Proton Efflux from Roots of Intact Spergularia marina Plants

Despite numerous considerations of the importance of protonextrusion to plant mineral nutrition, measurements of net H⁺efflux rates in photo-autotrophic plants growing in completemedium are rare, and the rates of efflux reported are considerablylower than that typical of low-salt seedlings. We present herethe first report of short term net proton efflux from rootsof a halophyte. and the highest rate of net efflux reportedto date for any higher plant, in both low-salt and completenutrient media. We also show responses of that efflux to a varietyof treatments, including NaCl fusicoccin, shoot excision anddarkness. Key words: Halophyte, Sea spurrey, H⁺ efflux, Roots     

Proton Electrochemical Gradients in Washed Cells of Nitrosomonas europaea and Nitrobacter agilis

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [³H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, ¹⁴C-benzoic and ¹⁴C-acetyl salicylic, and the weak base [¹⁴C]methylamine. Nitrosomonas europaea accumulated ¹⁴C-benzoic acid and ¹⁴C-acetyl salicylic acid when the external pH was below 7.0 and [¹⁴C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [¹⁴C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Δψ only contributed to Δp. In Nitrosomonas europaea the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.     

Competition between Picoplanktonic Cyanobacteria and Heterotrophic Bacteria along Crossed Gradients of Glucose and Phosphate

A laboratory experiment was performed to test whether differences in nutrient and energy demands between picophytoplankton and heterotrophic bacteria can explain the apparent inverse biomass relationship between these organisms in lakes along gradients of organic carbon and nutrients. Growth rates and final yield of cells were analyzed in crossed gradients of glucose and phosphate. Concentrations of phosphate (10, 25, and 60 microg P L(-1)) and glucose (0, 0.3, and 3 mg C L(-1)) were used in all possible combinations giving 9 different treatments. Heterotrophic bacteria had higher maximum growth rates in all treatments and became larger than picophytoplankton in many treatments. The variance in abundance of heterotrophic bacteria between treatments could almost completely be explained by the combined effects of glucose and P. In treatments where carbon limitation slowed down the growth of heterotrophic bacteria, picophytoplankton became abundant and these organisms showed a positive response to P in combination with a negative response to glucose. The negative effect of glucose on picophytoplankton is suggested to be indirect and caused by competition with bacteria that are favored by organic C. The results suggest that competition for phosphate between phytoplankton and bacteria is not size-dependent, that heterotrophic bacteria are superior competitors for P, and that organic C produced by picophytoplankton was of minor importance for heterotrophic bacteria.     

Proton Pump Activity of Mitochondria-rich Cells : The Interpretation of External Proton-concentration Gradients

We have hypothesized that a major role of the apical H⁺-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl⁻ via an apical Cl⁻/HCO₃⁻-exchanger. The activity of the H⁺ pump was studied by monitoring mucosal [H⁺]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 ± 0.07 (mean ± SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 ± 18 μm, corresponding to an estimated “unstirred layer” thickness of 329 ± 29 μm. Mucosal acidification was dependent on serosal pCO₂, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H⁺. The [H⁺] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H⁺]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H⁺ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace''s equation. Model calculations predicted that with a physiological density of MR cells, the [H⁺] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H⁺]–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H⁺]-gradient depended on the mucosal anion such as to indicate that base (HCO₃⁻) is excreted in exchange not only for Cl ⁻, but also for Br⁻ and I⁻, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.     

Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients

Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 x 10(6) daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 x 10(6) to 39 x 10(6) daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with (3)H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments.     

Cyanobacteria in rice soils

Cyanobacteria were recovered from each of 38 soil samples collected from local rice fields. Of the 84 species belonging to 31 genera that were isolated, 42 were heterocystous diazotrophic species belonging to 14 genera and the remaining were non-heterocystous. Fischerella, Nostoc and Calothrix were widespread.Z.U.M. Khan is with the Department of Botany, Jahangirnagar University, Savar, Dhaka, Bangladesh. Z.N. Tahmida Begum and M.Z. Hossain are with the Department of Botany and R. Mandal is with the Department of Soil Science, bot at the University of Dhaka, Dhaka-1000, Bangladesh;     

Cyanobacteria in mangrove ecosystems

Mangroves are subject to the effects of tides and fluctuations in environmental conditions, which may reach extreme conditions. These ecosystems are severely threatened by human activities despite their ecological importance. Although mangroves are characterized by a highly specialized but low plant diversity in comparison to most other tropical ecosystems, they support a diverse microbial community. Adapted microorganisms in soil, water, and on plant surfaces perform fundamental roles in nutrient cycling, especially nitrogen and phosphorus. Cyanobacteria contribute to carbon and nitrogen fixation and their cells act as phosphorus storages in ecosystems with extreme or oligotrophic environmental conditions such as those found in mangroves. As the high plant productivity in mangroves is only possible due to interactions with microorganisms, cyanobacteria may contribute to these ecosystems by providing fixed nitrogen, carbon, and herbivory-defense molecules, xenobiotic biosorption and bioremediation, and secreting plant growth-promoting substances. In addition to water, cyanobacterial colonies have been detected on sediments, rocks, decaying wood, underground and aerial roots, trunks, and leaves. Some mangrove cyanobacteria were also found in association to algae or seagrasses. Few studies on mangrove cyanobacteria are available, but together they have reported a substantial number of species in these ecosystems. However, the cyanobacterial diversity in this biome has been traditionally underestimated. Though mangrove communities generally host cyanobacterial taxa commonly found in marine environments, unique microhabitats found in mangroves potentially harbor several undescribed cyanobacterial taxa. The relevance of cyanobacteria for mangrove conservation is highlighted in their use for the recovery of degraded mangroves as biostimulants or in bioremediation.     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Ultrasensitive glycogen synthesis in Cyanobacteria

Cyanobacter ADPglucose pyrophosphorylase exhibits a ultrasensitive response in activity towards its allosteric effector 3-phosphoglycerate, elicited by orthophosphate and polyethyleneglycol-induced molecular crowding. The ultrasensitive response was observed either when the enzyme operates in the zero or first order region for its physiological substrates. The ultrasensitivity exhibited maximal amplification factors of 15-19-fold with respect to 1% of the maximal system velocity. Only a 2.4-3.8-fold increase in 3PGA concentration was necessary to augment the flux from 10% to 90% through AGPase as compared with 200-fold required for the control. The results are discussed in terms of finely tuned regulatory mechanisms of polysaccharide synthesis in oxygenic photosynthetic organisms.     

Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics)

The potency of various uncouplers for collapsing the light-induced pH gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane pH gradient ([delta]pH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable [delta]pH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the [delta]pH was determined using the empirical relationship between the [delta]pH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the pH gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast.     

Cyanobacteria toxins in the Salton Sea

Background

Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants.

Results

Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6–4 photoproducts from their DNA after irradiation with 150 J/m² of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry.

Conclusion

Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6–4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.     

Distribution of Plasmids in Heterocystous Cyanobacteria

The distribution of plasmids was studied in 15 different heterocystous cyanobacteria using agarose gel electrophoresis. Out of these, six were found to possess only one plasmid DNA of size ranging from 30–33 mD. A simplified and less time consuming protocol for studying the plasmids has been suggested.     

Aminopeptidase Activity in Marine Chroococcoid Cyanobacteria

Synechococci are important primary producers in the ocean and can also utilize some components of the dissolved organic matter (DOM). The readily utilizable DOM in seawater is mainly polymeric (e.g., protein, polysaccharide) or phosphorylated and requires hydrolysis prior to uptake. We examined whether synechococci express ectoenzymes to hydrolyze DOM components and considered the possible significance of ectohydrolases for Synechococcus ecology and organic matter cycling in the sea. Five strains of non-nitrogen-fixing synechococci in axenic cultures were tested for enzyme activities with fluorogenic substrates. All strains show ectocellular aminopeptidase activity, but other enzymes were undetectable. The aminopeptidase level was in the range determined for five marine heterotrophic bacterial isolates tested for comparison. Aminopeptidase was not secreted into the medium; the majority (74%; tested in WH 7803) was cell surface bound, and a small fraction was periplasmic. The periplasmic activity was not released by cold osmotic shock of WH 7803. Phenylmethylsulfonyl fluoride and EDTA, inhibitors of serine and metalloproteases, strongly or completely inhibited WH 7803 aminopeptidase. The enzyme seemed constitutive; per-cell activity did not change during incubations in unenriched seawater, bovine serum albumin, or nitrate-replete mineral medium. In natural planktonic assemblages in the Southern California Bight, aminopeptidase activity was correlated with Synechococcus abundance as well as the abundance of other bacteria. Ectocellular aminopeptidase may be common in marine synechococci and play roles in their nitrogen nutrition, particularly in low-nitrate and low-light environments. Since synechococci are much less abundant than heterotrophic bacteria in seawater, the impact of Synechococcus aminopeptidase on proteolysis in the sea is likely to be episodic and restricted to specialized microenvironments.     

Hydrotaxis of Cyanobacteria in Desert Crusts

We studied the migration of cyanobacteria in desert crusts from Las Bárdenas Reales (Spain). The crusts were almost exclusively colonized by the filamentous cyanobacterium Oscillatoria, which formed a dense layer approximately 600 µm thick located between 1.5 and 2.1 mm deep. Laboratory and field experiments showed that saturation of the crust with liquid water induced a migration of the cyanobacteria leading to a significant greening of the surface within a few minutes. Under light and rapid evaporation, the green color rapidly disappeared and the crust surface was completely devoid of filaments within 60 min. In contrast, 260 min was required to recover the original white color of the crust when slow evaporation was experimentally imposed. The up and down migration following wetting and drying occurred also in the dark. This demonstrates that light was not a required stimulus. Addition of ATP synthesis inhibitors prevented the cyanobacterium from migrating down into the crust, with filaments remaining on the surface. Therefore, the disappearance of the green color observed during desiccation can only be attributed to an active cyanobacterial motility response to the decrease in the water content. The simplest explanation that can account for the evidence gathered is the presence of a mechanism that links, directly or indirectly, these motility responses to gradients in water content, namely a form of hydrotaxis.