Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties

Amphitrite ornata dehaloperoxidase (DHP) and Notomastus lobatus chloroperoxidase (NCPO) catalyze the peroxide-dependent dehalogenation of halophenols and halogenation of phenols, respectively. Both enzymes have histidine (His) as their proximal heme iron ligand. Crystallographic examination of DHP revealed that it has a globin fold [M.W. LaCount, E. Zhang, Y.-P. Chen, K. Han, M.M. Whitton, D.E. Lincoln, S.A. Woodin, L. Lebioda, J. Biol. Chem. 275 (2000) 18712-18716] and kinetics studies established that ferric DHP is the active state [R.L. Osborne, L.O. Taylor, K. Han, B. Ely, J.H. Dawson, Biochem. Biophys. Res. Commun. 324 (2004) 1194-1198]. NCPO likely has these same properties. Previous work with His-ligated heme proteins has revealed characteristic spectral distinctions between dioxygen binding globins and peroxide-activating peroxidases. Since DHP, and likely NCPO, is a peroxide-activating globin, we have sought to determine in the present investigation whether the ferric resting states of these two novel heme-containing enzymes are myoglobin-like or peroxidase-like. To do so, we have examined their exogenous ligand-free ferric states as well as their azide, imidazole and NO bound ferric adducts (and ferrous-NO complexes) with UV-Visible absorption and magnetic circular dichroism spectroscopy. We have also compared each derivative to the analogous states of horse heart myoglobin (Mb) and horseradish peroxidase (HRP). The spectra observed for parallel forms of DHP and NCPO are virtually identical to each other as well as to the spectra of the same Mb states, while being less similar to the spectra of corresponding HRP derivatives. From these data, we conclude that exogenous ligand-free ferric DHP and NCPO are six-coordinate with water and neutral His as ligands. This coordination structure is distinctly different from the ferric resting state of His-ligated peroxidases and indicates that DHP and NCPO do not activate bound peroxide through a mechanism dependent on a push effect imparted by a partially ionized proximal His as proposed for typical heme peroxidases.     

Species-abundance-biomass responses by estuarine macrobenthos to sediment chemical contamination

Macrobenthic community responses can be measured through concerted changes in univariate metrics,including species richness, total abundance, and totalbiomass. The classic model of pollution effects onmarine macrobenthic communities recognizes thatspecies/abundance/biomass (SAB) curves varydistinctively in a nonlinear manner with the magnitudeof organic enrichment. For example, at moderatelevels of organic enrichment, small-bodiedopportunistic species boost the abundance curve, whilespecies richness falls. Ratios among the metrics formuseful indicators of how the community changes withorganic enrichment. However, the classic SAB model isbased on organic enrichment effects over small spatialand temporal scales, and the applicability of the SABmodel to sediment chemical contamination and acrossbroad natural estuarine gradients is largely unknown. Here, SAB responses were examined with respect toprimary gradients in metals and organic chemicalsbased on an extensive dataset comprising 319 estuarinesites from throughout the northern Gulf of Mexico. Each SAB metric was first adjusted with respect to thethree primary natural estuarine gradients, salinity,depth, and sediment silt/clay content. Adjusted SABrelationships varied in their details with respect todifferent classes of sediment contamination, but alltypes of SAB stress responses appear to exhibitsimilar basic characteristics. As in the SAB model,all three SAB metrics were notably low at the highestconcentrations of both metal and organic-chemicalcontaminants. Moreover, rapid decreases in the B/Aratio with increasing contamination supported theconcept that relatively long-lived, large-bodied,equilibrium taxa decline markedly at highconcentrations of toxicants.     

Dark respiration in the marine macroalga Chondrus crispus (Rhodophyceae)

Dark respiration in the red macroalga Chondrus crispus was studied under a variety of conditions. The components of respiration were examined using selective inhibitors in order to characterise pathways of respiration and examine regulation of respiration in marine macroalgae.In comparison to respiration rates generally reported for higher-plant leaves and roots, the steady-state rate of O₂ consumption by this alga, after 30 min dark pretreatment, was found to be quite low (three- to sixfold lower than in higher plants). The addition of uncoupler had only a slight effect on the basal respiration rate, indicating that in these conditions, substrate supply could be limiting respiration. The addition of KCN inhibited respiration by approx. 60%, indicating the presence of alternative oxidase activity. The coefficient of engagement of the alternative pathway (calculated from the data herein) showed that under normal conditions there was little participation of the alternative pathway in O₂ consumption. The response of respiration to O₂ tension was examined with and without inhibitors and the apparent K _m was 17 to 21 M. The addition of KCN plus salicylhydroxamic acid almost completely blocked respiration in C. crispus. The hypothesis that respiratory substrate limits respiration in this alga was investigated by measuring respiration rates immediately after periods of photosynthetic activity. It was found that the respiration rate was dependent on the duration of the light period and could increase up to twofold. This stimulated rate of respiration declined in a first-order fashion during the next 20 to 30 min, finally reaching the basal, zero-order rate measured before illumination. These results strongly indicate a change in the nature of the respiratory substrates during this period. No change in the contribution of the alternative pathway of respiration could be detected following light pretreatment.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - SHAM salicyl hydroxamic acid     

Recent patterns of sediment accumulation in a small closed eutrophic lake revealed by the sediment records

A short-core palaeolimnological investigation was undertaken with the aim ofacquiring knowledge of sediment deposition. Analyses of the lithological composition of sediments from the whole-lake basin were performed on the small eutrophic L. Linajärv (northern Estonia) and the concentrations of mineral and organic matter were measured on 647 sub-samples from 14 sediment cores. The accumulation rate of the sediment sequences was established and C/N ratios of organic matter in some cores were recorded. Results indicate that the water depth, basin slopes and distance to the shore have the most important impact on the physical sediment properties. It was shown that variations in the mineral matter concentrations were influenced by the changes in deposition conditions in the areas with steep slopes. The study indicated that more objective information about the sedimentation mechanisms is obtained using analysis of the concentration ratio of mineral and organic matter since it reduces the implied role of diagenetic compaction.     

Degradation of nonane by bacteria from Antarctic marine sediment

A microbial enrichment culture tolerant of petroleum hydrocarbons was developed from sediment collected near Casey Station, Antarctica. To select cold-adapted microbes that would degrade diesel, enrichments were cultured at 0°C during six successive transfers to fresh medium, with Special Antarctic Blend diesel (SAB) as the sole carbon source. Biodegradation of components of the SAB was then measured in microcosms inoculated with the enrichment culture. After 16 weeks, the amount of biodegradation was small, but nonane (a C9 alkane) had degraded significantly more in inoculated microcosms than in sterile controls. DNA was then extracted from the enrichment cultures and a fragment of the 16S rRNA gene was amplified for denaturing gradient gel electrophoresis. Bands were excised from the gel and, following sequencing, were found to belong to the genera Pseudomonas and Colwellia.     

Biodegradation of cyclic nitramines by tropical marine sediment bacteria

Undersea deposition of unexploded ordnance (UXO) constitutes a potential source of contamination of marine environments by hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). The goal of the present study was to determine microbial degradation of RDX and HMX in a tropical marine sediment sampled from a coastal UXO field in the region of Oahu Island in Hawaii. Sediment mixed cultures growing in marine broth 2216 (21°C) anaerobically mineralized 69% or 57% (CO₂, 25 days) of the total carbon of [UL-¹⁴ C]-RDX (100 M) or [UL-¹⁴ C]-HMX (10 M), respectively. As detected by PCR-DGGE, members of -proteobacteria (Halomonas), sulfate-reducing -proteobacteria (Desulfovibrio), firmicutes (Clostridium), and fusobacterium appeared to be dominant in RDX-enrichment and/or HMX-enrichment cultures. Among 22 sediment bacterial isolates screened for RDX and HMX biodegradation activity under anaerobic conditions, 5 were positive for RDX and identified as Halomonas (HAW-OC4), Marinobacter (HAW-OC1), Pseudoalteromonas (HAW-OC2 and OC5) and Bacillus (HAW-OC6) by their 16S rRNA genes. Sediment bacteria degraded RDX to N₂O and HCHO via the intermediary formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine. The present findings demonstrate that cyclic nitramine contaminants are likely to be degraded upon release from UXO into tropical marine sediment.     

Tolerance of the invasive marine alga Caulerpa taxifolia to burial by sediment

Beds of the invasive alga Caulerpa taxifolia were observed to be buried with sediment after heavy rainfall, and it was proposed that burial and subsequent uncovering could help explain rapid changes in the sizes of beds of C. taxifolia. Responses of C. taxifolia fragments to the different extents of burial and for different durations (2, 6, 17 days) were investigated in the laboratory. Once uncovered, fragments in all treatments began to grow, and by 92 days partially buried fragments were not significantly different from controls in terms of the numbers of stolon meristems or total stolon length. Fragments buried totally were severely affected, but nevertheless, 35% of the fragments survived total burial for 17 days, and began to recover slowly when the sediment was removed. C. taxifolia can, therefore, persist for long periods under sediments, meaning that, in areas where burial is likely, the actual distribution of the species may be underestimated. Moreover, any potential control technique needs to penetrate sediments to be totally effective.     

Effects of sediment anoxia and light on turion germination and early growth of Potamogeton crispus

Potamogeton crispus is a cosmopolitan aquatic species and is widely used as a pioneer species for vegetation restoration of eutrophic lakes. However, many restoration projects applying P. crispus turions have not been successful. Earlier studies focused on effects of light and temperature on turion germination. The purpose of this study was to determine whether sediment anoxia and light interactively affected the turion germination and early growth of P. crispus. Anoxic conditions in the experiment were produced by adding sucrose to the sediment. The germination rate of the turions was 68–73% lower in the highly anoxic condition treatment than in the control. Medium light intensity (10% of natural light at the water surface) was more favorable for germination under slightly anoxic conditions than either low or high light intensity. The growth of newly-formed sprouts was also significantly inhibited by sediment anoxia. Photosynthesis and shoot biomass were reduced under sediment anoxia, whereas total chlorophyll content, root biomass, and soluble protein content were highest in the low anoxic condition treatment. Medium light improved net photosynthesis and biomass production of the sprouts. We conclude that turion germination and sprout growth can be significantly inhibited by sediment anoxia. Medium light intensity may alleviate this inhibition by anoxia, but light has little effect when sediment anoxia is severe. For the purposes of vegetation restoration, more attention should be paid to the role of sediment anoxia, and it is necessary to improve sediment and light conditions for turion germination and early growth of P. crispus in eutrophic lakes. These results will contribute to a more complete understanding of turion germination dynamics of P. crispus and will be useful for future restoration programs. Handling editor: S. M. Thomaz     

Conspicuous veils formed by vibrioid bacteria on sulfidic marine sediment

We describe the morphology and behavior of a hitherto unknown bacterial species that forms conspicuous veils (typical dimensions, 30 by 30 mm) on sulfidic marine sediment. The new bacteria were enriched on complex sulfidic medium within a benthic gradient chamber in oxygen-sulfide countergradients, but the bacteria have so far not been isolated in pure culture, and a detailed characterization of their metabolism is still lacking. The bacteria are colorless, gram-negative, and vibrioid-shaped (1.3- to 2.5- by 4- to 10- micro m) cells that multiply by binary division and contain several spherical inclusions of poly-beta-hydroxybutyric acid. The cells have bipolar polytrichous flagella and exhibit a unique swimming pattern, rotating and translating along their short axis. Free-swimming cells showed aerotaxis and aggregated at ca. 2 micro M oxygen within opposing oxygen-sulfide gradients, where they were able to attach via a mucous stalk, forming a cohesive whitish veil at the oxic-anoxic interface. Bacteria attached to the veil kept rotating and adapted their stalk lengths dynamically to changing oxygen concentrations. The joint action of rotating bacteria on the veil induced a homogeneous water flow from the oxic water region toward the veil, whereby the oxygen uptake rate could be enhanced up to six times, as shown by model calculations. The veils showed a pronounced succession pattern. New veils were generated de novo within 24 h and had a homogeneous whitish translucent appearance. Bacterial competitors or eukaryotic predators were apparently kept away by the low oxygen concentration prevailing at the veil surface. Frequently, within 2 days the veil developed a honeycomb pattern of regularly spaced holes. After 4 days, most veils were colonized by grazing ciliates, leading to the fast disappearance of the new bacteria. Several-week-old veils finally developed into microbial mats consisting of green, purple, and colorless sulfur bacteria.     

Active transport and accumulation of iodide by newly isolated marine bacteria

Iodide (I(-))-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive (125)I(-). When they were grown in a liquid medium containing 0.1 microM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 microM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 +/- 3.6 (mean +/- standard deviation) pmol of iodide per mg of dry cells and 5.5 x 10(3), respectively. In the presence of much higher concentrations of iodide (1 microM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 muM and 0.55 pmol min(-1) mg of dry cells(-1), respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO(3)(-)), was not transported.     

Phosphorus accumulation by a marine photosynthetic bacterium, Chromatiumsp.

Among 8 strains of typical marine and freshwater photosynthetic bacteria, a Chromatium sp., which is known to flocculate upon the addition of seawater, had the highest intracellular phosphorus content of 0.865 mmol P/mg dry wt with a ratio of polyphosphate to phosphate (poly-Pi/Pi) of 2.14. Intracellular phosphorus content increased withincreasing NaCl concentration up to 260 mM. The poly-Pi/Pi value was also the highest in the medium containing 260 mM NaCl. Sodium cation in seawater enhanced intracellular phosphorus accumulation in this bacterium.     

Influence of the cell envelope on the accumulation of cadmium by a marine pseudomonad

The accumulation of cadmium by a sensitive marine pseudomonad was investigated on the basis of the physiological state of its cell envelope. The more impaired the envelope the greater the accumulation. This phenomenon was due to a greater number of adsorption sites for cadmium on the inner layers and/or to the impermeability of the cell envelope.