal properties of cowpea protein isolates were very sensitive to processing conditions, these behaviors would have implications in processing of CPI-containing foodstuff.     

Effect of Carbon and Energy Source on Bacterial Chromate Reduction

Studies were conducted to evaluate carbon and energy sources suitable to support hexavalent chromium (Cr(VI)) reduction by a bacterial consortium enriched from dichromate-contaminated aquifer sediments. The consortium was cultured under denitrifying conditions in a minimal, synthetic groundwater medium that was amended with various individual potential carbon and energy sources. The effects of these individual carbon and energy sources on Cr(VI) reduction and growth were measured. The consortium was found to readily reduce Cr(VI) with sucrose, acetate, L-asparagine, hydrogen plus carbon dioxide, ethanol, glycerol, glycolate, propylene glycol, or D-xylose as a carbon and energy source. Minimal Cr(VI) reduction was observed when the consortium was cultured with citrate, 2-ketoglutarate, L-lactate, pyruvate, succinate, or thiosulfate plus carbon dioxide as a carbon and energy source when compared with abiotic controls. The consortium grew on all of the above carbon and energy sources, with the highest cell densities reached using D-xylose and sucrose, demonstrating that the consortium is metabolically diverse and can reduce Cr(VI) using a variety of different carbon and energy sources. The results suggest that the potential exists for the enrichment of Cr(VI)-reducing microbial populations in situ by the addition of a sucrose-containing feedstock such as molasses, which is an economical and readily available carbon and energy source.     

The Effects of Hydrostatic Pressure on Living Aquatic Organisms VII. Size and Pressure Effects

A survey of the pressure resistance of aquatic animals in different stages of their life cycle shows that adults generally are more tolerant of pressure than the egg and nauplii, but older adults appear less pressure resistant than younger adults. Data on many species of aquatic animals of different size shows no correlation between size and pressure resistance. It is concluded that size is not a special determinant in the successful deep-sea colonization of shallow-water animals and this is consonant with the fact of occasional large deep-sea species whereas the average size is quite small in comparison with littoral species.     

Hydrogen and Formate Oxidation Coupled to Dissimilatory Reduction of Iron or Manganese by Alteromonas putrefaciens

The ability of Alteromonas putrefaciens to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory Fe(III) or Mn(IV) reduction was investigated. A. putrefaciens grew with hydrogen, formate, lactate, or pyruvate as the sole electron donor and Fe(III) as the sole electron acceptor. Lactate and pyruvate were oxidized to acetate, which was not metabolized further. With Fe(III) as the electron acceptor, A. putrefaciens had a high affinity for hydrogen and formate and metabolized hydrogen at partial pressures that were 25-fold lower than those of hydrogen that can be metabolized by pure cultures of sulfate reducers or methanogens. The electron donors for Fe(III) reduction also supported Mn(IV) reduction. The electron donors for Fe(III) and Mn(IV) reduction and the inability of A. putrefaciens to completely oxidize multicarbon substrates to carbon dioxide distinguish A. putrefaciens from GS-15, the only other organism that is known to obtain energy for growth by coupling the oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). The ability of A. putrefaciens to reduce large quantities of Fe(III) and to grow in a defined medium distinguishes it from a Pseudomonas sp., which is the only other known hydrogen-oxidizing, Fe(III)-reducing microorganism. Furthermore, A. putrefaciens is the first organism that is known to grow with hydrogen as the electron donor and Mn(IV) as the electron acceptor and is the first organism that is known to couple the oxidation of formate to the reduction of Fe(III) or Mn(IV). Thus, A. putrefaciens provides a much needed microbial model for key reactions in the oxidation of sediment organic matter coupled to Fe(III) and Mn(IV) reduction.     

The Effects of Hydrostatic Pressure on Living Aquatic Organisms VIII. Behavioral and Metabolic Responses of Uca pugilator to Variations in Hydrostatic Pressure and Temperature

In systematic examination of the pressure responses of a broad spectrum of organic life, it is very important to know the range of variation exhibited by a single species. As a consequence of extensive observations on the effects of pressure and temperature on behavioral responses, lethality, and metabolic responses, it is clear that the range of variation in pressure required to induce a response diminishes as the species taxon is approached. The rate of exposure to pressure does not influence the pressure required for reversible behavioral responses. In contrast, the duration of pressure dramatically influences the pressure required to achieve death with the shorter time periods requiring much higher pressure levels than the longer time periods. Notwithstanding this relationship there appears some evidence suggesting that short term acclimation to pressure does occur.     

Effects of Hydrostatic Pressure on Photosynthetic Systems. I. Preferential Destruction of the O2-Evolving Center

The effects of hydrostatic pressure treatments on the photosyntheticactivities of isolated thylakoids and PSII membranes were studied,and the following results were obtained. (1) The O₂-evolvingactivity was selectively inhibited by treatment at 200 MPa andabove, while the electron transport in the PSII reaction center,as measured by the photoreduction of 2,6-dichlorophenolindophenol(DCIP) with 1,5-diphenylcarbazide (DPC), was markedly enhanced.(2) The activity of PSI, as measured by the photoreduction ofmethylviologen with reduced DCIP, was not much affected evenafter treatment at 500 MPa, whereas this electron transportbecame uncoupled from phosphorylation at 200 MPa and above.(3) In pressure-treated PSII membranes, the EPR signal of Y⁺_zbecame photoinducible with the concomitant appearance of anEPR signal for Mn²⁺. (4) The 33-kDa extrinsic protein was retainedin inhibited PSII membranes, but the 17- and 23-kDa extrinsicproteins were lost. (5) Cross-linking of PSII proteins by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) suppressed the pressure-induced inactivation of the evolutionof O₂. (6) In pressure-treated PSII membranes, higher concentrationsof DCMU were required to inhibit the photoreduction of DCIP.Features of the pressure-induced inhibition of various reactionsin photosynthetic membranes are discussed on the basis of theseresults. ¹On leave from Advanced Research Laboratory, Hitachi Ltd., Hatoyama,Saitama, 350-03 Japan.