Niche-Partitioning of Prochlorococcus Populations in a Stratified Water Column in the Eastern North Atlantic Ocean

The in situ community structure of Prochlorococcus populations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL) Prochlorococcus genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning of Prochlorococcus genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted Prochlorococcus strains. DGGE analysis and the sequencing of Prochlorococcus 16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning of Prochlorococcus genotypes in a stratified water column provide a genetic basis for the dim and bright Prochlorococcus populations observed in flow cytometric signatures in several oceanic provinces.     

Levels of Daily Light Doses Under Changed Day-Night Cycles Regulate Temporal Segregation of Photosynthesis and N2 Fixation in the Cyanobacterium Trichodesmium erythraeum IMS101

While the diazotrophic cyanobacterium Trichodesmium is known to display inverse diurnal performances of photosynthesis and N₂ fixation, such a phenomenon has not been well documented under different day-night (L-D) cycles and different levels of light dose exposed to the cells. Here, we show differences in growth, N₂ fixation and photosynthetic carbon fixation as well as photochemical performances of Trichodesmium IMS101 grown under 12L:12D, 8L:16D and 16L:8D L-D cycles at 70 μmol photons m^-2 s^-1 PAR (LL) and 350 μmol photons m^-2 s^-1 PAR (HL). The specific growth rate was the highest under LL and the lowest under HL under 16L:8D, and it increased under LL and decreased under HL with increased levels of daytime light doses exposed under the different light regimes, respectively. N₂ fixation and photosynthetic carbon fixation were affected differentially by changes in the day-night regimes, with the former increasing directly under LL with increased daytime light doses and decreased under HL over growth-saturating light levels. Temporal segregation of N₂ fixation from photosynthetic carbon fixation was evidenced under all day-night regimes, showing a time lag between the peak in N₂ fixation and dip in carbon fixation. Elongation of light period led to higher N₂ fixation rate under LL than under HL, while shortening the light exposure to 8 h delayed the N₂ fixation peaking time (at the end of light period) and extended it to night period. Photosynthetic carbon fixation rates and transfer of light photons were always higher under HL than LL, regardless of the day-night cycles. Conclusively, diel performance of N₂ fixation possesses functional plasticity, which was regulated by levels of light energy supplies either via changing light levels or length of light exposure.     

PHYLOGENETIC ANALYSIS OF 32 STRAINS OF VAUCHERIA (XANTHOPHYCEAE) USING THE rbcL GENE AND ITS TWO FLANKING SPACER REGIONS1

The complete rbcL gene was sequenced for 21 species and 32 strains of Vaucheria and for five other Xanthophyceae (Asterosiphon dichotomus (Kützing) Rieth, Botrydium becharianum Vischer, B. cystosum Vischer, B. stoloniferum Mitra, Tribonema intermixtum Pascher). The psbA‐rbcL spacer, upstream of the rbcL gene, and the RUBISCO spacer between the rbcL and rbcS genes were also completely sequenced for the Vaucheria strains and Asterosiphon. The psbA‐rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for the rbcL gene, and a second analysis was conducted using a portion of the psbA‐rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed that Vaucheria species formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair‐wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain of V. terrestris that was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains of V. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist within Vaucheria because the rbcL gene is a conservative gene that is identical in other protists.     

Study on photosynthetic responses and chlorophyll fluorescence in Rhizophora mucronata seedlings under shade regimes

The photosynthetic performance of mangrove Rhizophora mucronata seedlings grown under seasonally full light (HL), 50 % shade (ML), and 80 % shade (LL) conditions was characterized by gas exchange, and chlorophyll fluorescence. The carboxylation efficiency significantly affected the seasonal change of the photosynthetic capacity. Temperature and light might have synergic effect on the carboxylation efficiency. The photosynthetic rate (PN) of R. mucronata seedlings under shade regimes, however, could not be attributed to variability in chlorophyll, C _i , ΦPSII, ETR or qP values but more to differences in carboxylation efficiency, g _max, and E _max. HL and ML plants had higher PN, g _s and E than the LL ones. Nevertheless, LL leaves exhibited low photoinhibition susceptibility. The high non-photochemical quenching in HL leaves may show that applied light intensity probably exceeded the photosynthetic capability. The findings indicate that ML treatments provided the best condition to obtain such carbon fixation capacity.     

Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs

Background

The marine cyanobacterium Prochlorococcus marinus, having multiple ecotypes of distinct genotypic/phenotypic traits and being the first documented example of genome shrinkage in free-living organisms, offers an ideal system for studying niche-driven molecular micro-diversity in closely related microbes. The present study, through an extensive comparative analysis of various genomic/proteomic features of 6 high light (HL) and 6 low light (LL) adapted strains, makes an attempt to identify molecular determinants associated with their vertical niche partitioning.

Results

Pronounced strand-specific asymmetry in synonymous codon usage is observed exclusively in LL strains. Distinct dinucleotide abundance profiles are exhibited by 2 LL strains with larger genomes and G+C-content ≈ 50% (group LLa), 4 LL strains having reduced genomes and G+C-content ≈ 35-37% (group LLb), and 6 HL strains. Taking into account the emergence of LLa, LLb and HL strains (based on 16S rRNA phylogeny), a gradual increase in average aromaticity, pI values and beta- & coil-forming propensities and a decrease in mean hydrophobicity, instability indices and helix-forming propensities of core proteins are observed. Greater variations in orthologous gene repertoire are found between LLa and LLb strains, while higher number of positively selected genes exist between LL and HL strains.

Conclusion

Strains of different Prochlorococcus groups are characterized by distinct compositional, physicochemical and structural traits that are not mere remnants of a continuous genetic drift, but are potential outcomes of a grand scheme of niche-oriented stepwise diversification, that might have driven them chronologically towards greater stability/fidelity and invoked upon them a special ability to inhabit diverse oceanic environments.     

Patterns and implications of gene gain and loss in the evolution of Prochlorococcus

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.     

Species identification of the medicinal plant Tulipa edulis (Liliaceae) by DNA barcode marker

Tulipa edulis (Liliaceae) is the botanical origin of the traditional Chinese medicine (TCM) “Guangcigu”. Due to overexploitation that induced a decline in natural sources, many dried bulbs from other species of Tulipa have been used, adulterating the medicine in recent years. This practice may cause a series of inconsistent therapeutic effects and quality control problems in the herbal medicine industry. Hence, three DNA regions (matK, psbA-trnH and rbcL) were evaluated as barcodes for identifying T. edulis and its adulterants. All candidate DNA barcodes were successfully amplified from leaf samples. Based on the sequence divergences, rbcL and psbA-trnH can assign T. edulis and its adulterants to the correct genus, while matK can accurately differentiate T. edulis and its adulterants. Thus, at the DNA level, the matK intergenic region is a more suitable, accurate and applicable identification of T. edulis and its adulterants than rbcL and psbA-trnH.     

Physiology and evolution of nitrate acquisition in Prochlorococcus

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is ∼17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.     

COMPARISON OF RESISTANCE TO LIGHT STRESS IN TOXIC AND NON‐TOXIC STRAINS OF MICROCYSTIS AERUGINOSA (CYANOPHYTA)1

Blooms of Microcystis aeruginosa (Kützing) Kützing occur frequently in many freshwater ecosystems around the world, but the role of environmental factors in promoting the growth and determining the proportion of toxic and non‐toxic strains still requires more investigation. In this study, four strains (toxic CPCC299 & FACHB905 and non‐toxic CPCC632 & FACHB315) were exposed to high light (HL) condition, similar to light intensity found at the surface of a bloom, to evaluate their sensitivity to photoinhibition. We also estimated their capacity to recover from this HL stress. For all strains, our results showed an increased inhibition of the photosynthetic activity with HL treatment time. When comparing the extent of photoinhibition between strains, both toxic strains were more resistant to the treatment and recovered completely their photosynthetic capacity after 3 h, while non‐toxic strains needed more time to recover. For toxic strains, the rETR under HL was higher compared to the rETR under low light (LL) control condition despite 50% photoinhibition. This suggests that the detrimental effect of high light (HL; up to 2 h) is outweighed by their higher photosynthetic potential. This conclusion did not stand for non‐toxic strains, and indicates their preference for LL environment. We also demonstrated that a LL/HL cycle induced a 259% increase in cell yield for a toxic strain and a decrease by 22% for a non‐toxic strain. This also indicates that toxic strains have higher tolerance to HL in a fluctuating light environment. Our data demonstrated that difference of sensitivity to HL between strains can modify the competitive outcome between toxic and non‐toxic strains and may affect bloom toxicity.     

Invasion of protein coding genes by green algal ribosomal group I introns

The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns.     

Comparative analysis of the constituents of two cultivars of Dioscorea dumetorum (Kunth) Pax. and their molecular barcoding

The edible and wild cultivars of Dioscorea dumetorum (Kunth) Pax. (Family: Dioscoreaceae) are commonly used as close substitutes in herbal markets due to their edibility and medicinal importance. Comparative study of the cultivars was done using chemical analysis of their methanol tuber extracts and by utilizing the DNA barcode regions, ribulose-1, 5-bisphosphate carboxylase subunits (rbcL) and the non-coding intergenic spacer gene (trnH-psbA) for nucleotide sequences comparison of cultivars. Of the 54 compounds identified from the methanol extract of the edible and wild cultivars using gas chromatography-mass spectrometry (GC-MS), 12 are similar, while 42 are remarkably different. In particular n-hexadecanoic (palmitic) acid had a higher percentage area in the edible cultivar (31.16%) compared to the wild cultivar (0.26%). The cultivars were successfully amplified using the universal primers gene rbcL and trnH-psbA. Their nucleotide sequences showed a slight variation when aligned with CodonCode Aligner V.9.0.1. They were identified by the Basic Local Alignment Search Tool (BLAST) through comparison with Genbank data, which shows close similarities with submitted data. The use of molecular barcoding in identifying the cultivars of Dioscorea dumetorum as well as the chemical composition of their tubers may form an important marker in the compilation of future herbal pharmacopoeia for its proper identification.     

Application of DNA Barcodes in Asian Tropical Trees – A Case Study from Xishuangbanna Nature Reserve,Southwest China

Background

Within a regional floristic context, DNA barcoding is more useful to manage plant diversity inventories on a large scale and develop valuable conservation strategies. However, there are no DNA barcode studies from tropical areas of China, which represents one of the biodiversity hotspots around the world.

Methodology and Principal Findings

A DNA barcoding database of an Asian tropical trees with high diversity was established at Xishuangbanna Nature Reserve, Yunnan, southwest China using rbcL and matK as standard barcodes, as well as trnH–psbA and ITS as supplementary barcodes. The performance of tree species identification success was assessed using 2,052 accessions from four plots belonging to two vegetation types in the region by three methods: Neighbor-Joining, Maximum-Likelihood and BLAST. We corrected morphological field identification errors (9.6%) for the three plots using rbcL and matK based on Neighbor-Joining tree. The best barcode region for PCR and sequencing was rbcL (97.6%, 90.8%), followed by trnH–psbA (93.6%, 85.6%), while matK and ITS obtained relative low PCR and sequencing success rates. However, ITS performed best for both species (44.6–58.1%) and genus (72.8–76.2%) identification. With trnH–psbA slightly less effective for species identification. The two standard barcode rbcL and matK gave poor results for species identification (24.7–28.5% and 31.6–35.3%). Compared with other studies from comparable tropical forests (e.g. Cameroon, the Amazon and India), the overall performance of the four barcodes for species identification was lower for the Xishuangbanna Nature Reserve, possibly because of species/genus ratios and species composition between these tropical areas.

Conclusions/Significance

Although the core barcodes rbcL and matK were not suitable for species identification of tropical trees from Xishuangbanna Nature Reserve, they could still help with identification at the family and genus level. Considering the relative sequence recovery and the species identification performance, we recommend the use of trnH–psbA and ITS in combination as the preferred barcodes for tropical tree species identification in China.     

Interactive effects of photon fluence rates and drought on CAM‐cycling in Delosperma tradescantioides (Mesembryanthemaceae)

The ability of photosynthesis and CAM to acclimate to low (220 µmol m^?2 s^?1; LL) and relatively high (550 µmol m^?2 s^?1; HL) photosynthetic photon flux densities (PPFD) was investigated in the CAM-cycling species Delosperma tradescantioides by means of CO₂ gas exchange and chlorophyll fluorescence analysis. Furthermore, the influence of short-term drought on malic acid accumulation and the activity of photosystem II (PSII) was studied to assess the possible interactions between drought and the prevailing PPFD in this species. HL plants showed features of sun versus shade acclimation relative to LL plants. Nocturnal malic acid accumulation (Δ-malate) and leaf water content also tended to be higher in HL plants. Irrespective of the PPFD during growth, the weak Δ-malate doubled within 3 days of drought. Despite largely restricted CO₂ uptake, photosynthetic activity as estimated from fluorescence analysis declined only ca 5%. After 7 days of drought, when plants showed CAM-idling and Δ-malate had decreased again, potential carbon assimilation was still ca 84% of that in well-watered plants and remained relatively constant throughout the day. Decarboxylation of malic acid accounted for ca 23% of potential assimilation assuming total oxidation of a maximum portion of this organic acid. Drought did not affect predawn maximum photochemical efficiency (F_v/F_m). Nonphotochemical quenching (qN) increased (24%) in response to desiccation and resulted in a more or less constant reduction state of PSII. This increase in qN resulted mainly from the change in its fast-relaxing component (qNF), while the slow component (qNS) was significant only at or above saturating PPFD in both HL and LL plants. The photon response characteristics of PSII, which differed between LL and HL plants, were unaffected by short-term drought. Photon harvesting and photon use were always adjusted to guarantee a low reduction state of PSII. Results suggest that in both LL and HL plants CAM-cycling may help to stabilize photosynthesis but to a large extent by other means than simply providing internally derived CO₂.     

5个油松种源光合特性的比较研究

为了揭示自然条件下不同种源油松光合特性的差异,为优良种源选择提供依据,在黄土高原种源实验地,对5个油松种源（陕西洛南LN,陕西黄陵HL,山西吕梁山LL,山西灵空山LK,辽宁千山QM）幼苗的光合生理特征及其与环境因子的关系进行了研究。结果表明：5个油松种源的净光合速率（P_n）呈双峰型,蒸腾速率（T_r）、气孔导度（G_s）亦基本呈双峰型,第1峰值均出现在上午8:00,有明显的光合午休现象。种源QM的水分利用效率（WUE）日变化呈单峰性,峰值出现在10:00,而种源LN、LL、LK和HL呈双峰型,第一峰值出现在8:00,第二峰值出现在12:00~16:00。种源LN、LL、LK和QM午间净光合速率的下降主要由气孔因素引起的,而种源HL则主要由叶肉细胞同化能力的降低引起的。种源LK的日均P_n、T_r和WUE均高于其他种源,是高光合、高蒸腾和高水分利用效率的种源,而种源HL属于低光合、低蒸腾和低水分利用效率的种源。T_a是影响各种源的P_n的首要因子,其次是VPD;RH是影响种源LN、LL和LK的T_r的首要因子;而T_a和VPD是影响种源HL和QM的T_r的主要因子。     

Molecular analysis of green-tide-forming macroalgae in the Yellow Sea

In the summer of 2008, free-floating green algae bloomed in the Yellow Sea. Samples were collected in a wide area (119°32′-122°00′E, 32°25′-36°49′N). We calculated the sequence divergences of nuclear ITS, chloroplast rbcL, and psbA data of free-floating samples collected from the Yellow Sea and Ulvaceae from Europe and Japan. In the ITS sequence, 19 out of the 21 Yellow Sea samples of 2008 were identical to those of a sample taken at Qingdao in 2007. A low divergence (0.2%) was found in remaining two samples. Similar evidence was shown by pairwise distances of rbcL and psbA gene sequence data, implying the uniformity of the Yellow Sea blooms in 2007 and 2008. The ITS sequence of the Yellow Sea samples differed 8.1-10.8% from free-floating Enteromorpha or Ulva reported worldwide. ITS-based molecular phylogenetic results and rbcL sequence data grouped the free-floating alga in the Yellow Sea into one clade with Enteromorpha procera, Enteromorpha linza and Enteromorpha prolifera. Furthermore, both morphological characteristics and ribotype network of the ITS sequences imply that the blooming algae in 2007 and 2008 were E. prolifera. The haplotypes of the Yellow Sea free-floating E. prolifera are closely related to those from the Japanese coast but less to European and American algae.