Isolation of endophytic bacteria from arboreal species of the Amazon and identification by sequencing of the 16S rRNA encoding gene

Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla), were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species.     

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.     

Bacterial characterization of Beijing drinking water by flow cytometry and MiSeq sequencing of the 16S rRNA gene

Flow cytometry (FCM) and 16S rRNA gene sequencing data are commonly used to monitor and characterize microbial differences in drinking water distribution systems. In this study, to assess microbial differences in drinking water distribution systems, 12 water samples from different sources water (groundwater, GW; surface water, SW) were analyzed by FCM, heterotrophic plate count (HPC), and 16S rRNA gene sequencing. FCM intact cell concentrations varied from 2.2 × 10³ cells/mL to 1.6 × 10⁴ cells/mL in the network. Characteristics of each water sample were also observed by FCM fluorescence fingerprint analysis. 16S rRNA gene sequencing showed that Proteobacteria (76.9–42.3%) or Cyanobacteria (42.0–3.1%) was most abundant among samples. Proteobacteria were abundant in samples containing chlorine, indicating resistance to disinfection. Interestingly, Mycobacterium, Corynebacterium, and Pseudomonas, were detected in drinking water distribution systems. There was no evidence that these microorganisms represented a health concern through water consumption by the general population. However, they provided a health risk for special crowd, such as the elderly or infants, patients with burns and immune‐compromised people exposed by drinking. The combined use of FCM to detect total bacteria concentrations and sequencing to determine the relative abundance of pathogenic bacteria resulted in the quantitative evaluation of drinking water distribution systems. Knowledge regarding the concentration of opportunistic pathogenic bacteria will be particularly useful for epidemiological studies.     

Phylogenetic Analysis of Pasteuria penetrans by 16S rRNA Gene Cloning and Sequencing

Pasteuria penetrans is an endospore-forming bacterial parasite of Meloidogyne spp. This organism is among the most promising agents for the biological control of root-knot nematodes. In order to establish the phylogenetic position of this species relative to other endospore-forming bacteria, the 16S ribosomal genes from two isolates of P. penetrans, P-20, which preferentially infects M. arenaria race 1, and P-100, which preferentially infects M. incognita and M. javanica, were PCR-amplified from a purified endospore extraction. Universal primers for the 16S rRNA gene were used to amplify DNA which was cloned, and a nucleotide sequence was obtained for 92% of the gene (1,390 base pairs) encoding the 16S rDNA from each isolate. Comparison of both isolates showed identical sequences that were compared to 16S rDNA sequences of 30 other endospore-forming bacteria obtained from GenBank. Parsimony analyses indicated that P. penetrans is a species within a clade that includes Alicyclobacillus acidocaldarius, A. cycloheptanicus, Sulfobacillus sp., Bacillus tusciae, B. schlegelii, and P. ramosa. Its closest neighbor is P. ramosa, a parasite of Daphnia spp. (water fleas). This study provided a genomic basis for the relationship of species assigned to the genus Pasteuria, and for comparison of species that are parasites of different phytopathogenic nematodes.     

Searching for gene flow from cultivated to wild strawberries in Central Europe

Background and Aims

Experimental crosses between the diploid woodland strawberry (Fragaria vesca L.) and the octoploid garden strawberry (F. × ananassa Duch.) can lead to the formation of viable hybrids. However, the extent of such hybrid formation under natural conditions is unknown, but is of fundamental interest and importance in the light of the potential future cultivation of transgenic strawberries. A hybrid survey was therefore conducted in the surroundings of ten farms in Switzerland and southern Germany, where strawberries have been cultivated for at least 10 years and where wild strawberries occur in the close vicinity.

Methods

In 2007 and 2008, 370 wild F. vesca plants were sampled at natural populations around farms and analysed with microsatellite markers. In 2010, natural populations were revisited and morphological traits of 3050 F. vesca plants were inspected. DNA contents of cell nuclei of morphologically deviating plants were estimated by flow cytometry to identify hybrids. As controls, 50 hybrid plants from interspecific hand-crosses were analysed using microsatellite analysis and DNA contents of cell nuclei were estimated by flow cytometry.

Key Results

None of the wild samples collected in 2007 and 2008 contained F. × ananassa microsatellite markers, while all hybrids from hand-crosses clearly contained markers of both parent species. Morphological inspection of wild populations carried out in 2010 and subsequent flow cytometry of ten morphologically deviating plants revealed no hybrids.

Conclusions

Hybrid formation or hybrid establishment in natural populations in the survey area is at best a rare event.     

Remarkable coexistence of multiple cytotypes of the Gymnadenia conopsea aggregate (the fragrant orchid): evidence from flow cytometry

Background and Aims

One of the prerequisites for polyploid research in natural systems is knowledge of the geographical distribution of cytotypes. Here inter- and intrapopulational ploidy diversity was examined in the Gymnadenia conopsea aggregate in central Europe and potential explanations and evolutionary consequences of the observed spatial patterns investigated.

Methods

DAPI flow cytometry supplemented by confirmatory chromosome counts was used to determine ploidy in 3581 samples of the G. conopsea aggregate from 43 populations. The fine-scale spatial pattern of cytotype distribution (intra- and interploidy associations) was analysed with univariate and bivariate K-functions.

Key Results

Gymnadenia tissues undergo a progressively partial endoreplication, which accounts for about 60 % and 75 % of the total genome in G. conopsea and G. densiflora, respectively. Flow cytometric profiles are therefore species-specific and can be used as a marker for rapid and reliable species recognition. Two majority (4x, 8x) and three minority (6x, 10x, 12x) cytotypes were found, often in mixed-ploidy populations (harbouring up to all five different ploidy levels). The scarcity of the minority cytotypes (about 2·7 %) suggests the existence of strong pre- or postzygotic mating barriers. Spatial structure was observed in plots of populations with the highest cytotype variation, including clumping of individuals of the same ploidy and negative association between tetra- and octoploids.

Conclusions

The remarkable ploidy coexistence in the G. conopsea aggregate has reshaped our perception of intrapopulational ploidy diversity under natural conditions. This system offers unique opportunities for studying processes governing the formation and establishment of polyploids and assessing the evolutionary significance of the various pre- and postzygotic mating barriers that maintain this ploidy mixture.     

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function.Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 Å lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.     

Evolution of cryptic gene pools in Hypericum perforatum: the influence of reproductive system and gene flow

Background and Aims Hypericum perforatum

(St. John''s wort) is a widespread Eurasian perennial plant species with remarkable variation in its morphology, ploidy and breeding system, which ranges from sex to apomixis. Here, hypotheses on the evolutionary origin of St. John''s wort are tested and contrasted with the subsequent history of interspecific gene flow.

Methods

Extensive field collections were analysed for quantitative morphological variation, ploidy, chromosome numbers and genetic diversity using nuclear (amplified fragment length polymorphism) and plastid (trnL-trnF) markers. The mode of reproduction was analysed by FCSS (flow cytometric seed screen).

Key Results

It is demonstrated that H. perforatum is not of hybrid origin, and for the first time wild diploid populations are documented. Pseudogamous facultative apomictic reproduction is prevalent in the polyploids, whereas diploids are predominantly sexual, a phenomenon which also characterizes its sister species H. maculatum. Both molecular markers characterize identical major gene pools, distinguishing H. perforatum from H. maculatum and two genetic groups in H. perforatum. All three gene pools are in close geographical contact. Extensive gene flow and hybridization throughout Europe within and between gene pools and species is exemplified by the molecular data and confirmed by morphometric analyses.

Conclusions Hypericum perforatum

is of a single evolutionary origin and later split into two major gene pools. Subsequently, independent and recurrent polyploidization occurred in all lineages and was accompanied by substantial gene flow within and between H. perforatum and H. maculatum. These processes are highly influenced by the reproductive system in both species, with a switch to predominantly apomictic reproduction in polyploids, irrespective of their origin.     

Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA

DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae.     

Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p

In eukaryotes, nuclear export of the large (60S) ribosomal subunit requires the adapter protein Nmd3p to provide the nuclear export signal. Here, we show that in yeast release of Nmd3p from 60S subunits in the cytoplasm requires the ribosomal protein Rpl10p and the G-protein, Lsg1p. Mutations in LSG1 or RPL10 blocked Nmd3-GFP shuttling into the nucleus and export of pre-60S subunits from the nucleus. Overexpression of NMD3 alleviated the export defect, indicating that the block in 60S export in lsg1 and rpl10 mutants results indirectly from failing to recycle Nmd3p. The defect in Nmd3p recycling and the block in 60S export in both lsg1 and rpl10 mutants was also suppressed by mutant Nmd3 proteins that showed reduced binding to 60S subunits in vitro. We propose that the correct loading of Rpl10p into 60S subunits is required for the release of Nmd3p from subunits by Lsg1p. These results suggest a coupling between recycling the 60S export adapter and activation of 60S subunits for translation.     

Study on the age-dependent tissue expression of FUT1 gene in porcine and its relationship to E. coli F18 receptor

Escherichia coli (E. coli) that produces adhesin F18 is the main pathogen responsible for porcine post-weaning diarrhea and edema disease. The receptor for E. coli F18 has not been described in pigs, however the alpha (1,2)-fucosyltransferase (FUT1) gene on chromosome 6 has been proposed as a candidate. The objective of this study, therefore, was to investigate the relationship between FUT1 gene expression and E. coli F18 receptor in Sutai pigs of different ages (8-, 18-, 30- and 35-day-old). FUT1 gene expression was detected in 11 pig tissues with the highest level in lung, and expressed consistently at the four time points. In most tissues, FUT1 gene expression levels decreased from days 8 to 18, then continually increased on days 30 and 35, with expression around weaning time higher than that on day 8. Gene ontology and pathway analysis showed that FUT1 was involved in 32 biological processes, mainly those integral to the membrane, or involved in glycosylation, as well as regulation of binding, interestingly participating in three pathways related to glycosphingolipid biosynthesis. From this analysis and the high linkage disequilibrium between the FUT1 gene and the E. coli F18 receptor locus, we can speculate that higher expression of the FUT1 gene in small intestine is beneficial to the formation of receptors to the E. coli F18 strain and is related to the sensitivity to the pathogen.     

Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry

Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.     

Multiple colonizations from Madagascar and converged acquisition of dioecy in the Mascarene Dombeyoideae (Malvaceae) as inferred from chloroplast and nuclear DNA sequence analyses

Background and Aims

In the Mascarenes, a young oceanic archipelago composed of three main islands, the Dombeyoideae (Malvaceae) have diversified extensively with a high endemism rate. With the exception of the genus Trochetia, Mascarene Dombeyoideae are described as dioecious whereas Malagasy and African species are considered to be monocline, species with individuals bearing hermaphrodite/perfect flowers. In this study, the phylogenetic relationships were reconstructed to clarify the taxonomy, understand the phylogeographic pattern of relationships and infer the evolution of the breeding systems for the Mascarenes Dombeyoideae.

Methods

Parsimony and Bayesian analysis of four DNA markers (ITS, rpl16 intron and two intergenic spacers trnQ-rsp16 and psbM-trnD) was used. The molecular matrix comprised 2985 characters and 48 taxa. The Bayesian phylogeny was used to infer phylogeographical hypotheses and the evolution of breeding systems.

Key Results

Parsimony and Bayesian trees produced similar results. The Dombeyoideae from the Mascarenes are polyphyletic and distributed among four clades. Species of Dombeya, Trochetia and Ruizia are nested in the same clade, which implies the paraphyly of Dombeya. Additionally, it is shown that each of the four clades has an independent Malagasy origin. Two adaptive radiation events have occurred within two endemic lineages of the Mascarenes. The polyphyly of the Mascarene Dombeyoideae suggests at least three independent acquisitions of dioecy.

Conclusions

This molecular phylogeny highlights the taxonomic issues within the Dombeyoideae. Indeed, the limits and distinctions of the genera Dombeya, Trochetia and Ruizia should be reconsidered. The close phylogeographic relationships between the flora of the Mascarenes and Madagascar are confirmed. Despite their independent origins and a distinct evolutionary history, each endemic clade has developed a different breeding systems (dioecy) compared with the Malagasy Dombeyoideae. Sex separation appears as an evolutionary convergence and may be the consequence of selective pressures particular to insular environments.     

Molecular evolution of pteridophytes and their relationship to seed plants: Evidence from complete 18S rRNA gene sequences

Complete 18S ribosomal RNA sequence data from representatives of all extant pteridophyte lineages together with RNA sequences from different seed plants were used to infer a molecular phylogeny of vascular plants that included all major land plant lineages. The molecular data indicate that lycopsids are monophyletic and are the earliest diverging group within the vascular land plants, whereasPsilotum nudum is more closely related to the seed plants than to other pteridophyte lineages. The phylogenetic trees based on maximum likelihood, parsimony and distance analyses show substantial agreement with the evolutionary relationships of land plants as interpreted from the fossil record.     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO₂ concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.