Estimation of the contribution of halobacterial to the bacterial biomass and activity in solar salterns by the use of bile salts

Abstract Bile salts (deoxycholate, taurocholate) were used to estimate the contribution of bacteria of the Halobacterium group to bacterial community size and activity at different salinities as found in a multi-pond saltern. Low concentrations of bile salts cause lysis of halophilic archaebacteria of the Halobacterium group, while halophilic eubacteria and halococci remain microscopically intact. Upon addition of bile salts, total bacterial numbers (as estimated microscopically) in saltern ponds at salinities below 250 g/l did not decrease, while above this salinity bacterial numbers decreased by 30–50%. To estimate the contribution of halobacteria to overall heterotrophic activity, the effect of bile salt addition was tested on the incorporation of labelled amino acids. In saltern ponds of a salinity below 250 g/l activity was not greatly inhibited by taurocholate, while at salinity above 300 g/l taurocholate completely abolished incorporation of amino acids.     

The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments

Abstract Antibiotics affecting protein synthesis were used to differentiate between the activity of different groups of organisms (halophilic archaebacteria, eubacteria and eukaryotes) in water samples from hypersaline ecosystems. Anisomycin (inhibiting both archaebacterial halophiles and eukaryotes) can be used to quantitate the contribution of the archaebacterial halophiles to amino acid incorporation by the microbial community, when cycloheximide (inhibiting eukaryotic protein synthesis, but not affecting halobacteria) is used as a control. Both in saltern ponds at salinities above 300 g/1 and in Dead Sea surface water more than 95% of the amino acid incorporation activity was abolished by anisomycin, but not by cycloheximide. Inhibition by anisomycin was well correlated with inhibition by low concentrations of bile salts, which specifically affect bacteria of the Halobacterium group. Chloramphenicol (an inhibitor of eubacterial protein synthesis) quantitatively inhibited amino acid incorporation in saltern brines of relatively low salinity, but also caused significant (28–42%) inhibition at high salinities. Erythromycin was also found valuable in the estimation of activities of the different bacterial groups.     

Variation of environmental features and microbial populations with salt concentrations in a multi-pond saltern

A multi-pond saltern that creates a gradient of salt concentrations has been studied with respect to some characteristics of the resulting environments and their microbial populations. The increase in salt concentration was correlated with increase in diurnal temperature and biomass present and with decrease in oxygen concentrations. Many types of organisms below 15% (w/v) total salts, were found, many of them normal inhabitants of seawater and even freshwater. Most organisms over 15% salts were halophilic. The salt concentrations comprised two ranges, each characterized by different microbial populations. First, between 15 and 30% salts, the populations ofDunaliella increased, reaching large numbers; moderately halophilic eubacteria and some fast-growing halobacteria predominated as heterotrophic microorganisms and, among the first, thePseudomonas-Alteromonas-Alcaligenes group andVibrio were the more abundant taxonomic groups; and gram-positive cocci appeared mainly over 25% salts. Phototrophic bacteria, both oxygenic and anoxygenic, were also found in this range, and among the anoxygenic type,Chromatium species andRodospirillum salexigens were probably predominant. Second, over 30% salts the diversity decreased greatly, all organisms found at the lower salt concentrations disappeared, and instead large populations of halobacteria developed. Over 50% salts, only three species of halobacteria were found.     

DNA,RNA, and protein synthesis in the cyanobacteriumSynechocystis sp. PCC 6803 adapted to different salt concentrations

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content with increasing salt concentrations up to 684mm NaCl was found, whereas the lowest DNA content was measured around 342mm NaCl, i.e., the salinity at which maximal growth occurred. With the uracil and thymidien incorporation technique, maxima in DNA and RNA synthesis were detected in control cells. Comparing these rates with nucleic acid synthesis rates calculated from the contents of DNA and RNA and the growth rates indicated that adaptation to 1026mm NaCl seemed to lead to an increased RNA turnover inSynechocystis. Analysis of protein synthesis with³⁵S-methionine labeling showed alterations in salt-adapated cells ofSynechocystis. At least three proteins (20.5, 25.8, and 35.8 kDa) were synthesized with highest rates at salinities leading to maximal growth, the synthesis of nine proteins (12.5, 16.9, 19.2, 22.2, 24.7, 28.5, 30.5, 50.3, and 63.5 kDa) increased and that of several other proteins decreased with increasing salinity; but only three proteins (12.5, 22.2, and 30.5 kDa) accumulated under these conditions. The adaptation ofSynechocystis to enhanced salt concentrations led also to increased contents of glucosylglycerol, glycogen, and significant amounts of K⁺ as well as Na⁺ ions.     

Halocins: are they involved in the competition between halobacteria in saltern ponds?

Many representatives of the family Halobacteriaceae ("halobacteria") excrete halophilic bacteriocins (halocins) that inhibit the growth of other halobacteria. In spite of the fact that halocin production is widespread among the Halobacteriaceae, no information is available on their ecological significance. To test whether halocins may play a role in the interspecies competition between dif-ferent types of halobacteria in saltern crystallizer ponds inhabited by dense communities of these red halophiles, we assayed for halocins active against a variety of halobacteria in salterns from different locations worldwide. Detection of halocin activity was based on the inhibition of growth of indicator organisms on agar plates, the decreased incorporation of radiolabeled substrates, and microscopic examinations. No halocin activity was detected in any of the brines examined, in spite of the fact that halocin production was demonstrated in cultures of most microorganisms isolated from these brines. Thus, the contribution of halocins in the competition between different halobacteria in hypersaline aquatic environments is probably negligible. Received: July 29, 1999 / Accepted: October 18, 1999     

The bioenergetic basis for the decrease in metabolic diversity at increasing salt concentrations: implications for the functioning of salt lake ecosystems

Examination of the microbial diversity in hypersaline lakes of increasing salt concentrations shows that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis from hydrogen and carbon dioxide or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. The observations can be explained on the basis of the energetic cost of haloadaptation used by the different metabolic groups and the free-energy change associated with the dissimilatory reactions. All halophilic microorganisms spend large amounts of energy to maintain steep gradients of Na⁺ and K⁺concentrations across their cytoplasmic membrane. Most Bacteria and also the methanogenic Archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. The halophilic aerobic Archaea (order Halobacteriales) and the halophilic fermentative Bacteria (order Halanaerobiales) use KCl as the main intracellular solute. This strategy, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic compatible solutes. By combining information on the amount of energy available to each physiological group and the strategy used to cope with salt stress, a coherent model emerges that provides explanations for the upper salinity limit at which the different microbial conversions occur in hypersaline lakes.     

Purification and properties of glutamine synthetase from the archaebacterium Halobacterium salinarium

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the halophilic archaebacterium Halobacterium salinarium. The enzyme was purified 300-fold to homogeneity with 30% yield. By gel filtration and SDS gel electrophoresis, it was shown that the enzyme has a native molecular weight of 495,000 and a subunit molecular weight of 62,000. This indicates an octameric quaternary structure. The amino acid composition and the isoelectric point of 4.9 are similar to other GSs. The enzyme shows highest stability in 4 M NaCl or KCl and at temperatures up to 45°C. Lower salt concentrations or higher temperatures lead to rapid and irreversible denaturation. By low concentrations of Mg²⁺ or Mn²⁺, the salt dependence was decreased and the thermostability increased. Mg²⁺ or Mn²⁺ are essential cofactors. The two resulting activities show differences in pH and salt concentrations required for optimal activity, different K _m-values and different sensitivity to inhibition by amino acids. The enzyme is not adenylylated like the GS from some eubacteria but cytidylylated. The covalently bound CMP increases Mn²⁺-and Mg²⁺-dependent activities at a different extent.     

Bioenergetic Aspects of Halophilism

Examinination of microbial diversity in environments of increasing salt concentrations indicates that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis for H₂ + CO₂ or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. Occurrence of the different metabolic types is correlated with the free-energy change associated with the dissimilatory reactions. Life at high salt concentrations is energetically expensive. Most bacteria and also the methanogenic archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. All halophilic microorganisms expend large amounts of energy to maintain steep gradients of NA⁺ and K⁺ concentrations across their cytoplasmic membrane. The energetic cost of salt adaptation probably dictates what types of metabolism can support life at the highest salt concentrations. Use of KCl as an intracellular solute, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic-compatible solutes. This may explain why the anaerobic halophilic fermentative bacteria (order Haloanaerobiales) use this strategy and also why halophilic homoacetogenic bacteria that produce acetate from H₂ + CO₂ exist whereas methanogens that use the same substrates in a reaction with a similar free-energy yield do not.     

Inhibition of Growth and of Thymidine Incorporation into DNA in Tetrahymena by Chlorpromazine,Pimozide and Penfluridol1

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 μM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 μmoles of drug were bound per 10⁶ cells. A 20-min preincubation of cells with chlorpromazine (14.7 μM) inhibited DNA synthesis by 46%, and with penfluridol (4 μM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Adaptation of thymidine utilization to changing rates of DNA synthesis in the cell cycle

Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (³H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (³H)thymidine (10^–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (³H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10^–8 to 10^–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (³H)thymidine supplied at 5 × 10^–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.     

The salt relations of Dunaliella

Dunaliella tertiolecta (marine) and D. viridis (halophilic) were each trained by serial transfer to growth at salt concentrations previously regarded as the other's domain. D. viridis then had a salt optimum at 1.0–1.5 M sodium chloride whereas that for D. tertiolecta was less than 0–2 M. Nevertheless D. tertiolecta grew faster than the halophil at all salt concentrations up to 3.5 M, the highest at which they were compared.Both species accumulate glycerol, which is necessary for growth at elevated salinities and which responds in its content to water activity (a _w ) rather than specifically to salt concentration. Variation in glycerol content is a metabolic process which occurs in the dark from accumulated starch as well as photosynthetically. Regulation of glycerol content by a _w does not require protein synthesis. The NADP-specific glycerol dehydrogenase of each of the algae is likely to be directly involved in the regulation of glycerol content. Kinetic studies, together with those described in an earlier publication, show that the enzyme has regulatory properties, and that both glycerol and dihydroxyacetone act as effectors as well as reactants. A mechanism of the reaction is tentatively proposed.     

A bacteriophage of a moderately halophilic bacterium

A bacteriophage of an aerobic, gram-negative, rod-shaped halophilic bacterium, provisionally named Pseudomonas sp. G3, is described. The phage has a head and a tail and is similar in appearance to Salmonella phage Beccles. It infects its bacterial host at all salt concentrations in which the bacteirum is able to grow. In contrast to phages of halophilic archaebacteria, the newly-described phage is relatively stable in the absence of salt. It also infects Vibrio costicola and two unidentified halophilic eubacteria.Abbreviations PPT proteose peptone-tryptone medium - pfu plaque-forming unit - G+C guanine + cytidine content, mol %     

Lysis of Halobacteria in Bacto-Peptone by Bile Acids

All tested strains of halophilic archaebacteria of the genera Halobacterium, Haloarcula, Haloferax, and Natronobacterium lysed in 1% Bacto-Peptone (Difco) containing 25% NaCl, whereas no lysis was observed with other strains belonging to archaebacteria of the genera Halococcus, Natronococcus, and Sulfolobus, methanogenic bacteria, and moderately halophilic eubacteria. Substances in Bacto-Peptone which caused lysis of halobacteria were purified and identified as taurocholic acid and glycocholic acid. High-performance liquid chromatography analyses of peptones revealed that Bacto-Peptone contained nine different bile acids, with a total content of 9.53 mg/g, whereas much lower amounts were found in Peptone Bacteriological Technical (Difco) and Oxoid Peptone. Different kinds of peptones can be used to distinguish halophilic eubacteria and archaebacteria in mixed cultures from hypersaline environments.     

DNA synthesis in cultured human fibroblasts: Regulation by 3′ : 5′-cyclic amp

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [³H] thymidine incorporation into acid-precipitable material, beginning after 8–12 hr and reaching maximum levels at 16–24 hr. Addition of dibutyryl-3′ : 5′-cyclic AMP (DBcAMP) together with serum inhibited [³H] thymidine incorporation by 75–95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3′ : 5′-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [³H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G₁ was inhibited by high concentrations of 3′ : 5′-cyclic AMP.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

A comparison of the utilization of thymine and thymidine for the synthesis of DNA-thymine in novikoff hepatoma cells

A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10⁻⁷ M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the K_m was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells.     

Depth Distribution of Bacterial Production in a Stratified Lake with an Anoxic Hypolimnion

The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both [³H]thymidine and [³H]leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, [³H]thymidine incorporation into DNA was highest in the epilimnion. The ratios of incorporation into DNA/protein averaged 2.2, 0.49, and 0.95 for the epilimnion, metalimnion, and hypolimnion, respectively. Low incorporation into DNA was not due to artifacts associated with the DNA isolation procedure. Recovery of added [³H]DNA was about 90% in waters in which the portion of [³H]thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. Our results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.     

Physical chemistry and evolution of salt tolerance in halobacteria

The cellular constituents of extremely halophilic bacteria not only tolerate high salt concentration, but in many cases require it for optical functioning. The characteristics affected by salt include enzyme activity, stability, allosteric regulation, conformation and subunit association. The salt effects are of two major kinds: electrostatic shielding of negative charges by cations at low salt concentration, and hydrophobic stabilization by salting-out type salts at high salt concentration. The composition of halobacterial proteins shows an excess of acidic amino acids and a deficiency of nonpolar amino acids, which accounts for these effects. Since the cohesive forces are weaker and the repulsing forces are stronger in these proteins, preventing aggregation in salt, these structures are no longer suited for functioning in the absence of high salt concentrations. Unlike these nonspecific effects, ribosomes in halobacteria show marked preference for potassium over sodium ions. To ensure the proper intracellular ionic composition, powerful ion transport systems have evolved in the halobacteria, resulting in the extrusion of sodium ions and their replacement by potassium. It is likely that such membrane transport system for ionic movements is a necessary requisite for salt tolerance.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

Biological effects of bovine glia maturation factor on glial cells in culture

Optimal bioassay conditions for bovine glia maturation factor (GMF) were determined among glial cells from normal glioblasts to glioma cells. Rat glioblasts 4–8 days after subculture show the highest response to GMF with regard to morphological transformation and mitogenic activity. Bovine GMF enhances DNA synthesis of rat glioblasts at 12 hr after stimulation; maximum incorporation of [methyl-³H]thymidine was detected at 18 hr. GMF increases twofold the saturation density of rat glioblasts but does not alter that of C6 astrocytoma cells. The apparent inhibition of mitogenic activity of high doses of GMF is seen in both normal and malignant glial cells.     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.