Auto-/heterotrophic endosymbiosis evolves in a mature stage of ecosystem development in a microcosm composed of an alga,a bacterium and a ciliate

We investigate an ecological mechanism by which endosymbiotic associations evolve, with a particular focus on the relationship between the evolution of endosymbiosis between auto- and heterotrophic organisms, and the stages of ecosystem development. For this purpose we conducted a long-term microcosm culture composed of three species, a green alga (Chlorella vulgaris), a bacterium (Escherichia coli), and a ciliated protozoan (Tetrahymena thermophila) for 3 years. During this culture T. thermophila cells harboring Chlorella cells emerged by phagocytotic uptake, and increased in frequency, reaching ca. 80–90%. This level was maintained in the late stage of ecosystem dynamics. Analysis of the ecosystem dynamics in the microcosm revealed that a complex causal process through direct/indirect interactions among ecosystem components led to reduction in dissolved O₂ and food (E. coli) available to the T. thermophila, which gave a selective advantage to the organisms in the endosymbiotic association. This result suggests that the endosymbiosis evolves in a mature stage of ecosystem development, where reproduction and survival of prospective partner organisms is highly resource-limited and density-dependent, favoring efficient matter/energy transfers among participating organisms due to physical proximity. Consequently, a complex web of interactions and pathways of matter/energy flow in ecosystem evolves from an initially simple one.     

Physiological changes of a green alga (Micractinium sp.) involved in an early-stage of association with Tetrahymena thermophila during 5-year microcosm culture

Endosymbioses between phototrophic algae and heterotrophic organisms are an important symbiotic association in that this association connects photo- and heterotrophic metabolism, and therefore, affects energy/matter pathways and cycling in the ecosystem. However, little is known about the early processes of evolution of an endosymbiotic association between previously non-associated organisms. In previous studies, we analyzed an early process of the evolution of an endosymbiotic association between an alga and a ciliate by using a long-term culture of an experimental model ecosystem (CET microcosm) composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). The results revealed that an algal type, isolated from 5-year cultures of the microcosm, prolonged the longevity of the ancestral and derived clones of T. thermophila in the absence of bacteria, suggesting that a cooperative algal phenotype that benefited the ciliate had evolved in the microcosm. Here, we investigated the physiological changes of the derived Micractinium clones that benefited Tetrahymena, focusing on the release of carbohydrates by and abundance of photopigments in the ancestral and 2 derived algal clones (SC10-2 and SC9-1) isolated from inside Tetrahymena cells. Analyses using HPLC revealed that the algal isolates released glycerol and sucrose at higher concentrations per cell and also contained higher levels of photopigments per cell at pH 7.2, in comparison with the ancestral strain. These phenotypic characters were considered responsible for the increased longevity of Tetrahymena cells, and thus supported the cooperator alga hypothesis.     

Direct and indirect interactions for coexistence in a species-defined microcosm

Mechanisms for coexistence among micro-organisms were studied by using a species-defined microcosm, consisting of the bacterium Escherichia coli, the ciliate Tetrahymena thermophila and the alga Euglena gracilis. These organisms were chosen as representative of ecological functional groups i.e. decomposer, consumer and producer, respectively. Direct and indirect interactions among these organisms were evaluated by comparisons of their population dynamics in culture with different combinations of the three species. There was an E. coli cell density dependent predator–prey interaction between T. thermophila and E. coli which was only established when there were more than 10⁶ cells ml^–1 of E. coli. Indirect interactions were evaluated from the cultivation of each organism in media containing metabolites of the others. Metabolites from each population strongly accelerated the growth of their own populations and those of the others except for the self-toxicity effect of E. coli metabolites. These observations suggested that not only the cell–cell contact of direct interactions, but also metabolite-mediated indirect interactions supported the maintenance of the populations of each micro-organism and their coexistence. In natural ecosystems, there are many interactions and it is difficult to evaluate all those regulating community dynamics. The gnotobiotic microcosm used in this study was shown to be suitable for examining the specific, species–species microbial interactions.     

Exploitation or cooperation? Evolution of a host (ciliate)-benefiting alga in a long-term experimental microcosm culture

Controversy persists as to whether the acquisition of beneficial metabolic functions via endosymbiosis can occur suddenly on an evolutionary time scale. In this study, an early stage of endosymbiotic associations, which evolved from previously unassociated auto (photo)- and heterotrophic unicellular organisms was analyzed using an experimental ecosystem model, called CET microcosm. This ecosystem model was composed of a green alga (Micractinium sp.; formerly described as Chlorella vulgaris), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). Our previous study using a CET microcosm that was cultured 3–5 years revealed that fitness of the ciliate increased by harboring algal cells within its own cells. This fact suggested three possibilities: (i) the ciliate evolved the ability to exploit intracellular algal cells (“exploiter ciliate hypothesis”), (ii) the alga evolved the ability to benefit the host ciliate by providing photosynthates (“cooperator alga hypothesis”), and (iii) a combination of (i) and (ii). To test these hypotheses, two-by-two co-cultures were conducted between the ancestral or derived ciliate and the ancestral or derived alga. The experimental results demonstrated that a cooperative alga evolved in the microcosm, although the possibility remains that an exploitative genotype of the ciliate might also exist in the population as a polymorphism. Remarkably, an algal isolate prolonged the longevity of not only the isolated ciliate, but also the ancestral ciliate. This result suggests that once a cooperative algal genotype evolves in a local population, it can then be transmitted to other individuals of the prospective host species and spread rapidly beyond the local range due to its positive effect on the host fitness. Such transmission suggests the possibility of a sudden acquisition of beneficial autotrophic function by the pre-associated host.     

Effects of acidic conditions on the physiology of a green alga (Micractinium sp.) before and after a 5-year interaction with Tetrahymena thermophila in an experimental microcosm

The mechanisms through which algae evolve physiological characteristics related to endosymbiotic associations with heterotrophic organisms remain unclear. We previously showed that a green alga (Micractinium sp.) was able to evolve a host (ciliate)-benefiting phenotype that prolonged the longevity of Tetrahymena thermophila in the absence of bacteria during a 5-year culture period in an experimental microcosm. Comparative experiments between the ancestral alga (i.e. original) and evolved algal clones of the same lineage can be performed to analyse the mechanisms that underlie algal evolution during interactions with ciliates. Here, we investigated the effects of acidic conditions on algal physiology because the acidic conditions within the food vacuoles of Tetrahymena could potentially affect algal evolution during long-term interactions. It might be expected that algal clones isolated from T. thermophila hosts would have developed the ability to resist acidic conditions in order to establish within the host, when compared with the ancestral strain. Unexpectedly, comparative analyses demonstrated that acidic conditions with pH values ranging from 3.9 to 4.3 limited the growth of the isolated algal clones SC9-1 and SC10-2, whereas the ancestral strain could grow under these conditions. Acidic conditions were responsible for significantly lower chlorophyll a/chlorophyll b ratios in the isolated algal clones, and the isolated clone SC9-1 exhibited a high cellular content of chlorophyllide a during a short exposure to acidic conditions, suggesting that degradation of chlorophyll a occurred. Furthermore, acidic conditions increased the release of extracellular glycerol and sucrose in the SC9-1 clone. These results indicated that the ancestral strain showed phenotypic changes related to acidic conditions, which may reflect the ongoing development of an algal phenotype suited to symbiosis. Hypotheses are proposed to explain the limited growth of the isolated clones, and implications for the Micractinium–Tetrahymena association are discussed.     

Effect at the ecosystem level of elevated atmospheric CO2in an aquatic microcosm

We studied the responses of an aquatic microcosm in two different eutrophic conditions to elevated atmospheric CO₂concentration. We used microcosms, consisting of Escherichia coli(bacteria), Tetrahymena thermophila(protozoa) and Euglena gracilis(algae), in salt solution with 50 and 500 mg l^–1of proteose peptone (eutrophic and hypereutrophic conditions, respectively) under ambient and elevated CO₂(1550±100 l l^–1) conditions. The density of E. gracilisincreased significantly under elevated CO₂in both eutrophic and hypereutrophic microcosms. In the eutrophic microcosm, the other elements were not affected by elevated CO₂. In the hypereutrophic microcosm, however, the concentrations of ammonium and phosphate decreased significantly under elevated CO₂. Furthermore, the density of T. thermophilawas maintained in higher level than that in the microcosm with ambient CO₂and the density of E. coliwas decreased by CO₂enrichment. Calculating the carbon biomasses of T. thermophilaand E. colifrom their densities, the changes in their biomasses by CO₂enrichment were little as compared with large increase of E. graciliscarbon biomass converted from chlorophyll a. From the responses to elevated CO₂in the subsystems of the hypereutrophic microcosm consisting of either one or two species, the increase of E. graciliswas a direct effect of elevated CO₂, whereas the changes in the density of E. coliand T. thermophilaand the decreases in the concentration of ammonium and phosphate are considered to be indirect effects rather than direct effects of elevated CO₂. The indirect effects of elevated CO₂were prominent in the hypereutrophic microcosm.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Increased Expression ofBrevibacterium sterolicumCholesterol Oxidase inEscherichia coliby Genetic Modification

To improve expression ofBrevibacterium sterolicumcholesterol oxidase inEscherichia coli,we utilized theT7lacpromoter and modified the gene to encode the first 21 amino acids with high-expressionE. colicodons. These changes resulted in a 60-fold improvement of expression level. N-terminal sequencing revealed that theE. coliproduced cholesterol oxidase signal peptide is cleaved 6 amino acids closer to the N-terminus than inB. sterolicum.The recombinantE. coliproduced protein is composed of 513 amino acids with a calculatedM_rof 55,374. The kinetic rate constants of the recombinant protein and theB. sterolicumproduced cholesterol oxidase are identical.     

A T7 promoter-specific,inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli β-galactosidase, as well as a 1,4-β-glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. Theα-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibitedα-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Genetics of colicin E susceptibility inCitrobacter freundii

The insensitivity ofCitrobacter freundii to the E colicins is based on tolerance to colicin E1 and resistance to colicins E2 and E3. Spontaneous colicin A resistant mutants ofC. freundii also lost their colicin E1 receptor function. Sensitivity to colicin E1 can be induced by F′gal ⁺ tol ⁺ plasmids, thetol A⁺ gene product of which is responsible for this effect. Receptor function for colicins E2 and E3 is induced by theE. coli F′14bfe ⁺ plasmid, which is also able to enhance notably the receptor capacity for colicin E1. Thebfe ⁺ gene product ofE. coli, which is responsible for these phenomena, also restores the receptor function for colicin A and E1 in colicin A resistant mutants ofC. freundii. All results show that there is a remarkable difference between theE. coli bfe ⁺ gene product and thebfe ⁺ gene product ofC. freundii and also between thetol A⁺ gene products of these strains. The sensitivity to phage BF23 parallels the sensitivity to colicins E2 and E3 and is also induced by the F′14bfe ⁺ plasmid.     

A community model of ciliateTetrahymena and bacteriaE. coli: Part II. Interactions in a batch system

Premised on relatively simple assumptions, mathematical models like those of Monod, Pirt or Droop inadequately explain the complex transient behavior of microbial populations. In particular, these models fail to explain many aspects of the dynamics of aTetrahymena pyriformis-Escherichia coli community. In this study an alternative approach, an individual-based model, is employed to investigate the growth and interactions ofTetrahymena pyriformis andE. coli in a batch culture. Due to improved representation of physiological processes, the model provides a better agreement with experimental data of bacterial density and ciliate biomass than previous modeling studies. It predicts a much larger coexistence domain than rudimentary models, dependence of biomass dynamics on initial conditions (bacteria to ciliate biomasses ratio) and appropriate timing of minimal bacteria density. Moreover, it is found that accumulation ofE. coli sized particles andE. coli toxic metabolites has a stabilizing effect on the system.     

Ribosome Specificity of Transfer Factors from Unicellular Algae

Abstract

Ribosome specificity of polymerizing enzymes prepared from Nostoc muscorum and from photosynthesizing and non-photosynthesizing strains of Euglena gracilis and Chlorella vulgaris was determined by assaying protein synthesis in vitro with Escherichia coli (70 S type) and Saccharomyces cerevisiae (80 S type) ribosomes. Polymerizing enzymes prepared from the prokaryote N. muscorum are active only on E. coli ribosomes, while the enzymes prepared from the photosynthesizing strains of E. gracilis and C. vulgaris are active on both 70 S and 80 S type ribosomes. Polymerizing enzymes from dark-grown cells of E. gracilis and of an achloric strain are active only on 80 S type ribosomes, and the activity on E. coli ribosomes may be restored by adding E. coli transfer factor T. In addition, activity on 70 S ribosomes becomes evident a few hours after exposure to light of dark-grown cells of E. gracilis. On the contrary, polymerizing enzymes prepared from a non-photosynthetic strain of C. vulgaris, and from the naturally occuring achloric alga Prototheca zopfii are active on both types of ribosomes. C. vulgaris and its achloric strain contain both 70 S and 80 S type ribosomes, while in P. zopfii only 80 S type ribosomes are evident.     

Breakdown ofd-glucose by mixed cultures ofEscherichia coli,Desulfovibrio vulgaris,andChromatium vinosum

Experiments with mixed cultures ofEscherichia coli, Desulfovibrio vulgaris, andChromatium vinosum were conducted using a synthetic medium with glucose as the substrate. The bacterial number and the changes in the chemical factors were determined during the development of the mixed culture. (i) Fermentation of glucose byE. coli produces organic acids (formic, acetic, lactic, and succinic) and alcohol. The growth-yield constant K (cell dry weight per weight solid substrate) does not exceed 0.05. (ii) In the mixed culture ofE. coli andD. vulgaris, the reduction of sulfate is accompanied by a total consumption of formate, lactate, and alcohol and an increase in the sulfide and acetate content. (iii) When the three physiologically different species are allowed to grow simultaneously, there is no accumulation of catabolites in the medium and the growth yield constant increases to 0.46. Maximum phototrophic production requires the presence of bothE. coli andD. vulgaris. A low substrate concentration and the simultaneous growth of the three organisms are other factors that contribute towards a high output. The biochemical parameters of the medium are influenced to a large extent by the glucose level. The results suggest that the behavior of the strains is different in pure and mixed cultures.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Identification of thepurA gene encoding adenylosuccinate synthetase inThiobacillus ferrooxidans

ThepurA gene ofThiobacillus ferrooxidans encoding adenylosuccinate synthetase [EC 6.3.4.4] was identified in the upstream region of theiro gene encoding Fe(II)-oxidase (J. Biol. Chem 267:11242–11247, 1992). ThepurA gene consisted of 1290 base-pairs, which translated into a 29-amino-acid protein. The gene is functionally active, because it is able to complement anEscherichia coli purA-deficient strain. The deduced gene product has a high degree (60.9%) of sequence identity with that (432 aa) ofE. coli purA gene, and both the products share GDEGKGK-DETG-TKLD sequences which are supposed to be GTP-binding domain. The downstream region of theiro gene contained another open-reading frame (ORF) of 1218 bp, and this showed high homlogy (56.6% over 249 bp) withE. coli ORF-II, which is found as a second ORF and truncated form in the downstream region of thepurA gene. Comparison of the gene organization in the flanking region ofpurA gene betweenT. ferrooxidans andE. coli is also described.     

Members of the pogo superfamily of DNA-mediated transposons in the human genome

Bacillus subtilis, likeEscherichia coli, possesses several sets of genes involved in the utilization of-glucosides. InE. coli, all these genes are cryptic, including the genes forming thebgl operon, thus leading to a Bgl^– phenotype. We screened forB. subtilis chromosomal DNA fragments capable of reverting the Bgl⁺ phenotype associated with anE. coli hns mutant to the Bgl^– wild-type phenotype. OneB. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from thebglP gene. Deletion studies as well as subcloning experiments allowed us to prove that the putativeB. subtilis bglP RAT sequence was responsible for the repression of theE. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein ofE. coli bgl operon by our putativeB. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.     

A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles

A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.     

Cloning of the gene coding for aromatic amino acid aminotransferase from anE. Coli B strain

Summary AnE. coli B strain showing high activity in the transamination of phenylpyruvate to phenylalanine was used as the DNA source for the construction of a cosmid library inE. coli DG30, a strain which is known to be defective in all three major transaminase genes (aspC, ilvE andtyrB). By complementation analysis, cosmid clones could be identified with inserts carrying atyrB gene. The DNA inserts were further subcloned into pAT153 and thetyrB gene fromE. coli B was found to be similar to the gene reported forE. coli K12. Plasmids containing theE. coli BtyrB gene were transformed into the originalE. coli B strain and the recombinant strains assayed for transaminase activity and plasmid stability.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday.