New subgroup of Bacteroidetes and diverse microorganisms in Tibetan plateau glacial ice provide a biological record of environmental conditions.

We recovered microorganisms from five ice core samples from three glaciers (Puruogangri, Malan, and Dunde) located in the Tibetan Plateau in China and analyzed their small subunit rRNA gene sequences. Most of the bacterial sequences were unknown previously; the most closely related known sequences were from bacteria of the Proteobacteria, Bacteroidetes, and Actinobacteria phyla. Chlorophyta, Streptophyta, Ciliophora, and fungal groups were represented among the 18S rRNA gene sequences that we obtained. The most abundantly represented glacial bacteria were Bacteroidetes, and Chlamydomonas was the predominant eukaryote. Comparative analysis showed that the Bacteroidetes sequences obtained from this study were highly similar to one another but most were only distantly related to previously characterized Bacteroidetes (<92% identity). We propose that our Bacteroidetes sequences represent two novel subgroups: one at the family level and one at the genus level. The unique ice environment and the high abundance of Bacteroidetes, combined with the coexistence of a high abundance of psychrophilic Chlamydomonas, strongly suggests that there is a viable ecosystem on the surface of Tibetan glaciers. Comparisons of microbial community structures in the five ice samples showed distinct differences, likely due to environmental differences in the locations in which the samples were obtained.

Morphological stasis of protists in lower cretaceous amber.

Paleomicrobiological studies of terrestrial and freshwater protists are extremely rare in comparison with studies of eukaryotic microfossils from marine ecosystems. Using optical and electron microscopy (SEM-BSE) for hard substrates, we have examined protists trapped in Lower Cretaceous amber from Peñacerrada (Alava, Spain). We present the earliest reasonably confident microfossils of three taxa: Excavata (Euglenozoa), that are similar to the extant genera Euglena and Phacus; Chlorophyceae identified as members of the genus Chlamydomonas, and finally, in the taxon Ciliophora (Chromalveolata), two ciliated protozoa identified as Colpoda (Class Colpodea) and Prorodon (Class Prostomatea). Morphological stasis is evident, and identification based on phenotypic traits indicates the existence of conservative phenotypes persisting over geological time scales.

Use of ammoniacal nitrogen tolerant microalgae in landfill leachate treatment.

Two microalgae, Chlorella pyrenoidosa and Chlamydomonas snowiae, were isolated from a high ammonia leachate pond in Li Keng Landfill, Guangzhou, China. Their growth and nutrient removal rates were determined in a serial dilution of landfill leachate under laboratory conditions, and their growth rates were compared with that of a C. pyrenoidosa strain isolated from a clean river. The results indicated the growth of all three algae was inhibited by high leachate concentrations, and the inhibition appears linked to high ammonia (ammoniacal-N670mgL(-1)). Significant amounts of ammoniacal-N, ortho-P and COD in the leachate were removed by the algae, with a positive correlation between algal growth and nutrient consumption. Not enough data are available to conclude that one strain was less inhibited by ammoniacal nitrogen or more effective at treating it. Phytotoxicity of leachate was reduced after algal growth, as demonstrated by a seed germination experiment with Brassica chinensis. The germination rates in 10%, 30% and 50% concentrations of algal-treated leachate were significantly higher than those in the same concentration but algal-free leachate.

Seasonal and diel changes in photosynthetic activity of the snow alga Chlamydomonas nivalis (Chlorophyceae) from Svalbard determined by pulse amplitude modulation fluorometry.

The seasonal and diel dynamics of the physiological state and photosynthetic activity of the snow alga Chlamydomonas nivalis were investigated in a snowfield in Svalbard. The snow surface represents an environment with very high irradiation intensities along with stable low temperatures close to freezing point. Photosynthetic activity was measured using pulse amplitude modulation fluorometry. Three types of cell (green biflagellate vegetative cells, orange spores clustered by means of mucilaginous sheaths, and purple spores with thick cell walls) were found, all of them photosynthetically active. The pH of snow ranged between 5.0 and 7.5, and the conductivity ranged between 5 and 75 microS cm(-1). The temperature of snow was stable (-0.1 to +0.1 degrees C), and the incident radiation values ranged from 11 to 1500 micromol photons m(-2) s(-1). The photosynthetic activity had seasonal and diel dynamics. The Fv/Fm values ranged between 0.4 and 0.7, and generally declined over the course of the season. A dynamic response of Fv/Fm to the irradiance was recorded. According to the saturating photon fluence values Ek, the algae may have obtained saturating light as deep as 3 cm in the snow when there were higher-light conditions, whereas they were undersaturated at prevalent low light even if on the surface.

Simultaneous control of turbidity and dilution rate through adjustment of medium composition in semi-continuous Chlamydomonas cultures.

For production of starch in algal cultures, a growth rate limited by a nutrient is an important factor. Under phototrophic conditions, turbidity must be also paid attention, as the shading effect may affect its productivity. Semi-continuous cultivation methods, which enable control of turbidity and dilution rate (D) at the same time, have been developed for evaluation of those factors on starch production in Chlamydomonas sp. A specific feature of the methods is in a process of alternately feeding medium adjusted at two different nitrogen (N) concentrations. In the turbidostat-based method, a turbidostat culture was operated repeating three steps of determining D within a preset interval, alternating media by comparing the D with a preset value, and adjusting D in the next interval by feeding the selected medium. In the chemostat-based method, turbidity of a chemostat culture was controlled by repeating two steps of alternating media by comparing transmitted photon flux intensity (I) with a preset value and adjusting I by feeding the selected medium. D controlled by the turbidostat-based method reached quickly a preset value as low as 0.010/h, and then it was dispersed around but above the preset value. On the other hand, mean N concentrations of fed media formed a plateau. In the chemostat-based method, I was well controlled to a preset value while the mean N concentrations were a bit fluctuated. Starch concentration varied from 0.052 to 0.41 g/L with turbidity and D defined by these methods.

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.

A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self-splicing and genetic evidence for maturase activity.

The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp. from Antarctica that self-splices accurately in vitro. Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E. myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria. In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg(2+) (>12 mM) and NH(4)(+). Self-splicing was improved by deleting most of the ORF and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription. Self-splicing of Chs.psbA1 pre-RNAs showed temperature optima of ~44 degrees C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50 degrees C, indicative of thermolability. Splicing of wild-type Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work provides the first description of a ribozyme from a psychrophilic organism. It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts.

Fatty acid patterns in Chlamydomonas sp. as a marker for nutritional regimes and temperature under extremely acidic conditions.

Fatty acid profiles were used to characterize nutritional pathways in Chlamydomonas sp. isolated from an acidic mining lake (pH 2.7). Surprisingly, profiles of Chlamydomonas sp. grown in the lab under photoautotrophic, mixotrophic, and heterotrophic conditions at in situ deep strata lake water temperatures (8 degrees C) were very similar, polyunsaturated fatty acids including alpha-linolenic acid (18:3omega3) and 16:4omega3 along with palmitic acid (16:0) being most abundant. Therefore, heterotrophic growth of Chlamydomonas sp. at low temperatures can result in high concentrations of polyunsaturated fatty acids, as previously only described for some psychrophilic bacteria. By contrast, the cultivation of isolated Chlamydomonas sp. at 20 degrees C, reflecting surface water temperatures, provided fatty acid patterns characteristic of the nutrition strategy applied: the concentration of polyunsaturated fatty acids decreased when the growth pathway changed from photoautotrophic via mixotrophic to heterotrophic. Total fatty acid concentration also diminished in this order. Principal component analysis confirmed the significance of FA profiling to mirror nutritional pathways. Lake-water analysis revealed low concentrations of dissolved organic carbon, mainly consisting of polymeric fulvic acids that are unable to support heterotrophic growth of Chlamydomonas sp. Polymeric fulvic acids present in the deeper strata of the lake turned out to be formed in situ on the basis of organic monomers including reduced sulfur-containing ones, as revealed by thermochemolysis and pyrolysis. Growth of Chlamydomonas sp. in the deep chlorophyll maximum is therefore assumed to mainly result from photosynthesis, despite very low photon densities. Phytol-including metabolites proved to be significant biomarkers to indicate the nutritional pathway of Chlamydomonas sp. alpha, omega-Dicarboxylic acids-light-induced degradation products of unsaturated fatty acids-appeared to be good indicators of photooxidative alterations to the algal species under study.

Surface gas-exchange processes of snow algae.

The red-colored chlorophyte Chlamydomonas nivalis is commonly found in summer snowfields. We used a modified Li-Cor gas-exchange system to investigate surface gas-exchange characteristics of snow colonized by this alga, finding rates of CO(2) uptake up to 0.3 micromol.m(-2).s(-1) in dense algal blooms. Experiments varying the irradiance resulted in light curves that resembled those of the leaves of higher plants. Red light was more effective than white and much more effective than green or blue, because of the red astaxanthin that surrounds and masks the algal chloroplasts. Integrating daily course measurements of gas exchange showed CO(2) uptake around 2,300 micromol.m(-2).day(-1) in heavily colonized patches, indicating that summer snowfields can be surprisingly productive.

Detection and quantification of snow algae with an airborne imaging spectrometer.

We describe spectral reflectance measurements of snow containing the snow alga Chlamydomonas nivalis and a model to retrieve snow algal concentrations from airborne imaging spectrometer data. Because cells of C. nivalis absorb at specific wavelengths in regions indicative of carotenoids (astaxanthin esters, lutein, beta-carotene) and chlorophylls a and b, the spectral signature of snow containing C. nivalis is distinct from that of snow without algae. The spectral reflectance of snow containing C. nivalis is separable from that of snow without algae due to carotenoid absorption in the wavelength range from 0.4 to 0.58 microm and chlorophyll a and b absorption in the wavelength range from 0.6 to 0.7 microm. The integral of the scaled chlorophyll a and b absorption feature (I(0.68)) varies with algal concentration (C(a)). Using the relationship C(a) = 81019.2 I(0.68) + 845.2, we inverted Airborne Visible Infrared Imaging Spectrometer reflectance data collected in the Tioga Pass region of the Sierra Nevada in California to determine algal concentration. For the 5.5-km(2) region imaged, the mean algal concentration was 1,306 cells ml(-1), the standard deviation was 1,740 cells ml(-1), and the coefficient of variation was 1.33. The retrieved spatial distribution was consistent with observations made in the field. From the spatial estimates of algal concentration, we calculated a total imaged algal biomass of 16.55 kg for the 0.495-km(2) snow-covered area, which gave an areal biomass concentration of 0.033 g/m(2).

Isolation, growth, ultrastructure, and metal tolerance of the green alga, Chlamydomonas acidophila (Chlorophyta).

An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance. Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5-4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50%, EC50 were measured, after 72 h of static exposure. EC50s were 14.4 microM Cd2+, 81.3 microM Co2+, 141 microM Cu2+, and 1.16 mM Zn2+ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas.

Snow algae of the Sierra Nevada, Spain, and High Atlas mountains of Morocco.

Snow algae (Chlorophyta) are reported from the Sierra Nevada mountains in southern Spain and the High Atlas mountains of Morocco. Populations of the snow algae Chlamydomonas sp., coloring the snow orange-red, were collected from Pico de Veleta, Spain, while snow samples from Mt. Neltner in the High Atlas mountains, contained resting spores of an orange-green colored Chloromonas sp. Other microbes observed in snow samples include bacteria, fungi, heterotrophic euglenids, diatoms, nematodes, and heterotrophic mastigotes (flagellated protists). This is the first report of snow algae from the Sierra Nevada mountains of Spain and from the Afro-alpine environment.

Isolation and characterization of a Chlamydomonas reinhardtii mutant lacking the gamma-subunit of chloroplast coupling factor 1 (CF1).

Several nonphotoautotrophic mutants of Chlamydomonas reinhardtii were generated by transforming strain nit1-305 (cw 15) with exogenous DNA. An enrichment for potential photophosphorylation mutants was performed on medium containing arsenate, and acetate-requiring auxotrophs were then identified by replica plating. Strains containing a potential mutation in the nuclear DNA encoding the chloroplast coupling factor 1 (CF1) gamma-subunit (the atpC gene) were first identified serologically with a monospecific antiserum directed against the CF1 gamma-subunit polypeptide. Of several mutants isolated, one, designated T1-54, was characterized at the protein, DNA, and RNA levels. Mutant strain T1-54 lacks anti-CF1 gamma-subunit cross-reacting material, exhibits polymorphism at the atpC locus compared with the parental strain, and lacks the mRNA transcript for the CF1 gamma-subunit. The data are consistent with there being an insertion of exogenous DNA, a deletion of DNA, or both at the 5' end of the gene encoding the CF1 gamma-subunit.

Isolation and characterization of Chlamydomonas temperature-sensitive mutants affecting gametic differentiation under nitrogen-starved conditions.

Two conditional mutants of Chlamydomonas reinhardtii, dif-1 and dif-2, affecting gametic differentiation under conditions of nitrogen (N)-starvation, have been isolated. These mutant cell remain "vegetative" at the restrictive temperature (35 degrees C) in -N medium, as defined by assays of cell body-agglutinin and cell wall lytic enzyme activities in the soluble fractions of cell homogenates. Moreover, the mutants fail to form mating structures at the restrictive temperature, but do so at the permissive temperature (25 degrees C). Temperature-shift experiments show that mutant cells which have differentiated into gametes at 25 degrees C dedifferentiate into "vegetative" cells under N-starvation conditions after transfer to 35 degrees C, but differentiate again into gametes at 25 degrees C. Genetic analyses indicate that the dif-1 and dif-2 genes are recessive and unlinked to each other or to the mating-type locus; the dif-1 phenotype cosegregates with a conditional flagellaless phenotype expressed in both +N and -N medium at the restrictive temperature.

Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium.

In Chlamydomonas reinhardtii, cadmium induces reduction of growth, reduction of chlorophyll content, and lethality. The toxicity was higher in a cell wall-deficient strain than in the wild type. By growing the cells on agar medium containing cadmium at concentrations inducing high lethality, stable resistant clones were isolated. The resistance was due to a nuclear mutation (cadAR) which probably preexisted in the wild-type cell population, as suggested by the fluctuation test. A double mutant (cadAR cadBR) was selected on media containing higher concentrations of cadmium. The cadBR mutation, which is unlinked to cadAR, determines a resistance intermediate between the CadAR mutant and the wild-type strain. Both cadAR and cadBR mutations are partially dominant.

Extensive restriction fragment length polymorphisms in a new isolate of Chlamydomonas reinhardtii.

A new field isolate of the unicellular green alga Chlamydomonas reinhardtii with useful properties for restriction fragment length polymorphism mapping is described in this report. The isolate, S1-D2 (mating type -), was the only strain found among 24 Chlamydomonas isolates taken from many locations which was interfertile with laboratory strains of C. reinhardtii. It mates at high efficiency, giving tetrads with excellent viability. Using cloned probes for both nuclear and chloroplast genes, we have found numerous restriction fragment length polymorphisms between S1-D2 and laboratory strains of C. reinhardtii.

Isolation of a photoreactivation-deficient mutant of Chlamydomonas.

A mutant deficient in photoreactivation has been isolated following mutagenesis of Chlamydomonas reinhardi with N-methyl-N'-nitro-N'-nitrosoguanidine. The mutant is deficient in the photorepair of pyrimidine dimers from nuclear DNA but appears to be normal in the rate of photorepair of dimers from chloroplast DNA. Cell-free extracts prepared from the photoreactivation-deficient mutant have about 17% of the DNA photolyase activity of wild-type cells. These results are consistent with the hypothesis that nuclear and chloroplast DNA photolyases are controlled by two separate genes.

Isolation and genetic analysis of mutant strains of Chlamydomonas reinhardi defective in gametic differentiation.

Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-l, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt- or mt+ gametes and the mutations are shown to be unlinked to the mt locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-1 strain agglutinate well with mt- gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt+ locus, and fine-structural studies reveal that imp-1 gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.