Bacterial Communities in Acidic and Circumneutral Streams

The relationship between pH and the abundance and activity of bacteria in streams was examined as part of a study of the effect of acidification on stream communities. Of the bacterial communities examined, the epilithic community appeared to be the most significantly affected by acidification. Microbial biomass, as quantified by measuring the ATP level, on rock surfaces was significantly correlated with pH. Also, bacterial production by the epilithic bacteria, indicated by incorporation of tritiated thymidine into DNA, was always higher at high-pH sites than at low-pH sites of the same stream order and elevation. Bacterioplankton concentrations varied between 0.53 × 10⁵ and 9.42 × 10⁵ cells · ml⁻¹ in the first- to fourth-order streams examined. The bacterioplankton concentration in one sample from a spring was 0.17 × 10⁵ cells · ml⁻¹. Bacterioplankton concentrations were not correlated with pH but were significantly correlated with seston concentrations. The correlation with seston is a result of increases in particle-associated bacteria at high seston concentrations. The proportion of bacterioplankton attached to particles varied from 0 to 70%. Bacterial numbers and production in the sediments were significantly correlated with the organic content of the sediment rather than with the pH of the overlying water. Thus, reduced abundance and activity of bacteria as a result of acidification could be detected only for the relatively active community on rock surfaces; this community was exposed to the low pH because of the unbuffered nature of its environment.     

Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H+-ATPase Levels in Chemostat Cultures of Lactococcus lactis

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pH_o). A decrease in either the dilution rate or the pH_o led to a decrease in the cytoplasmic pH (pH_i) of the cells, and similar levels of acid tolerance were observed at any specific pH_i irrespective of whether the pH_i resulted from manipulation of the growth rate, manipulation of the pH_o, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pH_o of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pH_o of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pH_i. The rate of ATP formation was not correlated with pH_i, but a good correlation between the cellular level of H⁺-ATPase and pH_i was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pH_i was established. Each pH_i below 6.6 was characterized by unique levels of ATR, H⁺-ATPase, and ATP. High levels of H⁺-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H⁺-ATPase is one of the ATR proteins induced by acid pH_i through growth at an acid pH_o or a slow growth rate.     

Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Changes in intracellular pH during repeated exercise

The rates of change in intracellular pH during repeated exercise sessions with rest periods was determined by 31 phosphorus-nuclear magnetic resonance spectroscopy (³¹P-MRS). Five long-distance runners and six healthy male subjects as controls performed a 2-min femoral flexion at 20 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore and repeated this exercise four times with 2-min rest periods intervening. In all cases during exercise the inorganic phosphate (P_i) peak split into two, the earlier increased rapidly (high-pH P_i) and the later (low-pH P_i) increased more slowly. The P_i peaks were separated by a fitting procedure using the least square mean method. The high-pH P_i area during exercise decreased as the number of repeated exercise periods increased, while the low-pH P_i area gradually increased. Although the total P_i area decreased exponentially during the recovery period, the high-pH P_i area decreased first and then the low-pH P_i area reduced gradually. The pH values were estimated from the chemical shift between the phosphocreatine peak and each split peak in the P_i. The high-pH in pooled data ranged from 6.6 to 7.0 during exercise and recovery, while the low pH decreased to 6.2 during exercise. As the number of exercise periods increased, each pH value gradually became less acidic, although there was a tendency to more acidity in the control subjects than in the long-distance runners. In conclusion, it was possible to obtain by non-invasive, continuous³¹P-MRS, a split pattern of P_i peaks during exercise and there were at least tow different intracellular pH values during exercise, suggesting that each P_i peak might be attributed to the types of muscle fibre recruited.     

Impact of Water Column Acidification on Protozoan Bacterivory at the Lake Sediment-Water Interface

Although the impact of acidification on planktonic grazer food webs has been extensively studied, little is known about microbial food webs either in the water column or in the sediments. Protozoon-bacterium interactions were investigated in a chronically acidified (acid mine drainage) portion of a lake in Virginia. We determined the distribution, abundance, apparent specific grazing rate, and growth rate of protozoa over a pH range of 3.6 to 6.5. Protozoan abundance was lower at the most acidified site, while abundance, in general, was high compared with other systems. Specific grazing rates were uncorrelated with pH and ranged between 0.02 and 0.23 h^-1, values similar to those in unacidified systems. The protozoan community from an acidified station was not better adapted (P = 0.95) to low-pH conditions than a community from an unacidified site (multivariate analysis of variance on growth rates for each community incubated at pHs 4, 5, and 6). Both communities had significantly lower (P < 0.05) growth rates at pHs 4 and 5 than at pH 6. Reduced protozoan growth rates coupled with high grazing rates and relatively higher bacterial yields (ratio of bacterial-protozoan standing stock) at low pH indicate reduced net protozoan growth efficiency and a metabolic cost of acidification to the protozoan community. However, the presence of an abundant, neutrophilic protozoan community and high bacterial grazing rates indicates that acidification of Lake Anna has not inhibited the bacterium-protozoon link of the sediment microbial food web.     

Production of Italian Dry Salami: Effect of Starter Culture and Chemical Acidulation on Staphylococcal Growth in Salami Under Commercial Manufacturing Conditions

The effect of starter culture and chemical acidulation on the growth and enterotoxigenesis of Staphylococcus aureus strain S-6 in Italian dry salami under commercial manufacturing conditions was studied. The experimental design included two levels of S. aureus (10⁴ and 10⁵/g), three levels of starter culture (0, 10⁵, and 10⁶/g), three levels of initial pH (pH₀) (6.1, 5.5, and 4.8), two manufacturing plants, and three replications. S. aureus growth in the salami was affected significantly (P < 0.005) by pH₀, initial levels of S. aureus (staph₀) and lactic acid bacteria (LAB₀), day of fermentation, and by the interactions of pH₀ × day, pH₀ × LAB₀, LAB₀ × staph₀, pH₀ × staph₀, and pH₀ × location of fermentation. In general, the lower the pH₀ and the higher the LAB₀, the greater the inhibition of S. aureus. The LAB levels during the fermentation were affected significantly (P < 0.005) by pH₀, LAB₀, day of fermentation, location, LAB₀ × pH₀, and LAB₀ × day. Derived regression equations related level of S. aureus and LAB at any day of fermentation to a number of microbiological and chemical variables. Close similarity of observed and predicted levels of S. aureus and LAB growth demonstrated the usefulness of the experimental approach in evaluating the safety of a process. No detectable enterotoxin or thermonuclease was found at any stage of processing even when S. aureus reached levels of 10⁷/g of salami.     

Acclimation of aquatic microbial communities to Hg(II) and CH3Hg+ in polluted freshwater ponds

The relationship of mercury resistance to the concentration and chemical speciation of mercurial compounds was evaluated for microbial communities of mercury-polluted and control waters. Methodologies based on the direct viable counting (DVC) method were adapted to enumerate mercury-resistant communities. Elevated tolerance to Hg(II) was observed for the microbial community of one mercury-polluted pond as compared to the community of control waters. These results suggest an in situ acclimation to Hg(II). The results of the methylmercury resistance-DVC assay suggested that minimal acclimation to CH₃Hg⁺ occurred since similar concentrations of CH₃HgCl inhibited growth of 50% of organisms in both the control and polluted communities. Analyses of different mercury species in pond waters suggested that total mercury, but not CH₃Hg⁺ concentrations, approached toxic levels in the polluted ponds. Thus, microbial acclimation was specific to the chemical species of mercury present in the water at concentrations high enough to cause toxic effects to nonacclimated bacterial communities.     

Anaerobic alkalithermophiles, a novel group of extremophiles

Although some anaerobic and aerobic mesophiles have long been known to grow at alkaline pH (above 9.5), little was known until recently about thermophilic alkaliphiles, termed now alkalithermophiles. This minireview describes presently known and recently validly described anaerobic alkalithermophilic bacteria (pH_opt ^55C > 8.5; T_opt > 55°C) and alkalitolerant thermophiles (pH_opt ^55C < 8.5 but pH_max ^55C above 9.0). Some of these are widely distributed, but others have been isolated (thus far) only from one specific location. This novel group of anaerobic bacteria is comprised of physiologically different genera and species which, so far, all belong to the Gram-type positive Bacillus-Clostridium phylogenetic subbranch. An interesting feature of these anaerobic alkalithermophiles is that most of the isolates have short doubling times. The fastest growing among them are strains of Thermobrachium celere, with doubling times as short as 10 min while growing above pH 9.0 and above 55°C. Received: January 22, 1998 / Accepted: February 16, 1998     

Role of Listeria monocytogenes σB in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σ^B, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σ^B independent. σ^B-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σ^B reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σ^B does not appear to contribute to pH_i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH_i buffering by the GAD system under the conditions examined in this study. In summary, a functional σ^B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.     

HCO3?-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO₃⁻-independent intracellular pH (pH_i) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pH_i in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H⁺ pump by Cl⁻ removal or via the selective inhibitor bafilomycin had only a small effect upon resting pH_i and recovery following an acid load. In contrast, resting pH_i in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pH_i recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO₃⁻-independent pH_i regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.     

Temperature and pH optima of extremely halophilic archaea: a mini-review

Archaeal microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review encompasses extremely halophilic archaea and their growth characteristics with respect to the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance. The focus is on poly-extremophiles, i.e., taxa growing optimally at a Na⁺ concentration at or above 1.7 M (approximately 10% w/v NaCl); alkaline pH, at or above 8.5; and elevated temperature optima, at or above 50°C. So far, only a very few extreme halophiles that are able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated. The distribution of extremely halophilic archaea growing optimally at 3.4 M Na⁺ (approximately 20% w/v NaCl) is bifurcated with respect to pH optima, either they are neutrophilic, with a pH_opt of approximately 7, or strongly alkaliphilic, with pH_opt at or above 8.5. Amongst these extreme halophiles which have elevated pH optima, only four taxa have an optimum temperature above 50°C: Haloarcula quadrata (52°C), Haloferax elongans (53°C), Haloferax mediterranei (51°C) and Natronolimnobius ‘aegyptiacus’ (55°C).     

Bacterial Community Affects Toxin Production by Gymnodinium catenatum

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell⁻¹) was similar to the parent cultures (169–206 fmol STX cell⁻¹), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell⁻¹) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell⁻¹). Specific toxin production rate (fmol STX day⁻¹) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell⁻¹ day⁻¹) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.     

Response of Bacterioplankton Communities to Cadmium Exposure in Coastal Water Microcosms with High Temporal Variability

Multiple anthropogenic disturbances to bacterial diversity have been investigated in coastal ecosystems, in which temporal variability in the bacterioplankton community has been considered a ubiquitous process. However, far less is known about the temporal dynamics of a bacterioplankton community responding to pollution disturbances such as toxic metals. We used coastal water microcosms perturbed with 0, 10, 100, and 1,000 μg liter⁻¹ of cadmium (Cd) for 2 weeks to investigate temporal variability, Cd-induced patterns, and their interaction in the coastal bacterioplankton community and to reveal whether the bacterial community structure would reflect the Cd gradient in a temporally varying system. Our results showed that the bacterioplankton community structure shifted along the Cd gradient consistently after a 4-day incubation, although it exhibited some resistance to Cd at low concentration (10 μg liter⁻¹). A process akin to an arms race between temporal variability and Cd exposure was observed, and the temporal variability overwhelmed Cd-induced patterns in the bacterial community. The temporal succession of the bacterial community was correlated with pH, dissolved oxygen, NO₃⁻-N, NO₂⁻-N, PO₄³⁻-P, dissolved organic carbon, and chlorophyll a, and each of these parameters contributed more to community variance than Cd did. However, elevated Cd levels did decrease the temporal turnover rate of community. Furthermore, key taxa, affiliated to the families Flavobacteriaceae, Rhodobacteraceae, Erythrobacteraceae, Piscirickettsiaceae, and Alteromonadaceae, showed a high frequency of being associated with Cd levels during 2 weeks. This study provides direct evidence that specific Cd-induced patterns in bacterioplankton communities exist in highly varying manipulated coastal systems. Future investigations on an ecosystem scale across longer temporal scales are needed to validate the observed pattern.     

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel,hHV1

Part of the “signature sequence” that defines the voltage-gated proton channel (H_V1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H_V1 genes. Replacing Trp²⁰⁷ in human H_V1 (hH_V1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, E_a, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp²⁰⁷ establishes the major energy barrier between closed and open hH_V1. Cation–π interaction between Trp²⁰⁷ and Arg²¹¹ evidently latches the channel closed. Trp²⁰⁷ mutants lost proton selectivity at pH_o >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H_V1 that is essential to its biological functions, was compromised. In the WT hH_V1, ΔpH-dependent gating is shown to saturate above pH_i or pH_o 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp²⁰⁷ mutants, ΔpH-dependent gating saturated at lower pH_o but not at lower pH_i. That Trp²⁰⁷ mutation selectively alters pH_o sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H_V1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH_o and pH_i in H_V1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.     

Genetic parameters of meat technological quality traits in a grand-parental commercial line of turkey

Genetic parameters for meat quality traits and their relationships with body weight and breast development were estimated for a total of 420 male turkeys using REML. The birds were slaughtered in a commercial plant and the traits measured included pH at 20 min (pH₂₀) and 24 h post-mortem (pHu) and colour of the breast and thigh meat. The heritabilities of the rate and the extent of the pH fall in the breast muscle were estimated at h² = 0.21 ± 0.04 and h² = 0.16 ± 0.04, respectively. Heritabilities ranging from 0.10 to 0.32 were obtained for the colour indicators in the breast muscle. A marked negative genetic correlation (r_g = -0.80 ± 0.10) was found between pH₂₀ and lightness (L*) of breast meat, both traits corresponding to PSE indicators. The pH₂₀ in the thigh muscle had a moderate heritability (h² = 0.20 ± 0.07) and was partially genetically related to pH₂₀ in the breast muscle (r_g = 0.45 ± 0.17). Body weight and breast yield were positively correlated with both initial and ultimate pH and negatively with the lightness of breast meat.     

Transport systems for carbonate in the extremely natronophilic cyanobacterium Euhalothece sp.

The effect of carbonate concentration, pH of the medium, and illumination intensity on the major physiological characteristics (growth rate and the intensities of CO₂ assimilation and oxygen photoproduction) of the natronophilic cyanobacterium Euhalothece sp. Z-M001 have been studied. It was established that the investigated microorganism has at least two transport systems (TS) for CO₂, which differ in both the pH optimum and substrate affinity: TS I has a pH_opt 9.4–9.5 and a K _{S 0.5} of 13–17 mM, whereas TS II has a pH_opt 9.9–10.2 and a K _{S 0.5} of 600–800 mM. The substrate affinity of these transport systems is several orders of magnitude lower than the substrate affinity of the transport systems of freshwater cyanobacteria. It is suggested that they are unique for extremely alkaliphilic cyanobacteria and reflect their adaptation to the seasonal cycles of the lake hydrochemistry.     

Effects of pH and Repellent Tactic Stimuli on Protein Methylation Levels in Escherichia coli

Intracellular pH (pH_int) and extracellular pH (pH_ext) of Escherichia coli were measured at 12-s time resolution by ³¹P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH_ext (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH_int decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s⁻¹ cm⁻² pH unit⁻¹. Addition of the membrane-permeant weak acid benzoate at constant pH_ext also caused a lowering of pH_int; at high concentrations it generated an inverted transmembrane pH gradient (ΔpH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pH_int was lowered by weak acid addition or when pH_ext was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pH_ext 7.0). The weak-acid concentration needed to reach that limit was dependent on pH_ext, as would be expected on the basis of ΔpH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH_int, demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pH_int and pH_ext. Evidence is presented that pH_int may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-³H- and ³⁵S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli.     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : I. Role of HCO3?

We studied the regulation of intracellular pH (pH_i) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH_i. Intrinsic buffering power (β_I) was 10.5 mM (pH unit)⁻¹ at pH_i 7.0, and decreased linearly with pH_i; the best-fit line to the data had a slope of −10.0 mM (pH unit)⁻². In the absence of HCO₃ ⁻, pH_i recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH_i 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH_i. Acid extrusion was inhibited 68% (pH_i 6.23) by substituting Li⁺ for Na⁺ in the bath solution. Switching from a CO₂/HCO₃ ⁻-free to a CO₂/HCO₃ ⁻-containing bath solution caused mean steady state pH_i to increase from 6.82 to 6.90, due to a Na⁺-driven HCO₃ ⁻ transporter. The HCO₃ ⁻-induced pH_i increase was unaffected by amiloride, but was inhibited 75% (pH_i 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pH_i 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO₂/HCO₃ ⁻-induced pH_i increase was blocked when external Na⁺ was replaced with N-methyl-d-glucammonium (NMDG⁺). In the presence of HCO₃ ⁻, the Na⁺-driven HCO₃ ⁻ transporter contributed to the pH_i recovery from an acid load. For example, HCO₃ ⁻ shifted the plot of acid-extrusion rate vs. pH_i by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO₃ ⁻ increased acid extrusion 3.8-fold (pH_i 6.20). When astrocytes were acid loaded in amiloride, with Li⁺ as the major cation, HCO₃ ⁻ failed to elicit a substantial increase in pH_i. Thus, Li⁺ does not appear to substitute well for Na⁺ on the HCO₃ ⁻ transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na⁺-driven HCO₃ ⁻ transporter are the predominant acid extruders in astrocytes.     

Do acid-tolerant picocyanobacteria exist? A study of two strains isolated from humic lakes in Poland

The occurrence of picocyanobacteria, the smallest cell-size fraction of cyanobacteria, in low-pH waters, is still poorly studied. In this study, we tested the hypothesis that picocyanobacteria found occasionally in low-pH environments are adapted to such water conditions. We isolated picocyanobacteria by means of the cytometric method from two humic lakes with pH ≤5. We obtained two strains belonging to two cosmopolitan phylogenetic clades of picocyanobacteria: Cyanobium gracile cluster and Subalpine cluster I. Experiments on filtered lake water from low-pH (≤5) and slightly alkaline (pH 8.2) lakes, and with an acidified cyanobacterium medium (pH 4.5) were conducted to test the growth of the isolated picocyanobacteria in various pH conditions. The experimental results of this study showed that some picocyanobacteria strains were acid tolerant, achieving higher growth rates and reaching higher maximum numbers in humic, naturally acidic waters rather than in alkaline waters. We show that despite a close phylogenetic relationship, strains of picocyanobacteria exhibit significant physiological and ecological diversity and that at least some picocyanobacteria have the evolutionary potential to cope with low pH. Characterization of the genetic basis of acid tolerance in picocyanobacteria is important to understand how these microorganisms function in aquatic ecosystems and how their communities may respond to a changing environment.     

Does long-term cultivation of saplings under elevated CO2 concentration influence their photosynthetic response to temperature?

Background and Aims Plants growing under elevated atmospheric CO₂ concentrations often have reduced stomatal conductance and subsequently increased leaf temperature. This study therefore tested the hypothesis that under long-term elevated CO₂ the temperature optima of photosynthetic processes will shift towards higher temperatures and the thermostability of the photosynthetic apparatus will increase.Methods The hypothesis was tested for saplings of broadleaved Fagus sylvatica and coniferous Picea abies exposed for 4–5 years to either ambient (AC; 385 µmol mol⁻¹) or elevated (EC; 700 µmol mol⁻¹) CO₂ concentrations. Temperature response curves of photosynthetic processes were determined by gas-exchange and chlorophyll fluorescence techniques.Key Results Initial assumptions of reduced light-saturated stomatal conductance and increased leaf temperatures for EC plants were confirmed. Temperature response curves revealed stimulation of light-saturated rates of CO₂ assimilation (A_max) and a decline in photorespiration (R_L) as a result of EC within a wide temperature range. However, these effects were negligible or reduced at low and high temperatures. Higher temperature optima (T_opt) of A_max, Rubisco carboxylation rates (V_Cmax) and R_L were found for EC saplings compared with AC saplings. However, the shifts in T_opt of A_max were instantaneous, and disappeared when measured at identical CO₂ concentrations. Higher values of T_opt at elevated CO₂ were attributed particularly to reduced photorespiration and prevailing limitation of photosynthesis by ribulose-1,5-bisphosphate (RuBP) regeneration. Temperature response curves of fluorescence parameters suggested a negligible effect of EC on enhancement of thermostability of photosystem II photochemistry.Conclusions Elevated CO₂ instantaneously increases temperature optima of A_max due to reduced photorespiration and limitation of photosynthesis by RuBP regeneration. However, this increase disappears when plants are exposed to identical CO₂ concentrations. In addition, increased heat-stress tolerance of primary photochemistry in plants grown at elevated CO₂ is unlikely. The hypothesis that long-term cultivation at elevated CO₂ leads to acclimation of photosynthesis to higher temperatures is therefore rejected. Nevertheless, incorporating acclimation mechanisms into models simulating carbon flux between the atmosphere and vegetation is necessary.