Direct PCR Amplification Research Articles

MOLECULAR CLONING AND CHARACTERIZATION OF HIGH‐AFFINITY NITRATE TRANSPORTERS IN MARINE PHYTOPLANKTON1

Nitrate transporter systems initiate nitrate uptake in photoautotrophic organisms including marine phytoplankton. The functional genes encoding high‐affinity nitrate transporters (Nrt2) in marine phytoplankton were investigated in this study. Direct PCR amplification with newly developed primers was used to detect Nrt2 genes in cultured marine phytoplankton species: Dunaliella tertiolecta Butcher (Chlorophyceae); Emiliania huxleyi (Lohmann) W. H. Hay et H. Mohler (Haptophyceae); and four diatoms, including Thalassiosira weissflogii (Grunow) G. A. Fryxell et Hasle, Skeletonema costatum (Grev.) Cleve, Ditylum brightwellii (T. West) Grunow, and Chaetoceros muelleri Lemmerm. (Bacillariophyceae). Phylogenetic analysis of the detected Nrt2 genes identified three distinct clusters grouped by each class of phytoplankton species. Transcriptional abundance (mRNA) of Nrt2 genes in D. tertiolecta, E. huxleyi, T. weissflogii, and C. muelleri were measured using quantitative reverse transcriptase‐PCR assays under various nitrogen conditions. The diatoms and E. huxleyi induced Nrt2 gene transcripts both in the presence of nitrate and under nitrogen (N) starvation, while the Nrt2 transcript in D. tertiolecta was induced only when nitrate was present. Chaetoceros muelleri exhibited the highest transcript induction of the Nrt2 gene. The transcriptional abundance of the Nrt2 genes in C. muelleri was further examined with short‐term nitrate incubation of cells grown under N‐sufficient or N‐starved conditions. The maximum induction of Nrt2 gene transcripts was observed within 1 h after nitrate was added. Thus, we report differential induction and regulation of Nrt2 genes in the selected phytoplankton species to varying conditions of external N.

Direct PCR for DNA Barcoding in the Genera Phytophopthora and Pythium

ABSTRACTA protocol for direct polymerase chain reaction (DPCR) amplification of commonly used phylogenetic markers of the genera Pythium and Phytophthora from mycelia was developed. This has proven useful for direct PCR amplification from hypha, zoospores and infected plant material, which is quicker and simpler, than other proposed attempts. Optimal reaction conditions are described allowing amplification of commonly used phylogenetic markers with a size of up to 1 kbp. This approach proved successful for amplification of selected markers from hundreds of Phytophthora, Pythium and fungal isolates and has been so far used in routine genotyping identification of oomycota and fungi in our laboratory.

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.

Uncultured Archaea in a hydrothermal microbial assemblage: phylogenetic diversity and characterization of a genome fragment from a euryarchaeote

The polychaete Alvinella pompejana lives in organic tubes on the walls of active hydrothermal chimneys along the East Pacific Rise. To examine the diversity of the archaeal community associated with the polychaete tubes, we constructed libraries by direct PCR amplification and cloning of 16S rRNA genes. Almost half of the sequences of the 16S rRNA gene libraries clustered with uncultured archaeal groups. In an effort to access genomic information from uncultured archaeal members we further constructed a fosmid library from the same DNA source. One of the clones, Alv-FOS5, was sequenced completely. Its sequence analysis revealed an incomplete rRNA operon and 32 predicted ORFs. Seventeen of these ORFs have been assigned putative functions, including transcription and translation, cellular processes and signalling, transport systems and metabolic pathways. Phylogenetic analyses of the 16S rRNA gene suggested that Alv-FOS5 formed a new lineage related to members of Deep-Sea Hydrothermal Vent Euryarchaeota group II. Phylogenetic analyses of predicted proteins revealed the existence of likely cases of horizontal gene transfer, both between Crenarchaeota and Euryarchaeota and between Archaea and Bacteria. This study is the first step in using genomics to reveal the physiology of an as yet uncultured group of archaea from deep-sea hydrothermal vents.

Genotyping of Toxoplasma gondii by Multiplex PCR and Peptide-Based Serological Testing of Samples from Infants in Poland Diagnosed with Congenital Toxoplasmosis

Toxoplasma gondii has a clonal population genetic structure with three (I, II, and III) lineages that predominate in North America and Europe. Type II strains cause most cases of symptomatic human infections in France and the United States, although few other regions have been adequately sampled. Here we determined the parasite genotype in amniotic fluid and cerebrospinal fluid samples from congenital toxoplasmosis cases in Poland. Nineteen confirmed congenital cases of toxoplasmosis were analyzed, including both severe and asymptomatic cases. The genotype of parasite strains causing congenital infection was determined by direct PCR amplification and restriction fragment length polymorphism analysis. Nested multiplex PCR analysis was used to type four independent polymorphic markers. The sensitivity of multiplex nested PCR was >/=25 parasites/ml in amniotic fluid and cerebral spinal fluid samples. Parasite DNA was successfully amplified in 9 of 19 samples (eight severely affected and one asymptomatic fetus). Only genotype II parasites were identified as the source of T. gondii infection based on restriction fragment length polymorphism analysis. Strains causing congenital infections were also typed indirectly based on detection of antibodies to strain-specific peptides. Serotyping indicated that 12 of 15 cases tested were caused by type II strains and these positives included both symptomatic and asymptomatic infections. Overall, the combined analysis indicated that 14 of the cases were caused by type II strains. Our results are consistent with the hypothesis that parasite burden is associated with severity of congenital toxoplasmosis and indicate that serological testing provides a promising method for genotypic analysis of toxoplasmosis.

A homozygous missense mutation in PEPD encoding peptidase D causes prolidase deficiency associated with hyper-IgE syndrome

Prolidase deficiency is a complex disease characterized by various skin manifestations accompanied by mental retardation, facial dysmorphism and susceptibility to pyogenic infections. We assessed a patient presenting a peculiar phenotype combining manifestations of prolidase deficiency with features typical of hyper-IgE syndrome. Mutation analysis was performed using direct PCR amplification and PCR restriction fragment length polymorphism analysis. We identified a novel homozygous recessive mutation in the PEPD gene, which was found to segregate in the family of the patient with the disease and was not found in a panel of DNA samples representative of all major Druze families living in northern Israel. Our results suggest that prolidase deficiency associated with hyper-IgE syndrome, a rare disorder, can be caused by mutations in PEPD.

Bacterial endophytes of the wildflowerCrocus albiflorusanalyzed by characterization of isolates and by a cultivation-independent approach

The presence and taxonomy of endophytic bacteria of the entire aerial parts of crocus (Crocus albiflorus), a wildflower native in the Alps, were investigated. A combination of plating of plant macerates, isolation and sequence identification of isolates, and direct 16S rDNA PCR amplification followed by whole-community fingerprinting (T-RFLP) and by construction of a bacterial clone library was used. The results clearly indicated that a wide range of bacteria from diverse phylogenetic affiliation, mainly gamma-Proteobacteria and Firmicutes, live in association with plants of C. albiflorus. The community composition of the culturable component of the microflora was remarkably different from that of the clone library. Only three bacterial divisions were found in the culture collection, which represented 17 phylotypes, whereas six divisions were identified in the clonal analysis comprising 38 phylotypes. The predominant group in the culture collection was the low G+C Gram-positive group, whereas in the clone library, the gamma-Proteobacteria predominated. Interestingly, the most prominent bacterium within the uncultured bacterial community was a pseudo monad closely related to a cold-tolerant Pseudomonas marginalis strain. The results suggest that Crocus supports a diverse bacterial microflora resembling the microbial communities that have been described for other plants and containing species that have not been described in association with plants.

POTENTIAL ACTIVITY AND DIVERSITY OF METHANOTROPHIC BACTERIA IN FOREST SOIL, PEAT, AND SEDIMENTS FROM A HYDROELECTRIC RESERVOIR (ROBERT-BOURASSA) AND LAKES IN THE CANADIAN TAIGA

The potential activity and diversity of methanotrophic bacteria in samples from forest soil, peat, and sediment from a hydroelectric reservoir (Robert–Bourassa) and lakes located in the sub-arctic taiga were studied. Incubation experiments with soil slurries, methanotrophic bacterial counts, and direct PCR amplification of genes specific to methanotrophic bacteria were carried out on soil and sediment samples from three locations (nonflooded, periodically flooded, and flooded) from the reservoir. Sediments from three lakes located near the reservoir were also sampled to compare with nearby unperturbed aquatic systems. Only the uppermost part (5–10 cm) of soils and sediments was analyzed. Regardless of the incubation temperature, potential methane (CH4) oxidation rates were lower (0.028–0.066 6mol CH4 g j1 soil d j1 )i n nonflooded forest soil compared to lacustrine sediments that exhibited potential oxidation rates generally close to those observed in periodically flooded or flooded forest soil (0.04–0.14 6mol CH4 g j 1 soil d j1 ), but far below the range of values (0.20–0.58 6mol CH4 g j 1 soil d j1 ) recorded in peat systems. Samples from flooded forest soil and peat had higher CH4 uptake activity, but this could not be related to a greater number of methanotrophic bacteria (pmoA probe-positive colonies). Analysis of genes specific to methanotrophic bacteria revealed a low diversity in the methanotrophic community, with the genus Methylocystis being dominant. Overall, samples of flooded soil and peat with higher CH4 production rates also had higher rates of CH4 oxidation by methanotrophic bacteria. Flooding also seems to modify the resilience of the methanotrophic community to temperature fluctuations, an important consideration for future studies. (Soil Science 2006;171:127–137)

Functional Impact of Sequence Alterations Found in BRCA1 Promoter/5'UTR Region in Breast/Ovarian Cancer Families from Upper Silesia, Poland

The 5' region of BRCA1 contains multiple regulatory sequences flanking the two alternative promoters α and β and two alternative, non-coding exons, 1a and 1b. Aberrations within the 5' region BRCA1 (encompassing two alternative promoters α and β and exons 1a and 1b) may be associated with an increased risk of breast and ovarian cancer. In this study we screened 150 patients for polymorphism and mutations in this region of BRCA1. All probands came from familial breast and/or ovarian cancer that had been found to be mutation-negative in a previous search for founder mutations in BRCA1 (185delAG, C61G, 4153delA, 5382insC) or BRCA2 (6174delT, 9631delC). In our study we found several sequence alterations within the non-coding region of BRCA1 by using direct DNA sequencing and allele-specific PCR amplification. Three families with a polymorphic deletion in BRCA1 exon 1b (2223delAAAAA, Acc. U37574) were found. Moreover, two linked nucleotide substitutions (2642A>T, 2743T>C, Acc. U37574) in BRCA1 intron 1 were detected in 16 patients. In order to assess the functional significance of these two sequence variants, we constructed a reporter vector encoding firefly luciferase under the transcriptional and translational control of wild type and altered BRCA1 promoter region. The reporter assay was performed using a lung cancer cell line (NCI-H1299) and a breast cancer cell line (MCF7). We have demonstrated that the analysed sequence variants have no functional significance in our experimental system. However, we have found that the BRCA1 promoter has lower relative activity in the breast cancer cell line compared with the lung cancer cell line. Based on the results of our functional experiments we conclude that the polymorphic deletion 2223delAAAAA and two linked substitutions 2642A>T and 2743T>C do not significantly alter BRCA1 expression and are probably not disease-causing mutations.

Bacillary Angiomatosis Caused by Bartonella quintana

Bacillary angiomatosis is a disorder of neovascular proliferation involving skin and lymph nodes of immunosuppressed patients. Bartonella henselae or Bartonella quintana have been inculpated as causative by direct culture or PCR amplification of DNA sequences. Here, we report the clinical evolution of a patient infected with human immunodeficiency virus (HIV) whose B. quintana infection was diagnosed by PCR.

Blood Culture-Negative Endocarditis in a Reference Center

To identify the current etiologies of blood culture-negative infective endocarditis and to describe the epidemiologic, clinical, laboratory, and echocardiographic characteristics associated with each etiology, as well as with unexplained cases, we tested samples from 348 patients suspected of having blood culture-negative infective endocarditis in our diagnostic center, the French National Reference Center for Rickettsial Diseases, between 1983 and 2001. Serology tests for Coxiella burnettii, Bartonella species, Chlamydia species, Legionella species, and Aspergillus species; blood culture on shell vial; and, when available, analysis of valve specimens through culture, microscopic examination, and direct PCR amplification were performed. Physicians were asked to complete a questionnaire, which was computerized. Only cases of definite infective endocarditis, as defined by the modified Duke criteria, were included. A total of 348 cases were recorded-to our knowledge, the largest series reported to date. Of those, 167 cases (48%) were associated with C. burnetii, 99 (28%) with Bartonella species, and 5 (1%) with rare, fastidious bacterial agents of endocarditis (Tropheryma whipplei, Abiotrophia elegans, Mycoplasma hominis, Legionella pneumophila). Among 73 cases without etiology, 58 received antibiotic drugs before the blood cultures. Six cases were right-sided endocarditis and 4 occurred in patients who had a permanent pacemaker. Finally, no explanatory factor was found for 5 remaining cases (1%), despite all investigations.Q fever endocarditis affected males in 75% of cases, between 40 and 70 years of age. Ninety-one percent of patients had a previous valvulopathy, 32% were immunocompromised, and 70% had been exposed to animals. Our study confirms the improved clinical presentation and prognosis of the disease observed during the last decades. Such an evolution could be related to earlier diagnosis due to better physician awareness and more sensitive diagnostic techniques. As for Bartonella species, B. quintana was recorded more frequently than B. henselae (53 vs 17 cases). For 18 patients with Bartonella endocarditis, the responsible species was not identified. Species determination was achieved through culture and/or PCR in 49 cases and through Western immunoblotting in 22. Comparison of B. quintana and B. henselae endocarditis revealed distinct epidemiologic patterns. The 2 cases due to T. whipplei reflect the emerging role of this agent as a cause of infective endocarditis. Because identification of the bacterium was possible only through analysis of excised valves by histologic examination, PCR, and culture on shell vial, the prevalence of the disease might be underestimated. Among patients who received antibiotic drugs before blood cultures, 4 cases (7%) were found to be associated with Streptococcus species (2 S. bovis and 2 S. mutans) through 16S rDNA gene amplification directly from the valve, which shows the usefulness of this technique in overcoming the limitations of previous antibiotic treatment. Right-sided endocarditis occurred classically in young patients (mean age, 36 yr), intravenous drug users in 50% of cases, and suffering more often from embolic complications. Finally, 5 cases without etiology or explaining factors were all immunocompetent male patients with previous aortic valvular lesions, and 3 of the 5 presented with an aortic abscess. Further investigations should be focused on this group to identify new agents of infective endocarditis.

Pre-Operative Differentiation Between Pancreatic Cancer (PC) and Distal Bile Duct Cancer (DBDC) Based on Endoscopic Brushing/FNA Mutational Analysis

Background: Malignant distal CBD strictures may be related to PC or DBDC. Accurate diagnosis is of prognostic value given better survival with resected DBDC (∼50% 5 yr survival) vs. resected PC (∼25% 5 yr survival). Currently employed pre-operative diagnostic modalities cannot accurately differentiate between DBDC and PC in malignant CBD strictures. The role of molecular markers in this context remains unknown. Methods: Twenty-eight patients with surgically confirmed PC (16) or DBDC (12) were studied. Biliary brushings (17) or EUS guided FNA (11) had been performed on all. Representative malignant cells from stained cytology slides and corresponding resection specimen (malignant and benign tissue) from all cases were micro-dissected and subjected to direct PCR amplification. The PCR products were analyzed for (1) k-ras codon 12/13 mutations and (2) microsatellite losses targeting 1p36, 3p26, 5q23, 9p21, 10q23 and 17p13, utilizing fluorescent capillary electrophoresis. The pattern of mutation acquisition and microsatellite loss was compared to the surgical pathology. Results: All malignant cytology and surgical specimens carried multiple mutations whereas the benign (control) samples carried none. Multiple microsatellite losses were seen in both PC and DBDC but K-ras codon 12 mutation was seen in 14/16 PC cases and only 1/12 DBDC cases (p < 0.001). This mutational pattern differentiated between PC and CBDC with a sensitivity of 88% and specificity of 92%. Conclusions: Although patients with resected DBDC can expect better survival than those with resected PC, this information is only available post-resection. A detailed molecular analysis applied to endoscopically acquired cytologic samples can help in reaching an accurate diagnosis of PC or DBDC. Multiple microsatellite losses are seen in both PC and DBDC but the presence of k-ras mutation is an accurate predictor of malignancy of pancreatic ductal origin.

Direct analysis of ribosomal DNA in denaturing gradients: application on the effects of Phlebiopsis gigantea treatment on fungal communities of conifer stumps

The aim of this study was to test the usefulness of direct PCR-amplification in analysing fungal diversity in stumps. The analysis was conducted on stumps treated against Heterobasidion spp. using a commercial formulation of Phlebiopsis gigantea (Rotstop), and carried out using denaturing gradient gel electrophoresis (DGGE) of small subunit (SSU) ribosomal DNA (rDNA) fragments PCR-amplified directly from wood DNA samples using two separate fungus-specific primer pairs. On average, two (range 0-9) different amplification products were observed by DGGE in single wood samples of approximately 500 mm3. The PCR products were classified into operational taxonomical unit (OTU) groups based on their DGGE mobility. Six OTUs could be affiliated with a known species based on a reference fungal collection of 37 species: Heterobasidion annosum, H. parviporum. Hypholoma capnoides, P. gigantea, Resinicium bicolor and Stereum sanguinolentum. Sequence analyses did not give further identifications. OTU profiles from old (6 yr-old) and fresh (1-year-old) Scots pine and Norway spruce stumps from treated and untreated forest plots were compared statistically, and some significant differences were observed in the species composition between the treated and untreated plots. However, the frequency of most of the OTUs did not seem to be affected, and the treatment did not seem to have reduced the overall level of fungal diversity. Based on these results, direct PCR-amplification seems to be useful in analyses of fungal communities in decaying conifer stumps.

An updated view of kinetoplastid phylogeny using environmental sequences and a closer outgroup: proposal for a new classification of the class Kinetoplastea

Given their ecological and medical importance, the classification of the kinetoplastid protists (class Kinetoplastea) has attracted much scientific attention for a long time. Morphology-based taxonomic schemes distinguished two major kinetoplastid groups: the strictly parasitic, uniflagellate trypanosomatids and the biflagellate bodonids. Molecular phylogenetic analyses based on 18S rRNA sequence comparison suggested that the trypanosomatids emerged from within the bodonids. However, these analyses revealed a huge evolutionary distance between the kinetoplastids and their closest relatives (euglenids and diplonemids) that makes very difficult the correct inference of the phylogenetic relationships between the different kinetoplastid groups. Using direct PCR amplification of 18S rRNA genes from hydrothermal vent samples, several new kinetoplastid-like sequences have been reported recently. Three of them emerge robustly at the base of the kinetoplastids, breaking the long branch leading to the euglenids and diplonemids. One of these sequences belongs to a close relative of Ichthyobodo necator (a fish parasite) and of the 'Perkinsiella amoebae'-like endosymbiont of Neoparamoeba spp. amoebae. The authors have studied the reliability of their basal position and used all these slow-evolving basal-emerging sequences as a close outgroup to analyse the phylogeny of the apical kinetoplastids. They thus find a much more stable and resolved kinetoplastid phylogeny, which supports the monophyly of groups that very often emerged as polyphyletic in the trees rooted using the traditional, distant outgroup sequences. A new classification of the class Kinetoplastea is proposed based on the results of the phylogenetic analysis presented. This class is now subdivided into two new subclasses, Prokinetoplastina (accommodating the basal species I. necator and 'Perkinsiella amoebae') and Metakinetoplastina (containing the Trypanosomatida together with three additional new orders: Eubodonida, Parabodonida and Neobodonida). The classification of the species formerly included in the genus Bodo is also revised, with the amendment of this genus and the genus Parabodo and the creation of a new genus, Neobodo.

Natural community structure of cyanobacteria in the South China Sea as revealed by rpoC1 gene sequence analysis.

To examine the community structure of cyanobacterial populations of the South China Sea on the surface and at depth of 80 m. Direct PCR amplification of RNA polymerase (rpoC1) genes from environmental DNAs extracted from seawater, and cloning of the fragments and sequence analysis were used. A great diversity of Prochlorococcus and Synechococcus were detected at the investigation site. Genetically related Prochlorococcus were found in both layers while Synechococcus were found only on the surface. Prochlorococcus were clustered with the known high-light adapted II genotypes, and further divided into seven groups. Synechococcus could be divided into two groups, and the second group could be further subdivided into several clades. The dominant genotype of Prochlorococcus was high-light adapted II genotype, and Synechococcus were distributed basically on the surface. This is the first report about the cyanobacterial community structure in the South China Sea, and an important supplement to the current understanding of the relationship between genetic and ecological diversity and environments.

Molecular and biological characterization of molt-inhibiting hormone of Penaeus monodon

The action of molt-inhibiting hormone (MIH) on the inhibition of ecdysone release from the Y-organ of decapod crustacean keeps the animal in the intermolt stage that dominates its molting cycle. MIH is thus one of the major keys in mediating growth and reproduction. This study has isolated cDNA encoding two types of MIH, Pem-MIH1 and Pem-MIH2, from the black tiger shrimp, Penaeus monodon on the basis of sequence homology to MIH from two other shrimp species. The full-length cDNA of Pem-MIH1 was characterized. Pem-MIH1 cDNA harbored 318 bp open reading frame that coded for a translated product containing 28 amino acids of the signal peptide and a putative mature Pem-MIH of 77 amino acids. The recombinant Pem-MIH1 was expressed in Pichia pastoris as a secreted protein. After purification by gel filtration, the purified Pem-MIH1 exhibited the ability to extend molting duration of P. monodon from 11.8 days to 16.3 days suggesting that Pem-MIH1 be responsible for molt-inhibiting function in the shrimp. The attempt to clone Pem-MIH1 and Pem-MIH2 genes was achieved by direct PCR amplification and PCR-based genome walking strategy, respectively. The structure of both Pem-MIH genes, containing three exons interrupted by two introns, was similar to each other and also to that of MIH genes of other crustaceans reported so far. Expression study of Pem-MIH1 and Pem-MIH2 in various tissues of P. monodon revealed the difference in expression patterns. Pem-MIH1 expressed in both the eyestalk and the thoracic ganglia whilst Pem-MIH2 expression was limited to the eyestalk. The expression of MIH in non-eyestalk tissue may suggest additional role of this hormone.

MOLECULAR CHARACTERIZATION OF THE ASSIMILATORY NITRATE REDUCTASE GENE AND ITS EXPRESSION IN THE MARINE GREEN ALGA DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

Nitrate is used as a nutrient for growth by many organisms through the nitrate assimilation pathway. The key enzyme involved in nitrate assimilation is nitrate reductase (NR). Transcriptional regulation of NR genes has been studied in various phototrophic eukaryotes from freshwater or terrestrial habitats. Here, we describe the first NR gene from a marine phytoplankton, the green alga Dunaliella tertiolecta Butcher. Its sequence is very similar to that of the other green algae, but its intron structure and transcriptional regulation differ significantly. A partial genomic NR gene fragment was cloned using direct PCR amplification with degenerate primers, and rapid amplification of cDNA ends was performed to obtain a complete NR gene from a cDNA of D. tertiolecta. The complete NR gene of D. tertiolecta is 2.7 kb in size and is similar at the amino acid level to the NRs from freshwater green algae Chlorella vulgaris (50.6%), Chlamydomonas reinhardtii (58.4%), and Volvox carteri (59.4%). Transcriptional regulation of D. tertiolecta NR was examined in response to different nitrogen sources and during the diel cycle. Dunaliella tertiolecta NR transcript was induced by nitrate and repressed by ammonium. In addition, transcription was not induced under nitrogen depletion. Induction of NR transcript responded to the diel cycle, and transcript levels were maximized within an hour after the beginning of the light period. The induction and repression of NR transcript were much slower than reported in freshwater green algae, which may be related to the size of internal nitrogen pools.

Spatial distribution of bacterial communities and phenanthrene degradation in the rhizosphere of Lolium perenne L.

Rhizodegradation of organic pollutants, such as polycyclic aromatic hydrocarbons, is based on the effect of root-produced compounds, known as exudates. These exudates constitute an important and constant carbon source that selects microbial populations in the plant rhizosphere, modifying global as well as specific microbial activities. We conducted an experiment in two-compartment devices to show the selection of bacterial communities by root exudates and phenanthrene as a function of distance to roots. Using direct DNA extraction, PCR amplification, and thermal gradient gel electrophoresis screening, bacterial population profiles were analyzed in parallel to bacterial counts and quantification of phenanthrene biodegradation in three layers (0 to 3, 3 to 6, and 6 to 9 mm from root mat) of unplanted-polluted (phenanthrene), planted-polluted, and planted-unpolluted treatments. Bacterial community differed as a function of the distance to roots, in both the presence and the absence of phenanthrene. In the planted and polluted treatment, biodegradation rates showed a strong gradient with higher values near the roots. In the nonplanted treatment, bacterial communities were comparable in the three layers and phenanthrene biodegradation was high. Surprisingly, no biodegradation was detected in the section of planted polluted treatment farthest from the roots, where the bacterial community structure was similar to those of the nonplanted treatment. We conclude that root exudates and phenanthrene induce modifications of bacterial communities in polluted environments and spatially modify the activity of degrading bacteria.

Lyme borreliosis spirochetes in questing ticks from mainland Portugal.

In Portugal, Ixodes ricinus ticks have been shown to contain DNA of several spirochetes belonging to the Borrelia burgdorferi sensu lato complex, with major differences in the genetic diversity between ecozones. Some isolates have been obtained since 1999, confirming the circulation of pathogenic strains in these ticks. Ixodes ricinus is considered to be a widespread species, however, in Portugal it is found only in a few habitats. Here we present preliminary results from a nationwide survey of questing I. ricinus (n = 4,001) and other Ixodidae (n = 1,534) in Portugal, initiated in 2001. The sampling points (so far 41) were selected using a Geographic Information System, according to the type of vegetation, accessibility and prevalence of human cases. The spatial and temporal of tick abundance and the infection of B. burgdorferi sensu lato in ticks were determined in selected areas. Ticks were examined for the presence of B. burgdorferi sensu lato by culturing (719 out of 4,001 I. ricinus), and direct PCR amplification of the 5S-23S intergenic spacer region (1,870 out of 5,535) followed by RFLP analysis, the reverse line blot assay and nucleotide sequencing of PCR amplicons. The most abundant tick genus was Rhipicephalus (53%), followed by Dermacentor (34%), I. ricinus and Hyalomma (7%, each). The Mafra and Grândola sites, where a more intensive study was carried out, were excellent habitats for I. ricinus. However, a clear difference of the prevalence of Borrelia infection and the genetic diversity of circulating spirochetes was observed in these two sites. Genotyping of all I. ricinus isolates revealed 5 B. garinii, 8 B. lusitaniae and 1 B. valaisiana strains, which were obtained for the first time in these regions along with a considerable percentage of tick-derived PCR amplicons. Two hard-tick species other than I. ricinus in Grândola were also B. lusitaniae positive, thus seeming to take part in the transmission cycle of Borrelia. The seasonal dynamics of I. ricinus in Mafra was bimodal, more pronounced in nymphs than in adults. The present findings indicate that B. burgdorferi sensu lato agents are differentially maintained in nature in local tick populations in different geographic areas across Portugal and that the risk of acquiring Lyme borreliosis in certain areas of Portugal is higher than previously assumed.

Optimization of DNA Extraction and Molecular Detection of Cryptosporidium Oocysts in Natural Mineral Water Sources

The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65°C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65°C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.