Sequencing and comparative analysis of three Chlorella genomes provide insights into strain-specific adaptation to wastewater.

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 > 2 Mb and genomic completeness ≥91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a ~1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

Rapid and high efficiency transformation of Chlamydomonas reinhardtii by square-wave electroporation.

Chlamydomonas reinhardtii, the unicellular green algae, is the model organism for studies in various physiological processes and for bioindustrial applications. To explore the molecular mechanisms underlying physiological processes or to establish engineered cell lines, the exogenous DNA needs to be integrated into the genome for the insertional mutagenesis or transgene expression. However, the amount of selected marker DNA is not seriously considered in the existing electroporation methods for mutants library construction. Here, we reported a rapid-and-high-efficiency transformation technique for cell-walled strains using square-wave electroporation system. The final yield with this electroporation method was 2-6 × 103 transformants per µg exogenous DNA for cell-walled strains in a strain-dependent manner. In general, this electroporation technique was the easy and applicable way to build a mutant library for screening phenotypes of interest.

First record of a large-scale bloom-causing species Nannochloropsis granulata (Monodopsidaceae, Eustigmatophyceae) in China Sea waters.

Nannochloropsis is an ubiquitous genus, found in diverse aquatic environments including open ocean as well as fresh and brackish water. Recently, large-scale blooms occurred frequently along eutrophic coastal zone from the Bohai Sea to the northern Yellow Sea in China. The cell density reached 10(9) to 10(10)cells per liter during a bloom near Qinhuangdao, Hebei Province. The bloom forming species, a yellow-green microalgae was successfully isolated and cultivated in the laboratory. Microscopic observation indicated that the cells contained simple morphological characteristics with a diameter about 2 µm. Pigment analyses confirmed that the pigment composition of the newly isolated strain BDH02 was similar to that of Nannochloropsis granulata. Phylogenetic analyses of 18S rRNA gene, ITS, and rbcL gene indicated that the strain was closely related to N. granulata. This is the first record of a bloom caused by N. granulata in China.

N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.

N(6)-methyldeoxyadenosine (6mA or m(6)A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here, we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms.

Nuclear DNA content variation in life history phases of the Bonnemasoniaceae (Rhodophyta).

Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4', 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15-1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome.

Molecular tools for studying the radial spoke.

Studies of cilia and flagella often entail biochemical analysis of axonemal complexes that either associate with the nine outer doublet microtubules or the two singlet microtubules in the 9+2 axoneme. Each complex contains multiple subunits, a few of which are ubiquitous vital proteins, while many are novel with prevalent domains that remain to be characterized. Investigation of axoneme biochemistry will continue providing insights into flagellar biology as well as molecular complexes in general. Yet, the complicated contents and extensive molecular interactions pose significant challenges in experimentation. As such, most biochemical studies remain limited to dynein motors and often require extensive training and expensive equipment. The rapid accumulation of high-throughput database and versatile research tools has now lessened the obstacles significantly. Here, we describe the strategies and methods that were used to circumvent some of the common difficulties to characterize the radial spoke in Chlamydomonas axoneme, some of which were tailored to students with little research experience. They could be adapted for the study of many other axonemal complexes and for classroom settings as well.

Cloning and genomic nucleotide sequence of the matrix attachment region binding protein from the halotolerant alga Dunaliella salina.

In our previous study, the sequence of a matrix attachment region binding protein (MBP) cDNA was cloned from the unicellular green alga Dunaliella salina. However, the nucleotide sequence of this gene has not been reported so far. In this paper, the nucleotide sequence of MBP was cloned and characterized, and its gene copy number was determined. The MBP nucleotide sequence is 5641 bp long, and interrupted by 12 introns ranging from 132 to 562 bp. All the introns in the D. salina MBP gene have orthodox splice sites, exhibiting GT at the 5' end and AG at the 3' end. Southern blot analysis showed that MBP only has one copy in the D. salina genome.

Life cycle analysis of unicellular algae.

Unicellular green alga is a very convenient object for flow cytometric characterization. Flow cytometry has been proposed as a quick and reliable tool for studying life cycle and growth of unicellular algae. Cell size of vegetating algae can be monitored in association with their DNA and endogenous chlorophyll content. Cells of interest (e.g., group of cells of a certain stage of the life cycle) in an asynchronously proliferating cell population can be sorted out for further microscopical or molecular biology studies. This methodological approach can be helpful for researchers who are interested in algal proliferation.

Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene.

Chlamydomonas reinhardtii is the most powerful photosynthetic eukaryotic unicellular model organism. However, its potential is not fully exploitable since as in most green plants specific targeting of nuclear genes is not routinely possible. Recently, we have shown by repair of an introduced truncated model gene that transformation of Chlamydomonas with single stranded DNA greatly suppresses random integration of the DNA in the genome whereas homologous recombination (HR) is left unchanged. However, endogenous genes still could not be targeted. Here we present optimized transformation conditions that further improved HR and suppressed non-homologous DNA integration (NHI). The improved transformation strategy allowed us now to specifically inactivate in two different Chlamydomonas strains the nuclear PHOT gene, which encodes for the blue light photoreceptor phototropin (PHOT). The option to target moderately expressed Chlamydomonas nuclear genes with high efficiency now further improves the utility of this this alga for basic science and biotechnology.

Accumulation, activity and localization of cell cycle regulatory proteins and the chloroplast division protein FtsZ in the alga Scenedesmus quadricauda under inhibition of nuclear DNA replication.

Synchronized cultures of the green alga Scenedesmus quadricauda were grown in the absence (untreated cultures) or in the presence (FdUrd-treated cultures) of 5-fluorodeoxyuridine, the specific inhibitor of nuclear DNA replication. The attainment of commitment points, at which the cells become committed to nuclear DNA replication, mitosis and cellular division, and the course of committed processes themselves were determined for cell cycle characterization. FdUrd-treated cultures showed nearly unaffected growth and attainment of the commitment points, while DNA replication(s), nuclear division(s) and protoplast fission(s) were blocked. Interestingly, the FdUrd-treated cells possessed a very high mitotic histone H1 kinase activity in the absence of any nuclear division(s). Compared with the untreated cultures, the kinase activity as well as mitotic cyclin B accumulation increased continuously to high values without any oscillation. Division of chloroplasts was not blocked but occurred delayed and over a longer time span than in the untreated culture. The FtsZ protein level in the FdUrd-treated culture did not exceed the level in the untreated culture, but rather, in contrast to the untreated culture, remained elevated. FtsZ structures were both localized around pyrenoids and spread inside of the chloroplast in the form of spots and mini-rings. The abundance and localization of the FtsZ protein were comparable in untreated and FdUrd-treated cells until the end of the untreated cell cycle. However, in the inhibitor-treated culture, the signal did not decrease and was localized in intense spots surrounding the chloroplast/cell perimeter; this was in agreement with both the elevated protein level and persisting chloroplast division.

Unstable RNAi effects through epigenetic silencing of an inverted repeat transgene in Chlamydomonas reinhardtii.

RNA interferences in the unicellular green alga, Chlamydomonas reinhardtii, can be silenced. We have used the silencing of a transgene (aadA) that confers resistance to spectinomycin to investigate the mechanisms responsible for silencing by an artificial inverted repeat (IR) of the aadA gene. The IR construct provided strong silencing, but the RNAi efficiency varied among subclones of a single RNAi-transformed strain with successive cell divisions. Northern blot analyses revealed an inverse correlation between the copy number of the hairpin RNA and the spectinomycin resistance of the subclones. There is an inverse correlation between the efficiency of RNAi and the frequency of methylated CpG (*CpG) in the silenced region. No significant methylated cytosine was observed in the target aadA gene, which suggests the absence of RNA-directed DNA methylation in trans. Several experiments suggest the existence of an intrinsic IR sequence-dependent but a transcription-independent DNA methylation system in C. reinhardtii. The correlation between the *CpG levels and the IR transcript implies the existence of IR DNA-dependent DNA methylation. Treatment of RNAi-induced cells with a histone deacetylase inhibitor, Trichostatin A, rapidly increased the amount of the hairpin RNA and suggests that transcription of the silencer construct was repressed by *CpG-related silencing mechanisms.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I.

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

Isolation and characterization of high quality DNA from marine benthic macroalgae.

The isolation of high quality DNA is essential for many molecular biology applications including polymerase chain reaction (PCR) and endonuclease restriction digestion based techniques. An easy and inexpensive protocol has been developed for extracting genomic DNA from seven species of algae viz. Lola capillaries, Enteromorpha intestinalis, Ulva lactuca and Rhizoclonium sp belonging to Chlorophyceae, Catenella nipae, Polysiphonia mollis belonging to Rhodophyceae and Dictyota ceylanica belonging to Phaeophyceae group were collected from the coastal regions of Sunderban delta in West Bengal, India dominantly growing on mud flats, bark of different mangrove trees, pneumatophores, stilt roots, concrete surfaces, wooden and bamboo poles, sides of the boats and other water vehicles inundated during high tides. The DNA was found suitable for restriction endonuclease digestion and PCR amplification with randomely amplified polymorphic DNA (RAPD) primers. The A260/A280 ratio of 1.15 0.14 to 1.94 indicated little contamination from proteins and polysaccharides. The PCR amplification with RAPD primers showed its suitability in PCR based techniques and the restriction digestion with Eco RV confirmed its suitability for hybridization based techniques. The protocol is equally good for isolating DNA from both fresh as well as preserved materials.

Chlamydomonas reinhardtii: a convenient model system for the study of DNA repair in photoautotrophic eukaryotes.

The green alga Chlamydomonas reinhardtii is a convenient model organism for the study of basic biological processes, including DNA repair investigations. This review is focused on the studies of DNA repair pathways in C. reinhardtii. Emphasis is given to the connection of DNA repair with other cellular functions, namely the regulation of the cell cycle. Comparison with the results of repair investigations that are already available revealed the presence of all basic repair pathways in C. reinhardtii as well as special features characteristic of this alga. Among others, the involvement of UVSE1 gene in recombinational repair and uniparental inheritance of chloroplast genome, the specific role of TRXH1 gene in strand break repair, the requirement of PHR1 gene for full activity of PHR2 gene, or encoding of two excision repair proteins by the single REX1 gene. Contrary to yeast, mammals and higher plants, C. reinhardtii does not appear to contain the ortholog of RAD6 gene, which plays an important role in DNA translesion synthesis and mutagenesis. Completed genome sequences will be a basis for molecular analyses allowing to explain the differences that have been observed in DNA repair of this alga in comparison with other model organisms.

Evolution of the cutinase gene family: evidence for lateral gene transfer of a candidate Phytophthora virulence factor.

Lateral gene transfer (LGT) can facilitate the acquisition of new functions in recipient lineages, which may enable them to colonize new environments. Several recent publications have shown that gene transfer between prokaryotes and eukaryotes occurs with appreciable frequency. Here we present a study of interdomain gene transfer of cutinases -- well documented virulence factors in fungi -- between eukaryotic plant pathogens Phytophthora species and prokaryotic bacterial lineages. Two putative cutinase genes were cloned from Phytophthora brassicae and Northern blotting experiments showed that these genes are expressed early during the infection of the host Arabidopsis thaliana and induced during cyst germination of the pathogen. Analysis of the gene organisation of this gene family in Phytophthora ramorum and P. sojae showed three and ten copies in tight succession within a region of 5 and 25 kb, respectively, probably indicating a recent expansion in Phytophthora lineages by gene duplications. Bioinformatic analyses identified orthologues only in three genera of Actinobacteria, and in two distantly related eukaryotic groups: oomycetes and fungi. Together with phylogenetic analyses this limited distribution of the gene in the tree of life strongly support a scenario where cutinase genes originated after the origin of land plants in a microbial lineage living in proximity of plants and subsequently were transferred between distantly related plant-degrading microbes. More precisely, a cutinase gene was likely acquired by an ancestor of P. brassicae, P. sojae, P. infestans and P. ramorum, possibly from an actinobacterial source, suggesting that gene transfer might be an important mechanism in the evolution of their virulence. These findings could indeed provide an interesting model system to study acquisition of virulence factors in these important plant pathogens.

Nitrate signaling by the regulatory gene NIT2 in Chlamydomonas.

Positive signaling by nitrate in its assimilation pathway has been studied in Chlamydomonas reinhardtii. Among >34,000 lines generated by plasmid insertion, 10 mutants were unable to activate nitrate reductase (NIA1) gene expression and had a Nit(-) (no growth in nitrate) phenotype. Each of these 10 lines was mutated in the nitrate assimilation-specific regulatory gene NIT2. The complete NIT2 cDNA sequence was obtained, and its deduced amino acid sequence revealed GAF, Gln-rich, Leu zipper, and RWP-RK domains typical of transcription factors and transcriptional coactivators associated with signaling pathways. The predicted Nit2 protein sequence is structurally related to the Nin (for nodule inception) proteins from plants but not to NirA/Nit4/Yna proteins from fungi and yeast. NIT2 expression is negatively regulated by ammonium and is optimal in N-free medium with no need for the presence of nitrate. However, intracellular nitrate is required to allow Nit2 to activate the NIA1 promoter activity. Nit2 protein was expressed in Escherichia coli and shown to bind to specific sequences at the NIA1 gene promoter. Our data indicate that NIT2 is a central regulatory gene required for nitrate signaling on the Chlamydomonas NIA1 gene promoter and that intracellular nitrate is needed for NIT2 function and to modulate NIA1 transcript levels.

DNA deformation energy as an indirect recognition mechanism in protein-DNA interactions.

Proteins that bind to specific locations in genomic DNA control many basic cellular functions. Proteins detect their binding sites using both direct and indirect recognition mechanisms. Deformation energy, which models the energy required to bend DNA from its native shape to its shape when bound to a protein, has been shown to be an indirect recognition mechanism for one particular protein, Integration Host Factor (IHF). This work extends the analysis of deformation to two other DNA-binding proteins, CRP and SRF, and two endonucleases, I-CreI and I-PpoI. Known binding sites for all five proteins showed statistically significant differences in mean deformation energy as compared to random sequences. Binding sites for the three DNA-binding proteins and one of the endonucleases had mean deformation energies lower than random sequences. Binding sites for I-PpoI had mean deformation energy higher than random sequences. Classifiers that were trained using the deformation energy at each base pair step showed good cross-validated accuracy when classifying unseen sequences as binders or nonbinders. These results support DNA deformation energy as an indirect recognition mechanism across a wider range of DNA-binding proteins. Deformation energy may also have a predictive capacity for the underlying catalytic mechanism of DNA-binding enzymes.

Maintenance DNA methyltransferase (Met1) and silencing of CpG-methylated foreign DNA in Volvox carteri.

DNA methylation plays an important role in the gene-silencing network of higher eukaryotes. We have analyzed the 21.5-kb maintenance methyltransferase (M-MTase) gene, met1, of the multicellular green alga Volvox carteri. The met1 transcript was detected only during the period when DNA replication and cell division are taking place. It encodes a 238 kDa protein containing eight C-terminal activity domains typical of M-MTases, plus upstream DNA-binding domains including the ProDom domain PD003757, which experimental analyses in animal systems have indicated is required for targeting the enzyme to DNA-replication foci. Several insertions of unknown function make Volvox Met1 the largest known member of the Met1/Dnmt1 family. Here we also show that several endogenous transposon families are CpG-methylated in Volvox, which we think causes them to be inactive. This view is supported by the observation that an in vitro CpG-methylated gene introduced into Volvox was maintained in the methylated and silent state over >100 generations. Thus, we believe that Met1 recognizes and perpetuates the in vitro methylation signal, and that the silencing machinery is then able to transduce such a methylation-only signal into a stable heterochromatic (and silent) state.

Virion-associated restriction endonucleases of chloroviruses.

Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. The prototype of the genus is Paramecium bursaria chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.

Microsatellite cross-amplification in coccolithophores: application in population diversity studies.

The development and isolation of microsatellites entails a significant input of time and money. Therefore there is an interest in using existing microsatellites on species from which markers have not yet been developed. Conservation of six previously identified microsatellite loci in the marine coccolithophorid species Emiliana huxleyi was found in a survey of two bloom forming coccolithophorid species--Gephyrocapsa oceanica and Coccolithus pelagicus. The number of alleles per locus varied from 1 to 8, and half of the microsatellite loci tested showed 4 or more alleles. The microsatellite markers used in this study may be applied to other coccolithophorid species for population analysis, eliminating the time-consuming, costly development of microsatellite markers for other coccolithophorid species.