Experimental Microbial Populations Thirty-five Years Later: The Influence of Food on the Symbiosis of Paramecium aurelia Syngen 4, 51.7

Changes in the nutrition of Paramecium aurelia affect its ability to serve as host for the bacteroid parasite, kappa, and the presence or absence of kappa affects its ability to grow in axenic culture. Loss of kappa, tested by the presence or absence of killer reaction, occurred in cultures of P. aurelia growing at a reduced division rate on autoclaved Enterobacter aerogenes in suspensions of lettuce and yeast autolysate 14–17 days after they had been rendered bacteria-free by washing. Killer Paramecium sterilized of bacteria by treatment with an antibiotic mixture of penicillin-G and streptomycin in combination with a nonbacterial nonliving culture medium, lost the ability to kill after from 6 to 48 hours in the sterilizing medium. The ciliates from which kappa had been lost during exposure to antibiotics could be transferred immediately and maintained in axenic culture, but those washed free of bacteria could not be maintained axenically until kappa had been lost during cultivation in a medium containing killed bacteria. It is suggested that a knowledge of the nutritional requirements of symbiotic microorganisms is essential for understanding the ecological aspects of eutrophication of aquatic environments.     

The role of bacteria in the metal tolerance of the fouling diatom Amphora coffeaeformis Ag.

Components of “spent” axenic and non-axenic Amphora coffeaeformis Ag. culture media were tested for their effect on copper and tributyltin tolerance in axenic A. coffeaeformis. Growth of such algal cultures in the presence of either toxin was enhanced by the addition of enriched “spent” medium from exponentially growing non-axenic cultures. This response was seen whether or not the bacteria had been removed. It appears that bacteria or bacteria-algal interactions produce some soluble (<0.2 μm) factor which either decreases the toxicity of copper and tributyltinfluoride (TBTF) or increases the tolerance of A. coffeaeformis to these toxins.     

Analysis of the prestarvation response in growing cells of Dictyostelium discoideum

We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF-regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.     

ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

Ascophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed. Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus-free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria-free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.     

AN IMPROVED METHOD FOR OBTAINING AXENIC CLONES OF PLANKTONIC BLUE-GREEN ALGAE1,2

Axenic clones from 5 isolates of Anabaena flosaquae, 1 isolate of Microcystis acruginosa, and 1 isolate of Aphanizomenon flos-aquae were obtained by a combination of steps that provided a 1000-fold reduction in the bacteria-algae ratio and permitted bacteria-free filaments or cells to be isolated and grown from agar pour plates. The first step consisted of the addition of phenol to a dark-treated culture to selectively reduce the numbers of actively growing bacteria while leaving the resting algal cells viable. The next steps involved washing the treated algal suspension on a Millipore filter pad or membrane followed by plating in washed agar containing buffered mineral medium plus vitamins and soil extract. The final steps consisted of incubating the agar pour plates, coring bacteria-free filaments or cells, culturing the agar cores in a buffered mineral medium, and rigorously testing the resulting cultures for bacteriological contamination. Between 50 and 90% of the cores grew, and of these about 50% were judged axenic. The method, with appropriate adaptations, should be suitable for obtaining axenic clones of other freshwater and marine algae.     

The effect of bacteria on interspecific relationships between the euryhaline rotifer Brachionus rotundiformis and the harpacticoid copepod Tigriopus japonicus

Inconsistent results have been obtained on the population growth of Brachionus rotundiformis and Tigriopus japonicus, when results from single-species and two-species mixed cultures are compared. Bacteria growth was not regulated in these experiments, which could be the cause for this. In order to test this possibility, we conducted similar experiments under axenic and synxenic (with presence of one species of bacteria) conditions. The population growth of B. rotundiformis was suppressed by the presence of T. japonicus in axenic cultures. T. japonicus could not persist in axenic cultures, but its population increased when grown in synxenic cultures. T. japonicus used RT bacteria strain as a food source, while these bacteria were toxic to B. rotundiformis. These results suggest that bacteria can modify the interspecific relationship between B. rotundiformis and T. japonicus.     

Root exudates of plants

Summary The release of substances from wheat roots was found to be directly related to the growth of the root system. Plants whose root system did not grow released almost no exudates.When exudate concentration in the vicinity of the roots was lowered by frequent replacements of the nutrient solution or by a simultaneous cultivation of exudate-utilizing bacteria, the release of exudates was enhanced. In axenic wheat cultures, the amount of exudates during a 12-day cultivation with 2- or 4-day intervals between medium replacements represented 50% of root dry weight and 12% of whole plant dry weight.Wheat plants cultivated in the presence of the bacteriumPseudomonas putida released up to double the amount of exudates compared with axenic variants.     

Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium

Photoautotrophic biofilms play an important role in various aquatic habitats and are composed of prokaryotic and/or eukaryotic organisms embedded in extracellular polymeric substances (EPS). We have isolated diatoms as well as bacteria from freshwater biofilms to study organismal interactions between representative isolates. We found that bacteria have a strong impact on the biofilm formation of the pennate diatom Achnanthidium minutissimum. This alga produces extracellular capsules of insoluble EPS, mostly carbohydrates (CHO), only in the presence of bacteria (xenic culture). The EPS themselves also have a strong impact on the aggregation and attachment of the algae. In the absence of bacteria (axenic culture), A. minutissimum did not form capsules and the cells grew completely suspended. Fractionation and quantification of CHO revealed that the diatom in axenic culture produces large amounts of soluble CHO, whereas in the xenic culture mainly insoluble CHO were detected. For investigation of biofilm formation by A. minutissimum, a bioassay was established using a diatom satellite Bacteroidetes bacterium that had been shown to induce capsule formation of A. minutissimum. Interestingly, capsule and biofilm induction can be achieved by addition of bacterial spent medium, indicating that soluble hydrophobic molecules produced by the bacterium may mediate the diatom/bacteria interaction. With the designed bioassay, a reliable tool is now available to study the chemical interactions between diatoms and bacteria with consequences for biofilm formation.     

Wild-Type Strains ofDictyostelium discoideumCan Be Transformed Using a Novel Selection Cassette Driven by the Promoter of the Ribosomal V18 Gene

Almost all methods for transformation of the social amebaDictyostelium discoideumrely on axenic growth, that is, growth in a synthetic medium, for at least part of the procedure. Axenic growth requires several mutations. Here we describe a procedure that can be used to transform wild-type strains which are able to grow only on the natural food source, bacteria. The method relies on a new selection cassette driven by the V18 promoter, a promoter that we show is substantially more active during growth on bacteria than the actin-6 promoter, which is widely used for axenic transformation. The procedure gives transformation frequencies of about 10⁻⁵with both strains Ax2 (capable of axenic growth) and NC4 (capable of growth only on bacteria). Using this vector, we have obtained NC4 strains carrying several β-galactosidase reporter cassettes. Our vector can also be used in axenic transformations.     

Morphological and physiological changes in Microcystis aeruginosa as a result of interactions with heterotrophic bacteria

1. To reveal the role of aquatic heterotrophic bacteria in the process of development of Microcystis blooms in natural waters, we cocultured unicellular Microcystis aeruginosa with a natural Microcystis‐associated heterotrophic bacterial community. 2. Unicellular M. aeruginosa at different initial cell densities aggregated into colonies in the presence of heterotrophic bacteria, while axenic Microcystis continued to grow as single cells. The specific growth rate, the chl a content, the maximum electron transport rate (ETR_max) and the synthesis and secretion of extracellular polysaccharide (EPS) were higher in non‐axenic M. aeruginosa than in axenic M. aeruginosa after cell aggregation, whereas axenic and non‐axenic M. aeruginosa displayed the same physiological characteristic before aggregation. 3. Heterotrophic bacterial community composition was analysed by PCR–denaturing gradient gel electrophoresis (PCR–DGGE) fingerprinting. The biomass of heterotrophic bacteria strongly increased in the coinoculated cultures, but the DGGE banding patterns in coinoculated cultures were distinctly dissimilar to those in control cultures with only heterotrophic bacteria. Sequencing of DGGE bands suggested that Porphyrobacter, Flavobacteriaceae and one uncultured bacterium could be specialist bacteria responsible for the aggregation of M. aeruginosa. 4. The production of EPS in non‐axenic M. aeruginosa created microenvironments that probably served to link both cyanobacterial cells and their associated bacterial cells into mutually beneficial colonies. Microcystis colony formation facilitates the maintenance of high biomass for a long time, and the growth of heterotrophic bacteria was enhanced by EPS secretion from M. aeruginosa. 5. The results from our study suggest that natural heterotrophic bacterial communities have a role in the development of Microcystis blooms in natural waters. The mechanisms behind the changes of the bacterial community and interaction between cyanobacteria and heterotrophic bacteria need further investigations.     

Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879

ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.     

Purification and culture characteristics of 36 benthic marine diatoms isolated from the Solthörn tidal flat (Southern North Sea)

Marine benthic diatoms growing in biofilms on sediment surfaces generally occur associated with heterotrophic bacteria, whereas modern molecular techniques and analyses of species‐specific physiology create a demand for axenic cultures. Numerous benthic diatoms were isolated from surface sediments during a monitoring of the Solthörn tidal flat (southern North Sea, Germany) from May 2008 to May 2009. Of these, around 50% could be purified from the accompanying heterotrophic bacteria using different antibiotics combined with physical separation methods (vortexing, ultrasound). Overall, seven different antibiotics were tested at different concentrations, and a best working protocol was developed. The axenic strains were stable on average for only around 15 months, indicating a symbiotic interaction between the benthic diatoms and the associated bacteria. While most short‐term effects during the purification process were restricted to differences in growth rates among xenic and axenic diatom strains, long‐term cultivation led to distinct changes in cell volumes and growth characteristics of the axenic strains.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Isolation of strains of the cellular slime mold Dictyostelium discoideum capable of growing after a single passage in axenic medium.

Wild-type strains of the cellular slime mold Dictyostelium discoideum grow on bacteria. Axenic strains of D. discoideum capable of growing in a simple salts-yeast extract-proteose peptone-glucose medium have been isolated from wild-type strains fo both mating types after a single passage in this medium. This result is consistent with the theory that the axenic phenotype has a simple genetic basis.     

Axenic Cultivation of the Cellulolytic Flagellate Trichomitopsis termopsidis (Cleveland) from the Termite Zootermopsis*

SYNOPSIS. Trichomitopsis termopsidis (Cleveland), a cellulolytic hindgut symbiote of the termite Zootermopsis, has been cultivated axenically under anaerobic conditions. The medium consists of cellulose, reduced glutathione, fetal calf serum, yeast extract, and autoclaved rumen fluid or autoclaved rumen bacteria, in a buffered salt solution the composition of which is based on an analysis of Zootermopsis hindgut fluid. The hindgut contents of surface-sterilized termites were inoculated into anaerobic buffer-containing cellulose and serum. Repeated passages yielded mixed cultures of T. termopsidis and termite hindgut bacteria. Flagellates were then inoculated into complete medium containing antibiotics, and after 2 passages, axenic cultures of T. termopsidis were obtained. Various nutritional supplements, including clarified rumen fluid or heat-killed bacteria of several known species failed to support the growth of T. termopsidis when substituted for autoclaved rumen fluid. The flagellates did not grow when any of several carbohydrates were substituted for cellulose. Electron microscopy of flagellates from axenic cultures revealed that cellulose particles and partially digested bacteria were present in food vacuoles. No endosymbiotic bacteria were present in the cytoplasm indicating that T. termopsidis does not depend on living prokaryotes for cellulose digestion. The results suggest that T. termopsidis possesses the enzyme cellulase.     

MORPHOGENESIS OF MONOSTROMA OXYSPERMUM (KÜTZ.) DOTY (CHLOROPHYCEAE) IN AXENIC CULTURE,ESPECIALLY IN BIALGAL CULTURE1

The typical morphology of Monostroma oxyspermum (Kütz.) Doty is lost in axenic culture. In synthetic media of the ASP type, it grows as a colony-like mass composed of round cells with numerous rhizoids. Such a mass is a fragile structure which falls apart upon shaking, or slight touch, into small cell-groups and single cells or cells with a long rhizoid. Only temporary saccate or monostromatic fronds appear and reach 1–2 mm in length when grown in enriched seawater media, but disintegrate and become a colony-like mass. The typical morphology is easily restored by adding at specific intervals filtrates of bacterial cultures and supernatant medium from axenic brown and red algal cultures to the basal medium (ASP7), or by reinfecting the Monostroma with an appropriate bacterial flora. Furthermore, the typical morphology in also maintained by bialgal cultures between Monostroma and other axenic strains of various species of seaweeds except the species belonging to the Chlorophyceae. Monostroma thus appears to utilize some substances released by most species of brown and red algae for its typical growth. Active substances released by bacteria, brown and red algae have not yet been identified and purified. However, it is demonstrated that in axenic cultures many species of seaweeds produce active extracellular substances which play an important role in growth and Morphogenesis of other species of seaweeds.     

Intersyngenic variations in the esterases of axenic stocks of Paramecium aurelia

The esterase isozymes were surveyed in axenic stocks of syngens 1, 2, 4, 5, 6, and 8 of Paramecium aurelia by starch gel electrophoresis. In paramecia there appear to be four types of esterases which are clearer in axenic than in bacterized stocks. Each type differs in its substrate specificity and/or its response to the inhibitor eserine sulfate. Minor variations in type D esterases sometimes occur in different extracts of the same stock and may result from changes in the temperature of growth of the cells or growth cycle differences. Differences in the mobility of the A, B, or C (cathodal) types of esterases may occur in different syngens. They also occur for the A and B types among stocks within a syngen, but the frequency is low, except in the case of syngen 2. Since each of the types of esterases varies independently, at least four and possibly more genes appear to specify the esterases in the species complex. Some pairs of syngens vary in their electrophoretic positions for all types of esterases. Other pairs have identical zymograms. This observation suggests that some syngens may differ from each other by as many as four esterase genes, while others may not differ at all. The difference between P. aurelia and Tetrahymena pyriformis in the degree of intrasyngenic variation observed for enzymes is discussed in relation to other types of characters, the organization of the genetic material in the macronucleus, the presence of symbionts, and their breeding systems. It is suggested that enzyme variation is achieved by the action of different selective forces in these two groups of ciliated protozoa.Supported by research grants from the National Institute of General Medical Sciences (GM-15879), U.S. Public Health Service, and from the British Medical Research Council.     

Phenol degradation by Paenibacillus thiaminolyticus and Bacillus cereus in axenic and mixed conditions

In this study, seven aerobic bacterial strains were screened for phenol tolerance at different concentration of phenol. Bacterial strains were unable to utilize phenol in absence of glucose, indicated the phenomenon of co-metabolism. Among the seven isolated bacterial strains, only ITRC BK-4 and ITRC BK-7 found potential and identified as Paenibacillus thiaminolyticus (DQ435022) and Bacillus cereus (DQ435023), respectively. Phenol degradation was monitored routinely with spectrophotometer and further confirmed by HPLC analysis. ITRC BK-4, ITRC BK-7 and mixed culture degrade 700 ppm phenol up to 51.72, 70.00 and 84.57% respectively in mineral salt medium (MSM) at temperature 37 ± 1°C, pH 7.5 ± 0.2, 120 rpm in presence of 1% glucose (w/v) within 144 h incubation. The mix culture was found more potential for phenol degradation compared to axenic strains. Hence, the axenic and mixed strains of these bacteria would be useful for the removal/mineralization of phenol from industrial waste waters.     

Growth Factors of Bacterial Origin for the Culture of the Rumen Oligotrich Protozoon, Entodinium caudatum

Attempts were made to develop an artificial medium suitable for axenic culture of Entodinium caudatum. Agnotobiotic cultures of the protozoon were established as stock cultures for testing the suitability of various growth media. A cell-free extract of mixed bacteria isolated from the rumen was shown to contain one or more growth factors for the protozoon when supplied with activated charcoal as a carrier. The medium (CYSE medium), which supported the growth of the protozoon in the presence of 50 μg/ml each of penicillin and chloramphenicol, consisted of activated charcoal (20 mg), heat-treated yeast (Y) (80 mg), 13%β-sitosterol-coated rice starch (S) (120 mg), and cell-free extract of rumen bacteria (1 ml) in 40 ml buffer solution. When culturing the protozoon, the CYSE medium was supplemented daily with 20 mg each of Y and S and half of the medium was replaced with fresh medium once every 5 d. The possible use of this method to establish an axenic culture of E. caudatum is discussed.     

Hemicellulolytic enzymes of the ligninolytic white-rot fungus Phlebia radiata. Influence of phenolic compounds on the synthesis of hemicellulolytic enzymes

The ligninolytic fungus Phlebia radiata growing in a low-nitrogen medium with wheat bran as the sole carbon source was induced by some lignin monomers, e.g. vanillic, veratric and ferulic acids. In the medium these substances showed a mainly stimulating influence on the hemicellulolytic enzymes activity except for arabinofuranosidase and ferulic acid esterase.