Timing the Eastern Asian–Eastern North American Floristic Disjunction: Molecular Clock Corroborates Paleontological Estimates

Sequence data of the chloroplast gene rbcL were used to estimate the time of the well-known eastern Asian–eastern North American floristic disjunction. Sequence divergence of rbcL was examined for 22 species of 11 genera (Campsis, Caulophyllum, Cornus, Decumaria, Liriodendron, Menispermum, Mitchella, Pachysandra, Penthorum, Podophyllum, and Phryma) representing a diverse array of flowering plants occurring disjunctly in eastern Asia and eastern North America. Divergence times of putative disjunct species pairs were estimated from synonymous substitutions, using rbcL molecular clocks calibrated for Cornus. Relative rate tests were performed to assess rate constancy of rbcL evolution among lineages. Corrections of estimates of divergence times for each species pair were made based on rate differences of rbcL between Cornus and other species pairs. Results of these analyses indicate that the time of divergence of species pairs examined ranges from 12.56 ± 4.30 million years to recent (<0.31 million years), with most within the last 10 million years (in the late Miocene and Pliocene). These results suggest that the isolation of most morphologically similar disjunct species in eastern Asia and eastern North America occurred during the global climatic cooling period that took place throughout the late Tertiary and Quaternary. This estimate is closely correlated with paleontological evidence and in agreement with the hypothesis that considers the eastern Asian–eastern North American floristic disjunction to be the result of the range restriction of a once more or less continuously distributed mixed mesophytic forest of the Northern Hemisphere that occurred during the late Tertiary and Quaternary. This implies that in most taxa the disjunction may have resulted from vicariance events. However, long-distance dispersal may explain the disjunct distribution of taxa with low divergence, such as Menispermum.     

Phylogenetic relationships among early-diverging eudicots based on four genes: were the eudicots ancestrally woody?

Based on analyses of combined data sets of three genes (18S rDNA, rbcL, and atpB), phylogenetic relationships among the early-diverging eudicot lineages (Ranunculales, Proteales, Trochodendraceae, Sabiaceae, and Buxaceae) remain unclear, as are relationships within Ranunculales, especially the placement of Eupteleaceae. To clarify relationships among these early-diverging eudicot lineages, we added entire sequences of 26S rDNA to the existing three-gene data set. In the combined analyses of four genes based on parsimony, ML, and Bayesian analysis, Ranunculales are strongly supported as a clade and are sister to other eudicots. Proteales appear as sister to the remaining eudicots, which are weakly (59%) supported as a clade. Relationships among Trochodendraceae, Buxaceae (including Didymeles), Sabiaceae, and Proteales remain unclear. Within Ranunculales, Eupteleaceae are sister to all other Ranunculales, with bootstrap support of 70% in parsimony analysis and with posterior probability of 1.00 in Bayesian analysis. Our character reconstructions indicate that the woody habit is ancestral, not only for the basal angiosperms, but also for the eudicots. Furthermore, Ranunculales may not be ancestrally herbaceous, as long maintained. The woody habit appears to have been ancestral for several major clades of eudicots, including Caryophyllales, and asterids.     

Natural strategies for the spatial optimization of metabolism in synthetic biology

Metabolism is a highly interconnected web of chemical reactions that power life. Though the stoichiometry of metabolism is well understood, the multidimensional aspects of metabolic regulation in time and space remain difficult to define, model and engineer. Complex metabolic conversions can be performed by multiple species working cooperatively and exchanging metabolites via structured networks of organisms and resources. Within cells, metabolism is spatially regulated via sequestration in subcellular compartments and through the assembly of multienzyme complexes. Metabolic engineering and synthetic biology have had success in engineering metabolism in the first and second dimensions, designing linear metabolic pathways and channeling metabolic flux. More recently, engineering of the third dimension has improved output of engineered pathways through isolation and organization of multicell and multienzyme complexes. This review highlights natural and synthetic examples of three-dimensional metabolism both inter- and intracellularly, offering tools and perspectives for biological design.     

Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase

DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1-171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1-171 of the E. coli DnaB helicase with significant affinity.     

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpo B gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpo B-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpo B-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus , and Shewanella marinus . In contrast to 16S rRNA gene-TTGE, rpo B-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpo B leads to a single band in TTGE, the rpo B gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.     

Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.     

Alterations in cell membrane properties caused by perfluorinated compounds

The recent detection of perfluorinated compounds (PFCs) in wildlife from even remote locations has spurred interest in the environmental occurrence and effects of these chemicals. While the global distribution of PFCs is increasingly understood, there is still little information available on their effects on wildlife. The amphiphillic nature of PFCs suggests that their effects could be primarily on cell membranes. In this study we measured the effects of PFCs on membrane fluidity and mitochondrial membrane potential using flow cytometry and effects on membrane permeability using cell bioassay procedures (H4IIE, MCF-7, PLHC-1). Of the PFCs tested, only perfluorooctane sulfonic acid (PFOS) increased the permeability of cell membranes to the hydrophobic ligands used. Three PFCs were tested in the membrane fluidity assay: PFOS, perfluorohexane sulfonic acid (PFHS), and perfluorobutane sulfonic acid (PFBS). PFOS increased membrane fluidity in fish leukocytes in a dose-dependent fashion, while PFHS and PFBS had no effect in the concentration range tested. The lowest effective concentrations for the membrane fluidity effects of PFOS were 5-15 mg/l. Effects on mitochondrial membrane potential occurred in the same concentration range as effects on membrane fluidity. This suggests that PFOS effects membrane properties at concentrations below those associated with other adverse effects.     

Global approach to reducing lead exposure and poisoning

Lead poisoning is an important environmental disease that can have life-long adverse health effects. Most susceptible are children, and most commonly exposed are those who are poor and live in developing countries. Studies of children's blood-lead levels (BLLs) are showing cognitive impairment at increasingly lower BLLs. Lead is dangerous at all levels in children. The sources of lead exposure vary among and within countries depending on past and current uses. Sources of lead may be from historic contamination, recycling old lead products, or from manufacturing new products. In all countries that have banned leaded gasoline, average population BLLs have declined rapidly. In many developing countries where leaded gasoline is no longer used, many children and workers are exposed to fugitive emissions and mining wastes. Unexpected lead threats, such as improper disposal of electronics and children's toys contaminated with lead, continue to emerge. The only medical treatment available is chelation, which can save lives of persons with very high BLLs. However, chelating drugs are not always available in developing countries and have limited value in reducing the sequelae of chronic low dose lead exposure. Therefore, the best approach is to prevent exposure to lead. Because a key strategy for preventing lead poisoning is to identify and control or eliminate lead sources, this article highlights several major sources of lead poisoning worldwide. In addition, we recommend three primary prevention strategies for lead poisoning: identify sources, eliminate or control sources, and monitor environmental exposures and hazards.     

N-linked glycosylation of the liver cancer biomarker GP73

The association between elevated circulating levels of GP73 (and fucosylated GP73 in particular) and hepatocellular carcinoma suggests that a thorough analysis of the extent of GP73 glycosylation is warranted. Detailed analysis of the glycosylation patterns of such low abundance proteins are hampered by technical difficulties. Using conventional lectin affinity chromatography, we have established that three quarters of the GP73 secreted from a cell line derived from HCC is fucosylated. Using mass spectrometry, we have established that at least two of three potential sites of N-linked glycosylation are occupied on most molecules of GP73 secreted from cultured hepatoma cells. Furthermore, the oligosaccharides added to recombinant GP73 resemble those present in the bulk of secreted protein, mostly bi-antennary with core fucose, with a smaller fraction of tri- and tetra-antennary structures. The frequency of fucosylation observed on the recombinant protein agrees well with the pattern of lectin binding of the endogenous secreted protein. Finally, we have developed a method to interrogate the glycans added to either the near full length protein or at a particular sequon, providing proof of concept that a small peptide embedded in a heterologous context can preserve both fucosylation and a high level of branching of oligosaccharides added.     

Integrating Remote Sensing and Ground Methods to Estimate Evapotranspiration

Evapotranspiraton (ET) is the second largest term in the terrestrial water budget after precipitation, and ET is expected to increase with global warming. ET studies are relevant to the plant sciences because over 80% of terrestrial ET is due to transpiration by plants. Remote sensing is the only feasible means for projecting ET over large landscape units. In the past decade or so, new ground and remote sensing tools have dramatically increased our ability to measure ET at the plot scale and to scale it over larger regions. Moisture flux towers and micrometeorological stations have been deployed in numerous natural and agricultural biomes and provide continuous measurements of actual ET or potential ET with an accuracy or uncertainty of 10–30%. These measurements can be scaled to larger landscape units using remotely-sensed vegetation indices (VIs), Land Surface Temperature (LST), and other satellite data. Two types of methods have been developed. Empirical methods use time-series VIs and micrometeorological data to project ET measured on the ground to larger landscape units. Physically-based methods use remote sensing data to determine the components of the surface energy balance, including latent heat flux, which determines ET. Errors in predicting ET by both types of methods are within the error bounds of the flux towers by which they are calibrated or validated. However, the error bounds need to be reduced to 10% or less for applications that require precise wide-area ET estimates. The high fidelity between ET and VIs over agricultural fields and natural ecosystems where precise ground estimates of ET are available suggests that this might be an achievable goal if ground methods for measuring ET continue to improve.