Phylogeography and the geographic cline in the armament of a seed-predatory weevil: effects of historical events vs. natural selection from the host plant

Japanese camellia (Camellia japonica) and its seed predator, the camellia weevil (Curculio camelliae), provide a notable example of a geographic mosaic of coevolution. In the species interaction, the offensive trait of the weevil (rostrum length) and the defensive trait of the plant (pericarp thickness) are involved in a geographically-structured arms race, and these traits and selective pressures acting on the plant defence vary greatly across a geographical landscape. To further explore the geographical structure of this interspecific interaction, we tested whether the geographical variation in the weevil rostrum over an 800-km range along latitude is attributed to local natural selection or constrained by historical (phylogeographical) events of local populations. Phylogeographical analyses of the mitochondrial DNA sequences of the camellia weevil revealed that this species has experienced differentiation into two regions, with a population bottleneck and subsequent range and/or population expansion within each region. Although these phylogeographical factors have affected the variation in rostrum length, analyses of competing factors for the geographical variation revealed that this pattern is primarily determined by the defensive trait of the host plant rather than by the effects of historical events of populations and a climatic factor (annual mean temperature). Thus, our study suggests the overwhelming strength of coevolutionary selection against the effect of historical events, which may have limited local adaptation.     

ATP-induced shrinkage of DNA with MukB protein and the MukBEF complex of Escherichia coli

Fluorescence microscopic observation of individual T4 DNA molecules revealed that the MukBEF complex (bacterial condensin) and its subunit, the MukB (a member of the SMC [structural maintenance of chromosomes] superfamily) homodimer, of Escherichia coli markedly shrunk large DNA molecules in the presence of hydrolyzable ATP. In contrast, in the presence of ADP or ATP-gammaS, the conformation of DNA was almost not changed. This suggests that the ATPase activity of subunit MukB is essential for shrinking large DNA molecules. Stretching experiments on the shrunken DNA molecules in the presence of ATP and MukBEF indicated a cross-bridging interaction between DNA molecules.     

A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.     

Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

A non-polar patch on the surface of a protein can cause a reduction in the solubility and stability of the protein, and thereby induce aggregation. However, a non-polar patch may be required so that the protein can bind to another molecule. The mutant 6L—derived from the acidic, dimeric α-helical protein sulerythrin and containing six additional leucines arranged to form a non-polar patch on its surface when properly folded—has a substantially reduced solubility in comparison with that of wild-type sulerythrin. This reduced solubility appears to cause 6L to aggregate. To reverse this aggregation, we mutated 6L so that it contained three to six additional glutamates or aspartates that we predicted would surround the non-polar leucine patch on natively folded 6L. Although the introduction of three glutamates or aspartates increased solubility, the mutants still aggregate and have a reduced α-helical content. Conversely, mutants with six additional glutamates or aspartates appear to exist mostly as dimers and to have the same α-helical content as that of wild-type sulerythrin. Notably, the introduction of five lysines or five arginines at the positions held by the glutamates or aspartates did not recover solubility as effectively as did the negatively charged residues. These results demonstrate that negatively charged residues, but not positively charged ones, surrounding a non-polar patch on an acidic protein can completely reverse the decrease in its solubility caused by the patch of non-polar surface residues.     

Adaptive plasmid evolution results in host-range expansion of a broad-host-range plasmid

Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this "long-term host range" can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained in strain P21, strain H2, and alternatingly in P21 and H2. Plasmids that evolved in P21 and in both hosts showed increased stability and decreased cost in ancestral host P21. However, the latter group showed higher variability in stability patterns, suggesting that regular switching between distinct hosts hampered adaptive plasmid evolution. The plasmids evolved in P21 were also equally or more stable in other hosts compared to pB10, which suggested true host-range expansion. The complete genome sequences of four evolved plasmids with improved stability showed only one or two genetic changes. The stability of plasmids evolved in H2 improved only in their coevolved hosts, not in the ancestral host. Thus a BHR plasmid can adapt to an unfavorable host and thereby expand its long-term host range.     

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.     

Survival time and resistance to desiccation of diapause and non-diapause eggs of temperate Aedes (Stegomyia) mosquitoes

Time-related changes in the patterns of host discrimination may have a complex form depending on whether or not hosts are marked following first parasitisation, the persistence of chemicals in these marks, and changes in the host as a result of being parasitised. Here we present an experimental design capable of detecting temporal patterns in host discrimination of solitary parasitoids. This method is tested against experimental data from Aphidius ervi attacking the aphid, Acyrthosiphon kondoi. The results demonstrate that there are statistically significant changes in the patterns of egg distribution in aphid hosts held for intervals between 1 and 48 h between parasitisation. These changes appear to be attributable to increasing oviposition restraint by the superparasitising female as the time interval between attacks is increased. The form of this relationship is not significantly different to a sigmoid function and the rate of change of restraint was greatest between 6–8 h after the first oviposition.     

Host-specific factors determine the persistence of IncP-1 plasmids

Conjugative plasmids play a very important role in bacterial adaptation through the dissemination of useful traits. Incompatibility group P-1 (IncP-1) plasmids exhibit an extreme broad-host-range among Gram-negative bacteria and known to be one of the major agents to disseminate various phenotypic traits such as antibiotic resistance and xenobiotic degradation. Although the plasmids are believed to be very stable in most Gram-negative bacteria, little is known about the factors that affect their stability in various hosts, allowing their persistence in bacterial population. Here we show that the stability of the cryptic IncP-1β plasmid pBP136 differed greatly in four different Escherichia coli K12 host backgrounds (MG1655, DH5α, EC100, and JM109), whereas the closely related plasmid pB10 was stable in all four strains. The supply of the kleF gene, which is involved in the stability of IncP-1 plasmids but absent in pBP136, did not improve the stability of the plasmid. Our findings suggest that persistence of IncP-1 plasmids in the absence of selection is affected by strain-specific factors.     

Molecular phylogeny and historical biogeography of the Holarctic wetland leaf beetle of the genus Plateumaris

Leaf beetles of the genus Plateumaris inhabit wetlands across the temperate zone of the Holarctic region. To explore the phylogeographic relationships among North American, East Asian, and European members of this genus and the origin of the species endemic to Japan, we studied the molecular phylogeny of 20 of the 27 species in this genus using partial sequences of mitochondrial cytochrome oxidase subunit I (COI) and the 16S and nuclear 28S rRNA genes. The molecular phylogeny revealed that three species endemic to Europe are monophyletic and sister to the remaining 11 North American and six Asian species. Within the latter clade, North American and Asian species did not show reciprocal monophyly. Dispersal-vicariance analysis and divergence time estimation revealed that the European and North America-Asian lineages diverged during the Eocene. Moreover, subsequent differentiation occurred repeatedly between North American and Asian species, which was facilitated by three dispersal events from North America to Asia and one in the opposite direction during the late Eocene through the late Miocene. Two Japanese endemics originated from different divergence events; one differentiated from the mainland lineage after differentiation from the North American lineage, whereas the other showed a deep coalescence from the North American lineage with no present-day sister species on the East Asian mainland. This study of extant insects provides molecular phylogenetic evidence for ancient vicariance between Europe and East Asia-North America, and for more recent (but pre-Pleistocene) faunal exchanges between East Asia and North America.