Chlorella: 125 years of the green survivalist.

Chlorella, the archetype of unicellular green algae, is a high-performance primary producer in aquatic and terrestrial ecosystems. Under the simple spherical morphology of Chlorella, many other 'green balls' unfolded as independent phylogenetic lineages as a result of convergent evolution. By contrast, green algae with strikingly different phenotypes were unmasked as close relatives of Chlorella by modern molecular techniques. Here, we point to the increasing impact of these diverse protists on ecology, evolution, and biotechnology in the light of integrative taxonomy.

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID-TOLERANT, FREE-LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)(1).

The processes of CO2 acquisition were characterized for the acid-tolerant, free-living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO3 (-) uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO2 formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO2 uptake and O2 evolution by membrane-inlet mass spectrometry demonstrated that air-grown cells reduced the CO2 concentration in the medium to an equilibrium concentration of 15 µM, but AZA-treated cells caused a drop in extracellular CO2 concentration to a compensation concentration of 27 µM at pH 8.0. CO2 -pulsing experiments with cells in the light indicated that the cells do not actively take up CO2 . An internal pool of unfixed inorganic carbon was not detected at the CO2 compensation concentration, probably because of the lack of active CO2 uptake, but was detectable at times before compensation point was reached. These results indicate that this free-living Coccomyxa possesses a CO2 -concentrating mechanism (CCM) due to an active bicarbonate-uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.

Bovine mastitis associated with Prototheca blaschkeae.

Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae. These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.

Chlorella sorokiniana UTEX 2805, a heat and intense, sunlight-tolerant microalga with potential for removing ammonium from wastewater.

In the summer of 2003, a microalga strain was isolated from a massive green microalgae bloom in wastewater stabilization ponds at the treatment facility of La Paz, B.C.S., Mexico. Prevailing environmental conditions were air temperatures over 40 degrees C, water temperature of 37 degrees C, and insolation of up to 2400 micromol m2 s(-1) at midday for several hours at the water surface for four months. The microalga was identified as Chlorella sorokiniana Shih. et Krauss, based on sequencing its entire 18S rRNA gene. In a controlled photo-bioreactor, this strain can grow to high population densities in synthetic wastewater at temperatures of 40-42 degrees C and light intensity of 2500 micromol m2 s(-1) for 5h daily and efficiently remove ammonium from the wastewater under these conditions better than under normal lower temperature (28 degrees C) and lower light intensity (60 micromol m2 s(-1)). When co-immobilized with the bacterium Azospirillum brasilense that promotes growth of microalgae, the population of microalga grew faster and removed even more ammonium. Under exposure to extreme growth conditions, the quantity of four photosynthetic pigments increased in the co-immobilized cultures. This strain of microalga has potential as a wastewater treatment agent under extreme conditions of temperature and light intensity.

Direct evidence for redundant segmental replacement between multiple 18S rRNA genes in a single Prototheca strain.

Informational genes such as those encoding rRNAs are related to transcription and translation, and are thus considered to be rarely subject to lateral gene transfer (LGT) between different organisms, compared to operational genes having metabolic functions. However, several lines of evidence have suggested or confirmed the occurrence of LGT of DNA segments encoding evolutionarily variable regions of rRNA genes between different organisms. In the present paper, we show, for the first time to our knowledge, that variable regions of the 18S rRNA gene are segmentally replaced by multiple copies of different sequences in a single strain of the green microalga Prototheca wickerhamii, resulting in at least 17 genotypes, nine of which were actually transcribed. Recombination between different 18S rRNA genes occurred in seven out of eight variable regions (V1-V5 and V7-V9) of eukaryotic small subunit (SSU) rRNAs. While no recombination was observed in V1, one to three different recombination loci were demonstrated for the other regions. Such segmental replacement was also implicated for helix H37, which is defined as V6 of prokaryotic SSU rRNAs. Our observations provide direct evidence for redundant recombination of an informational gene, which encodes a component of mature ribosomes, in a single strain of one organism.

A cryptic intracellular green alga in Ginkgo biloba: ribosomal DNA markers reveal worldwide distribution.

Intracellular symbioses involving eukaryotic microalgae and a variety of heterotrophic protists and invertebrates are widespread, but are unknown in higher plants. Recently, we reported the isolation and molecular identification of a Coccomyxa-like green alga from in vitro cell cultures of Ginkgo biloba L. This alga resides intracellularly in an immature "precursor" form with a nonfunctional chloroplast, implying that algal photosynthetic activity has no role in this endosymbiosis. In necrotizing Ginkgo cells, precursors evolved into mature algae, proliferated, and were liberated into the culture medium after host cell bursting. In the present paper we demonstrate by molecular methods a worldwide distribution of the alga in planta. Endosymbiont-specific sequences of ribosomal DNA could be traced in Ginkgo tissues of each specimen examined from different geographic locations in Europe, North America, and Asia. The Ginkgo/Coccomyca association represents a new kind of intracellular, vertically inherited symbiosis. Storage bodies, probably of lipid nature, present in the cytoplasm of each partner suggest a possible involvement of the endosymbiont in metabolic pathways of its host.

Chlamydomonas pitschmannii Ettl, a little known species from thermoacidic environments.

Three Chlamydomonas strains were isolated from the soils of a hot spring located in the Campi Flegrei Caldera (Naples, Italy). Ecophysiological, morpho-cytological and molecular features were used to characterize these isolates and to compare them with chlamydomonax acidophila strains from algal culture collections. The strains were collected from three points of the volcanic site, differing in their physico-chemical conditions. Among the examined Chlamydomonas strains, only the isolates from Campi Flegrei could grow optimally at pH values < or =3.0. These isolates also showed a high tolerance to desiccation and high temperatures, not evidenced by the other Chlamydomonas strains included in the study. 18S rDNA phylogeny indicates that the isolates from Campi Flegrei are closely related to Chlamydomonas pitschmannii and two strains isolated in Canada and Europe, that have been designated as Chlamydomonas acidophila. A Chlamydomonas acidophila strain isolated from the type locality in Japan is less closely related according to its molecular phylogeny, and can also be discerned by light and electron microscopy. Moreover, vegetative cells and sporangia of Chlamydomonas acidophila from Japan showed a median trilaminar structure not observed in the other strains. Our results show that Chlamydomonas pitschmannii could represent a hitherto unknown extremophilic Chlamydomonas species.

Phylogenetic relationships and taxonomic position of Chlorella-like isolates from low pH environments (pH < 3.0).

BACKGROUND: Little is known about phytoplankton communities inhabiting low pH environments such as volcanic and geothermal sites or acidic waters. Only specialised organisms are able to tolerate such extreme conditions. There is, thus, low species diversity. We have characterised the previously isolated acid tolerant Chlorella-like microalgae Viridiella fridericiana and Chlorella protothecoides var. acidicola by microscopical and biomolecular methods in order to assess their phylogenetic relationships. RESULTS: Both isolates belong to the trebouxiophycean lineage of chlorophytes. 18S and ITS1 sequence data clearly confirm that Viridiella fridericiana constitutes a new genus apart from the morphologically similar and likewise acid tolerant microalga Chlorella saccharophila. Chlorella protothecoides var. acidicola on the other hand is not a variety of Chlorella protothecoides but falls within a heterogeneous cluster consisting of Nannochloris, "Chlorella" spec. Yanaqocha, and Koliella, and is most closely related to algae which were also isolated from extreme environments. CONCLUSIONS: The distribution of acid tolerant strains in the 18S rRNA tree shows that acquisition of acid tolerance was unlikely a monophyletic event in green microalgae. We propose that different strains have independently adapted to extreme environments. Some of them have spread worldwide and were able to colonise other extreme habitats. Considering the problems of successfully isolating acid tolerant strains, acidic soils could represent an unsuspected source of biological diversity with high potential for biotechnological utilisations.

Hyaloraphidium curvatum: a linear mitochondrial genome, tRNA editing, and an evolutionary link to lower fungi.

We have sequenced the mitochondrial DNA (mtDNA) of Hyaloraphidium curvatum, an organism previously classified as a colorless green alga but now recognized as a lower fungus based on molecular data. The 29.97-kbp mitochondrial chromosome is maintained as a monomeric, linear molecule with identical, inverted repeats (1.43 kbp) at both ends, a rare genome architecture in mitochondria. The genome encodes only 14 known mitochondrial proteins, 7 tRNAs, the large subunit rRNA and small subunit rRNA (SSU rRNA), and 3 ORFs. The SSU rRNA is encoded in two gene pieces that are located 8 kbp apart on the mtDNA. Scrambled and fragmented mitochondrial rRNAs are well known from green algae and alveolate protists but are unprecedented in fungi. Protein genes code for apocytochrome b; cytochrome oxidase 1, 2, and 3, NADH dehydrogenase 1, 2, 3, 4, 4L, 5, and 6, and ATP synthase 6, 8, and 9 subunits, and several of these genes are organized in operon-like clusters. The set of seven mitochondrially encoded tRNAs is insufficient to recognize all codons that occur in the mitochondrial protein genes. When taking into account the pronounced codon bias, at least 16 nuclear-encoded tRNAs are assumed to be imported into the mitochondria. Three of the seven predicted mitochondria-encoded tRNA sequences carry mispairings in the first three positions of the acceptor stem. This strongly suggests that these tRNAs are edited by a mechanism similar to the one seen in the fungus Spizellomyces punctatus and the rhizopod amoeba Acanthamoeba castellanii. Our phylogenetic analysis confirms with overwhelming support that H. curvatum is a member of the chytridiomycete fungi, specifically related to the Monoblepharidales.

Discovery of an endophytic alga in Ginkgo biloba.

Although intracellular associations with mycorrhizal fungi are known for Ginkgo biloba, no other endosymbiotic relationships have ever been reported for this "living fossil." A protoplast culture derived from haploid explants has now revealed the existence of a green alga in vitro, whose eukaryotic status was confirmed by transmission electron microscopic studies. Phylogenetic 18S rDNA sequence analyses showed this alga to be closely related to the lichen photobiont Coccomyxa. Algae, which in host cells exist as more or less undifferentiated "precursor" forms, proliferated within necrosing G. biloba cells of a subculture derived from a zygotic embryo and were finally released into the medium. Light and electron microscopic observations showed that G. biloba cells rapidly filled up with countless green particles whose number increased up to the bursting of the hypertrophic host cells. At the beginning of reproduction no algae were visible in the nutritive medium, demonstrating that the proliferation started inside the G. biloba cells and excluding the possibility of an exogenous contamination. Occasionally, mature algae together with their precursor forms were detected by transmission electron microscopy in intact host cells of a green callus. The algae were easily identified by their similarity to the cultured algae. Eukaryotic algae have never been reported to date to reside inside higher plant cells, whereas several algal associations are well known from the animal kingdom.