Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum

Background

The apicomplexan parasite Cryptosporidium parvum is an emerging pathogen capable of causing illness in humans and other animals and death in immunocompromised individuals. No effective treatment is available and the genome sequence has recently been completed. This parasite differs from other apicomplexans in its lack of a plastid organelle, the apicoplast. Gene transfer, either intracellular from an endosymbiont/donor organelle or horizontal from another organism, can provide evidence of a previous endosymbiotic relationship and/or alter the genetic repertoire of the host organism. Given the importance of gene transfers in eukaryotic evolution and the potential implications for chemotherapy, it is important to identify the complement of transferred genes in Cryptosporidium.

Results

We have identified 31 genes of likely plastid/endosymbiont (n = 7) or prokaryotic (n = 24) origin using a phylogenomic approach. The findings support the hypothesis that Cryptosporidium evolved from a plastid-containing lineage and subsequently lost its apicoplast during evolution. Expression analyses of candidate genes of algal and eubacterial origin show that these genes are expressed and developmentally regulated during the life cycle of C. parvum.

Conclusions

Cryptosporidium is the recipient of a large number of transferred genes, many of which are not shared by other apicomplexan parasites. Genes transferred from distant phylogenetic sources, such as eubacteria, may be potential targets for therapeutic drugs owing to their phylogenetic distance or the lack of homologs in the host. The successful integration and expression of the transferred genes in this genome has changed the genetic and metabolic repertoire of the parasite.     

Metabolic network analysis on Phaffia rhodozyma yeast using 13C-labeled glucose and gas chromatography-mass spectrometry

Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for 'all natural' products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-(13)C]glucose were conducted with the wildtype strain (CBS5905T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes. Amino acid labeling patterns, as determined by GC-MS, were in accordance with a metabolic network consisting of the Embden-Meyerhof-Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway ( approximately 65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower.     

Impact of transamination reactions and protein turnover on labeling dynamics in (13)C-labeling experiments

A dynamic model describing carbon atom transitions in the central metabolism of Saccharomyces cerevisiae is used to investigate the influence of transamination reactions and protein turnover on the transient behavior of (13)C-labeling chemostat experiments. The simulations performed suggest that carbon exchange due to transamination and protein turnover can significantly increase the required time needed for metabolites in the TCA cycle to reach isotopic steady state, which is in agreement with published experimental observations. On the other hand, transamination and protein turnover will speed-up the net rate of incorporation of labeled carbon into some free and protein-bound amino acids. The simulation results indicate that the pattern of labeled carbon incorporation into amino acids obtained from biomass hydrolysate shows significant deviation from the commonly assumed first-order kinetics behavior until after three residence times. These observations suggest that greater caution should be used while also pointing to new opportunities in the design and interpretation of (13)C-labeling experiments.     

Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks.In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.     

Rhythms in human bone marrow and blood cells

In 24h studies of bone marrow (BM), circadian stage-dependent variations were demonstrated in the proliferative activity of BM cells from subsets of 35 healthy diurnally active men. On an average, the percentage of total BM cells in deoxyribonucleic acid (DNA) synthesis phase was 188% greater at midday than at midnight (circadian rhythm: p = 0.018; acrophase or peak time of 13: 16h). Patients with malignant disease (n = 15) and a normal cortisol circadian rhythm showed higher fractions of BM cells in S-phase at midday. Colony-forming units--granulocyte/macrophage (CFU-GM), an indicator of myeloid progenitor cells, showed the same circadian variation as DNA S-phase (average range of change or ROC = 136%; circadian rhythm: p < 0.001; acrophase of 12:09h). Deoxyribonucleic acid S-phase and CFU-GM in BM both showed a circannual rhythm (p = 0.015 and 0.008) with an identical acrophase of August 12. The daily peak in BM glutathione content, a tripeptide involved in cellular defense against cytotoxic damage, preceded BM proliferative peaks by 4-5 h (ROC = 31-90%; circadian rhythm: p = 0.05; acrophase of 08:30h). Myeloid (ROC = 57%; circadian rhythm: p = 0.056; acrophase at 08:40h) and erythroid (ROC = 26%; circadian rhythm: p = 0.01; acrophase of 13:01h) precursor cells were positively correlated (r = 0.41; p < 0.001), indicating a circadian temporal relationship and equal influence on S-phase of total BM cells. Yield of positive selected CD34+ progenitor stem cells also showed significant circadian variation (ROC = 595%; circadian rhythm: p = 0.02; acrophase of 12:40h). Thus, the temporal synchrony in cell cycling renders BM cells more sensitive at specific times to hematopoietic growth factors and cell cycle-specific cytotoxic drugs. Moreover, proper timing of BM harvesting may improve progenitor cell yield. When using marker rhythms in the blood to allow for individualized timing of BM procedures, the times of low values in white blood corpuscles, neutrophils, and lymphocytes and high values in cortisol were predictive of the times of highest BM erythroid, myeloid, and total S-phase numbers occurring in the following 12 h.     

Irradiance-dependent senescence of isolated leaves

Isolated leaves from pea ( Pisum sativum L. cv. Alaska or the genotype L-1107), oat ( Avena sativa L. cv. Victory), and fuchsia ( Fuchsia triphylla L. cv. Koralle) were retained at irradiances between 0 and 130 μmol m^-2 s^-1 PAR (photosynthetic active radiation). Irradiance-dependent CO₂ fixation was measured when the leaves were excised, and time-dependent changes in light compensation point were determined. If the irradiance was below the light compensation point for CO₂ fixation, the respiratory quotient was low, indicating that lipids were respired. The isolated leaves senesced at these low light levels. At higher light intensities the decrease in chlorophyll level was not accompainied by diminished protein level, and the respiratory quotient was close to unity. Only an irradiance equal to or slightly above the light compensation point maintained a stable chlorophyll level for a long time. This suggested that depletion of reserves in darkness or at low levels of irradiance is important for the initiation of the senescence syndrome. At high levels of irradiance, the decrement in chlorophyll level may be caused by photooxidation. Only in leaves placed under an irradiance close to the compensation point, was CO₂ fixation able to prevent aging of the leaves.     

Establishment of New Genetic Traits in a Microbial Biofilm Community

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacI^q gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.     

Effects of long-term artificial acidification on the ground vegetation and soil in a 100 year-old stand of Scots pine (Pinus sylvestris)

A study on the effects of eight years application of artificial acid rain on the vegetation and soil in an old Scots pine forest is described. Artificial rain of pH 2.5 and 3.0 caused severe damage to mosses, especially Pleurozium schreberi and Dicranum polysetum. The presence of Melampyrum pratense decreased drastically in plots treated with rain of pH 2.5 and 3.0. In Vaccinium myrtillus reduced leaf production was found in plots treated with rain of pH 2.5. A considerable decrease in base saturation had taken place in plots treated with pH 2.5 and pH 3.0 rain. Exchangeable calcium and magnesium in particular had been reduced, and the content of mangnesium in tissue of Vaccinium myrtillus appeared also to be reduced in plots treated with water of pH 2.5. The study demonstrates the need for better methods in evaluating vegetation responses in field studies. The use of visual cover recording should be supplemented by frequency analysis and harvesting methods to get better estimates of changes in vegetation structure.     

Effects of artificial acid rain on decomposition of spruce needles and on mobilisation and leaching of elements

Summary Dry matter and chemical changes in decomposing spruce needles were investigated after 16 and 38 weeks in laboratory lysimeters treated with distilled water or distilled water acidified to pH 3 or 2 with sulphuric acid. The water was added twice weekly in quantities equal to 100 or 200 mm month^–1. The CO₂ evolution and leaching of P, K, Mg, Mn, and Ca was followed together with pH measurements of the leachate.The loss of dry matter was approximately 25% during the first 16 weeks and approximately 37% after 38 weeks. At the first samling, 16 weeks, the amount of material decomposed was greater from the lysimeters given 100 mm month^–1 of water. At this water quantity dilute sulphuric acid increased the decomposition. After 38 weeks sulphuric acid at pH 3 and 2 had decreased the decomposition at 200 mm month^–1. However, the effects of acid application were small. The effect of treatment using acidified water on the content of monosaccharides was not consistent, whereas there was an indication of reduced decomposition of lignin when treated with 200 mm water month^–1 at pH 3 and 2. Nitrogen was conserved in the lysimeters with small differences between the various treatments. The order of mobility of metal elements was K>Mg>Mn>Ca. Increasing the quantity of water increased the leaching of K especially, whereas addition of dilute sulphuric acid increased the leaching of Mg, Mn and particularly Ca. During the first 16 weeks of the experiment, sulphuric acid reduced the leaching of P while later on this treatment increased the leaching. The pH of the leachate from the lysimeters treated with distilled water was initially 4.0–4.6 increasing to approximately 6.6 after 22 weeks. The pH of the decomposed needle material was 4.6 and approximately 5.2 after 16 and 38 weeks respectively. When treated with water at pH 3 the pH of the leachate was between 4 and 5, and the pH of the needles 4.2–5.1. Treatment with water at pH 2 gave a leachate with pH just above 2 and decreased the pH of the needles that had received 200 mm rain month^–1 to 2.9.The effect of the artificial acid rain appears to be more pronounced on the leaching of metal elements than on the biological activity and the dynamics of N and P. The treatments must be considered extreme when compared with the acidity of natural rain.SNSF-contribution FA45/79.     

Multiple subsurface phytoplankton blooms occurring simultaneously in the Skagerrak

The distribution and characteristics of phytoplankton in theSkagerrak in August–September 2000 were analysed in orderto evaluate the importance of subsurface phytoplankton peaksto water column ecology and primary production. In areas affectedby outflow from the Baltic, enhanced chlorophyll concentrationswere found in the warm surface waters (i.e. upper 10–20m). However, for the central Skagerrak, the major part (50–80%)of the chlorophyll in the water column was found below the warmsurface waters. The highest chlorophyll concentrations (up to>18 µg l^-1) in the study area were also found belowthe warm surface waters and up to 95% of total water columnprimary production was recorded below the warm surface waterlayer. Measurements of variable fluorescence (F_v/F_m) indicatedthe greatest potential capacity for electron flow in photosystemII in phytoplankton was located below the warm surface waters.Spectrophotometrically determined pigment ratios suggest thatthe enhanced capacity for photosynthesis in the deeper watersmay be related to greater nutrient availability here than insurface waters. Subsurface chlorophyll distributions seen inrelation to the different water masses identified in the area,as well as community analysis of the phytoplankton present inthe subsurface peaks, indicate the presence of at least threedistinct subsurface phytoplankton blooms in the Skagerrak duringthe study period. Local oxygen saturation maxima recorded immediatelyabove the subsurface peaks provide in situ evidence that thesepeaks are photosynthetically active. This suggests that newproduction is taking place in these peaks, although quantificationof this production is hampered due to a lack of informationconcerning the initial conditions in and lifetime of the subsurfacepeaks. The subsurface phytoplankton peaks were, generally, foundimmediately above an oxygen minimum that covered the entirestudy area. In the relatively cold deep Atlantic water foundbelow the oxygen minimum layer, no or very little chlorophyllwas recorded and oxygen concentrations increased. Thus, it isargued that the respiration of the organic material producedin the upper part of the water column during late summer mayprimarily occur in the intermediate layers of the water column.