Comparison of Extracellular Enzyme Activities and Community Composition of Attached and Free-Living Bacteria in Porous Medium Columns

Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of β-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater β-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 × 10⁻⁷ nmol cell⁻¹ h⁻¹) than the free-living community (5.02 × 10⁻⁸ nmol cell⁻¹ h⁻¹). Conversely, the free-living community had a significantly higher cell-specific β-glucosidase activity (1.92 × 10⁻⁶ nmol cell⁻¹ h⁻¹) than the surface-associated community (6.08 × 10⁻⁷ nmol cell⁻¹ h⁻¹). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.     

Genotypic and Phenotypic Responses of a Riverine Microbial Community to Polycyclic Aromatic Hydrocarbon Contamination

The phenotypic and genotypic adaptation of a freshwater sedimentary microbial community to elevated (22 to 217 μg g [dry weight] of sediment⁻¹) levels of polycyclic aromatic hydrocarbons (PAHs) was determined by using an integrated biomolecular approach. Central to the approach was the use of phospholipid fatty acid (PLFA) profiles to characterize the microbial community structure and nucleic acid analysis to quantify the frequency of degradative genes. The study site was the Little Scioto River, a highly impacted, channelized riverine system located in central Ohio. This study site is a unique lotic system, with all sampling stations having similar flow and sediment characteristics both upstream and downstream from the source of contamination. These characteristics allowed for the specific analysis of PAH impact on the microbial community. PAH concentrations in impacted sediments ranged from 22 to 217 μg g (dry weight) of sediment⁻¹, while PAH concentrations in ambient sediments ranged from below detection levels to 1.5 μg g (dry weight) of sediment⁻¹. Total microbial biomass measured by phospholipid phosphate (PLP) analysis ranged from 95 to 345 nmol of PLP g (dry weight) of sediment⁻¹. Nucleic acid analysis showed the presence of PAH-degradative genes at all sites, although observed frequencies were typically higher at contaminated sites. Principal component analysis of PLFA profiles indicated that moderate to high PAH concentrations altered microbial community structure and that seasonal changes were comparable in magnitude to the effects of PAH pollution. These data indicate that this community responded to PAH contamination at both the phenotypic and the genotypic level.     

Extracellular Hydrolytic Enzyme Activities of the Heterotrophic Microbial Communities of the Rouge River: An Approach to Evaluate Ecosystem Response to Urbanization

The potential effects of urbanization on the bioavailability of dissolved organic carbon (DOC) were tested by determining the extracellular enzyme activities of the heterotrophic microbial communities of the Rouge River. The activities of 19 enzymes were monitored across two water samples (river water and groundwater) at different spatial and temporal scales. High phosphatase, esterase, and aminopeptidase activities was observed in site 9 (site most exposed to anthropogenic sources) showed higher concentrations of DOC compared to sites 1 and 8 (sites exposed to less anthropogenic sources), where moderate activities of diverse range of enzymes were observed. High relative contributions of phosphatase, esterase, and aminopeptidase activities to the overall enzyme activity as observed in site 9 stressed the increased importance of peptides as C source for heterotrophic communities and high in-stream carbon processing, which account for high nonspecific extracellular enzyme activities. In contrast, high contribution of glycosyl hydrolases occurred consistently across all sites, which highlights the significance of microbial detrital and plant biomass as carbon sources. Majority of the enzymes showed evidence of activity at various extents during spring and summer. However, higher activities of leucine aminopeptidase, valine aminopeptidase, β-glucosidase, and α-mannosidase were observed in the summer; and alkaline phosphatase and α-glucosidase in the spring. The results presented here suggest a shift in organic carbon bioavailability across all sites of contrasting urbanization, despite similarities in DOC concentrations. Hence, API ZYM technique can be used as an effective indicator of river water and groundwater system health across an urban gradient.     

Bacterial fluoride resistance,Fluc channels,and the weak acid accumulation effect

Fluoride ion (F⁻) is a ubiquitous environmental threat to microorganisms, which have evolved a family of highly selective “Fluc” F⁻ channels that export this inhibitory anion from their cytoplasm. It is unclear, however, how a thermodynamically passive mechanism like an ion channel can protect against high concentrations of external F⁻. We monitored external F⁻ concentrations in Escherichia coli suspensions and showed that, in bacteria lacking Fluc, F⁻ accumulates when the external medium is acidified, as a predicted function of the transmembrane pH gradient. This weak acid accumulation effect, which results from the high pKa (3.4) and membrane permeability of HF, is abolished by Fluc channels. We also found that, although bacterial growth is inhibited by high concentrations of F⁻, bacteria can withstand cytoplasmic F⁻ at levels a hundred times higher than those that inhibit proliferation, resuming growth when the F⁻ challenge is removed.     

Bacterioplankton Secondary Production Estimates for Coastal Waters of British Columbia, Antarctica, and California

The principal objective of this study was to quantify the rate of heterotrophic bacterioplankton production. Production was estimated by two approaches: (i) measurement of increasing bacterial abundance with time in filtered (3-μm pore size) seawater and (ii) estimation of bacterial deoxyribonucleic acid synthesis by tritiated thymidine incorporation in unfractionated seawater. The two approaches yielded comparable results when used at the Controlled Ecosystem Population Experiment (Saanich Inlet, British Columbia, Canada), at McMurdo Sound (Antarctica), and off Scripps Pier (La Jolla, Calif.). Estimated bacterioplankton production was lower in Antarctic samples (ranging from ~0 to 2.9 μg of C liter⁻¹ day⁻¹) than in those from the other two sites (ranging from 0.7 to 71 μg of C liter⁻¹ day⁻¹). In all three regions studied, it appeared that a significant fraction of the total primary production was utilized by the bacterioplankton and that substantial growth could occur in the absence of large particles. These results support the conclusion that bacterioplankton are a quantitatively important component of coastal marine food webs.     

Macrofauna regulate heterotrophic bacterial carbon and nitrogen incorporation in low-oxygen sediments

Oxygen minimum zones (OMZs) currently impinge upon >1 million km² of sea floor and are predicted to expand with climate change. We investigated how changes in oxygen availability, macrofaunal biomass and retention of labile organic matter (OM) regulate heterotrophic bacterial C and N incorporation in the sediments of the OMZ-impacted Indian continental margin (540–1100 m; [O₂]=0.35–15 μmol l⁻¹). In situ pulse-chase experiments traced ¹³C:¹⁵N-labelled phytodetritus into bulk sediment OM and hydrolysable amino acids, including the bacterial biomarker 𝒟-alanine. Where oxygen availability was lowest ([O₂]=0.35 μmol l⁻¹), metazoan macrofauna were absent and bacteria assimilated 30–90% of the labelled phytodetritus within the sediment. At higher oxygen levels ([O₂]=2–15 μmol l⁻¹) the macrofaunal presence and lower phytodetritus retention with the sediment occur concomitantly, and bacterial phytodetrital incorporation was reduced and retarded. Bacterial C and N incorporation exhibited a significant negative relationship with macrofaunal biomass across the OMZ. We hypothesise that fauna–bacterial interactions significantly influence OM recycling in low-oxygen sediments and need to be considered when assessing the consequences of global change on biogeochemical cycles.     

Enzymatic Activity on Sandy Beaches of the Ligurian Sea (NW Mediterranean)

Enzymatic activity was measured on two beaches of the Ligurian Sea (NW Mediterranean) during late spring and summer 2003. The detected activities (leucine aminopeptidase, β-glucosidase, α-glucosidase, and β-N-acetylglucosaminidase) were related to the available organic substrates (proteins and carbohydrates) and to the bacterial community (expressed in terms of abundance, biomass, and frequency of cell division). The very low chlorophyll a concentrations (never higher than 40 ng g⁻¹) suggested that heterotrophic microorganisms play a major role in the beach ecosystem. Enzymatic activities devoted to organic matter degradation were lower in the emerged part of the beaches and higher in the sites covered, permanently or temporarily, by seawater, suggesting that sea action enlivens the degradation processes. Leucine aminopeptidase ranged from 0.26 to 13.02 nmol g⁻¹h⁻¹, and β-glucosidase (the most expressed glycolytic enzyme) from 0.03 to 4.51 nmol g⁻¹h⁻¹. Strong changes in the proteolytic/glycolytic activity ratio were observed, with a sudden rise in glycolysis during summer, leading to ratio values from about 30 down to 1. Thus, beaches were identified as preferential degradation sites, where very refractory compounds such as cellulose may also be efficiently processed.     

Bacterial Biomass, Metabolic State, and Activity in Stream Sediments: Relation to Environmental Variables and Multiple Assay Comparisons

Bacterial biomass, metabolic condition, and activity were measured over a 16-month period in the surface sediments of the following four field sites with differing dissolved organic matter regimes: a woodlot spring seep, a meadow spring seep, a second-order stream, and a third-order stream. Total bacterial biomass was measured by lipid phosphate and epifluorescence microscopic counts (EMC), and viable biomass was measured by ¹⁴C most probable number, EMC with 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction, and ATP. Bacterial metabolic condition was determined from the percentage of respiring cells, poly-β-hydroxybutyrate concentrations, and adenylate energy charge. Activity measures included ¹⁴C-lipid synthesis, ³²P-phospholipid synthesis, the rate of uptake of algal lysate dissolved organic carbon, and respiration, from which biosynthesis was calculated (dissolved organic carbon uptake corrected for respiration). Total bacterial biomass (from EMC) ranged from 0.012 to 0.354 μg of C/mg of dry sediment and was usually lowest in the third-order stream. The percentage of cells respiring was less than 25% at all sites, indicating that most bacteria were dormant or dead. Adenylate energy charge was measured only in the third-order stream and was uniformly low. Poly-β-hydroxybutyrate concentrations were greater in the woodlot spring seep than in the second- and third-order streams. Uptake of algal lysate dissolved organic carbon ranged from undetectable levels to 166 mg of C · m⁻² · h⁻¹. Little community respiration could be attributed to algal lysate metabolism. Phospholipid synthesis ranged from 0.006 to 0.354 pmol · mg of dry sediment⁻¹ · h⁻¹. Phospholipid synthesis rates were used to estimate bacterial turnover at the study sites. An estimated 375 bacterial generations per year were produced in the woodlot spring seep, and 67 per year were produced in the third-order stream.     

A Unique Carboxyl-terminal Insert Domain in the Hematopoietic-specific,GTPase-deficient Rho GTPase RhoH Regulates Post-translational Processing

RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family that was first identified as a hypermutable gene in human B lineage lymphomas. RhoH remains in a constitutively active state and thus its effects are regulated by expression levels or post-translational modifications. Similar to other small GTPases, intracellular localization of RhoH is dependent upon the conserved “CAAX” box and surrounding sequences within the carboxyl (C) terminus. However, RhoH also contains a unique C-terminal “insert” domain of yet undetermined function. RhoH serves as adaptor molecule in T cell receptor signaling and RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia. Disease progression is attenuated in a Rhoh^−/− mouse model of chronic lymphocytic leukemia and treatment of primary human chronic lymphocytic leukemia cells with Lenalidomide results in reduced RhoH protein levels. Thus, RhoH is a potential therapeutic target in B cell malignancies. In the current studies, we demonstrate that deletion of the insert domain (LFSINE) results in significant cytoplasmic protein accumulation. Using inhibitors of degradation pathways, we show that LFSINE regulates lysosomal RhoH uptake and degradation via chaperone-mediated autophagy. Whereas the C-terminal prenylation site is critical for ZAP70 interaction, subcellular localization and rescue of the Rhoh^−/− T cell defect in vivo, the insert domain appears dispensable for these functions. Taken together, our findings suggest that the insert domain regulates protein stability and activity without otherwise affecting RhoH function.     

Spatial Differences in East Scotia Ridge Hydrothermal Vent Food Webs: Influences of Chemistry,Microbiology and Predation on Trophodynamics

The hydrothermal vents on the East Scotia Ridge are the first to be explored in the Antarctic and are dominated by large peltospiroid gastropods, stalked barnacles (Vulcanolepas sp.) and anomuran crabs (Kiwa sp.) but their food webs are unknown. Vent fluid and macroconsumer samples were collected at three vent sites (E2, E9N and E9S) at distances of tens of metres to hundreds of kilometres apart with contrasting vent fluid chemistries to describe trophic interactions and identify potential carbon fixation pathways using stable isotopes. δ¹³C of dissolved inorganic carbon from vent fluids ranged from −4.6‰ to 0.8‰ at E2 and from −4.4‰ to 1.5‰ at E9. The lowest macroconsumer δ¹³C was observed in peltospiroid gastropods (−30.0‰ to −31.1‰) and indicated carbon fixation via the Calvin-Benson-Bassham (CBB) cycle by endosymbiotic gamma-Proteobacteria. Highest δ¹³C occurred in Kiwa sp. (−19.0‰ to −10.5‰), similar to that of the epibionts sampled from their ventral setae. Kiwa sp. δ¹³C differed among sites, which were attributed to spatial differences in the epibiont community and the relative contribution of carbon fixed via the reductive tricarboxylic acid (rTCA) and CBB cycles assimilated by Kiwa sp. Site differences in carbon fixation pathways were traced into higher trophic levels e.g. a stichasterid asteroid that predates on Kiwa sp. Sponges and anemones at the periphery of E2 assimilated a proportion of epipelagic photosynthetic primary production but this was not observed at E9N. Differences in the δ¹³C and δ³⁴S values of vent macroconsumers between E2 and E9 sites suggest the relative contributions of photosynthetic and chemoautotrophic carbon fixation (rTCA v CBB) entering the hydrothermal vent food webs vary between the sites.     

Deletion of the Complement C5a Receptor Alleviates the Severity of Acute Pneumococcal Otitis Media following Influenza A Virus Infection in Mice

There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa^−/−) or factor B (Bf ^−/−) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection.     

Microelectrode Measurements of Nitrate Gradients in the Littoral and Profundal Sediments of a Meso-Eutrophic Lake (Lake Vechten, The Netherlands)

NO₃⁻ concentration profiles were measured in the sediments of a meso-eutrophic lake with a newly developed microelectrode. The depth of penetration of NO₃⁻ varied from only 1.3 mm in organic-rich profundal silty sediments to 5 mm in organic-poor littoral sandy sediments. The thickness of the zone of denitrification in the organic-rich sediments was <500 μm. Oxygen profiles measured simultaneously revealed that the zone of denitrification was directly adjacent to the aerobic zone. The results demonstrate high denitrification rates (0.26 to 1.31 mmol m⁻² day⁻¹) at in situ nitrate concentrations in the overlying water (0.030 mmol liter⁻¹) and limitation of denitrification by nitrate availability.     

Molecular Analysis of Geographic Patterns of Eukaryotic Diversity in Antarctic Soils

We describe the application of molecular biological techniques to estimate eukaryotic diversity (primarily fungi, algae, and protists) in Antarctic soils across a latitudinal and environmental gradient between approximately 60 and 87°S. The data were used to (i) test the hypothesis that diversity would decrease with increasing southerly latitude and environmental severity, as is generally claimed for “higher” faunal and plant groups, and (ii) investigate the level of endemicity displayed in different taxonomic groups. Only limited support was obtained for a systematic decrease in diversity with latitude, and then only at the level of a gross comparison between maritime (Antarctic Peninsula/Scotia Arc) and continental Antarctic sites. While the most southerly continental Antarctic site was three to four times less diverse than all maritime sites, there was no evidence for a trend of decreasing diversity across the entire range of the maritime Antarctic (60 to 72°S). Rather, we found the reverse pattern, with highest diversity at sites on Alexander Island (ca. 72°S), at the southern limit of the maritime Antarctic. The very limited overlap found between the eukaryotic biota of the different study sites, combined with their generally low relatedness to existing sequence databases, indicates a high level of Antarctic site isolation and possibly endemicity, a pattern not consistent with similar studies on other continents.     

Role of Sulfate Reduction Versus Methanogenesis in Terminal Carbon Flow in Polluted Intertidal Sediment of Waimea Inlet, Nelson, New Zealand

An investigation of the terminal anaerobic processes occurring in polluted intertidal sediments indicated that terminal carbon flow was mainly mediated by sulfate-reducing organisms in sediments with high sulfate concentrations (>10 mM in the interstitial water) exposed to low loadings of nutrient (equivalent to <10² kg of N · day⁻¹) and biochemical oxygen demand (<0.7 × 10³ kg · day⁻¹) in effluents from different pollution sources. However, in sediments exposed to high loadings of nutrient (>10² kg of N · day⁻¹) and biochemical oxygen demand (>0.7 × 10³ kg · day⁻¹), methanogenesis was the major process in the mediation of terminal carbon flow, and sulfate concentrations were low (≤2 mM). The respiratory index [¹⁴CO₂/(¹⁴CO₂ + ¹⁴CH₄)] for [2-¹⁴C]acetate catabolism, a measure of terminal carbon flow, was ≥0.96 for sediment with high sulfate, but in sediments with sulfate as little as 10 μM in the interstitial water, respiratory index values of ≤0.22 were obtained. In the latter sediment, methane production rates as high as 3 μmol · g⁻¹ (dry weight) · h⁻¹ were obtained, and there was a potential for active sulfate reduction.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Chemoautotrophic Carbon Fixation Rates and Active Bacterial Communities in Intertidal Marine Sediments

Chemoautotrophy has been little studied in typical coastal marine sediments, but may be an important component of carbon recycling as intense anaerobic mineralization processes in these sediments lead to accumulation of high amounts of reduced compounds, such as sulfides and ammonium. We studied chemoautotrophy by measuring dark-fixation of ¹³C-bicarbonate into phospholipid derived fatty acid (PLFA) biomarkers at two coastal sediment sites with contrasting sulfur chemistry in the Eastern Scheldt estuary, the Netherlands. At one site where free sulfide accumulated in the pore water right to the top of the sediment, PLFA labeling was restricted to compounds typically found in sulfur and ammonium oxidizing bacteria. At the other site, with no detectable free sulfide in the pore water, a very different PLFA labeling pattern was found with high amounts of label in branched i- and a-PLFA besides the typical compounds for sulfur and ammonium oxidizing bacteria. This suggests that other types of chemoautotrophic bacteria were also active, most likely Deltaproteobacteria related to sulfate reducers. Maximum rates of chemoautotrophy were detected in first 1 to 2 centimeters of both sediments and chemosynthetic biomass production was high ranging from 3 to 36 mmol C m⁻² d⁻¹. Average dark carbon fixation to sediment oxygen uptake ratios were 0.22±0.07 mol C (mol O₂)⁻¹, which is in the range of the maximum growth yields reported for sulfur oxidizing bacteria indicating highly efficient growth. Chemoautotrophic biomass production was similar to carbon mineralization rates in the top of the free sulfide site, suggesting that chemoautotrophic bacteria could play a crucial role in the microbial food web and labeling in eukaryotic poly-unsaturated PLFA was indeed detectable. Our study shows that dark carbon fixation by chemoautotrophic bacteria is a major process in the carbon cycle of coastal sediments, and should therefore receive more attention in future studies on sediment biogeochemistry and microbial ecology.     

Effects of Glucose Concentrations on Cadmium, Copper, Mercury, and Zinc Toxicity to a Klebsiella sp

The influence of glucose concentration on Cd, Cu, Hg, and Zn toxicity to a Klebsiella sp. was studied by following the degradation of ¹⁴C-labeled glucose at pH 6.0. Uptake of ¹⁴C into the cells was also determined. The carbon concentrations ranged from 0.01 to 40 mg liter⁻¹, which are equivalent to soluble C concentrations in natural environments. The toxicity of Cu, Cd, and Zn to a Klebsiella sp. was affected considerably by the C concentration. Copper at 10⁻⁵ M was toxic when the carbon concentration was 10 or 40 mg liter⁻¹, while at 0.01 to 1.0 mg liter⁻¹ no toxicity was observed. Cadmium and zinc were toxic at 10⁻² M in media containing 0.01 to 1.0 mg of C liter⁻¹. At C concentrations greater than 1.0 mg liter⁻¹, the inhibition of glucose degradation and carbon assimilation was observed at 10⁻³ M Cd and Zn. The toxicity of mercury seemed to be independent of the C concentration. Results of this study showed that the nutritional state of an organism may have a profound effect on its sensitivity to metals. Metals taken up by an energy-driven transport system may be less toxic under conditions of C starvation. The C concentration should be taken into account when evaluating results from toxicity studies, especially as most microorganisms in nature live under energy-limited conditions.     

Structural Effects of Cl and Other Anions on the Water Oxidizing Complex of Chloroplast Photosystem II

To further our understanding of the role of Cl⁻ and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl⁻ free media of pH ≈ 7 was enhanced by millimolar concentrations of the divalent anion SO₄²⁻ and also by divalent cations like Mg²⁺ and Ca²⁺; (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl⁻ = Br⁻ > NO₃⁻ > F⁻ > ClO₄⁻; (c) at molar concentrations, SO₄²⁻ stabilized the binding of the 23 kilodalton polypeptide, while Cl⁻ and Br⁻ became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0°C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl⁻, or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly, but is essential for the catalytic mechanism as well. Binding of, or charge screening by, divalent ions interferes with this process. At high salt concentrations, all these effects are overridden by “lyotropic” actions of the solutes that affect the integrity of the water oxidizing protein complex by stabilizing or disrupting critical hydrophobic domains.