iMARS--mutation analysis reporting software: an analysis of spontaneous cII mutation spectra

The sensitivity of any mutational assay is determined by the level at which spontaneous mutations occur in the corresponding untreated controls. Establishing the type and frequency at which mutations occur naturally within a test system is essential if one is to draw scientifically sound conclusions regarding chemically induced mutations. Currently, mutation-spectra analysis is laborious and time-consuming. Thus, we have developed iMARS, a comprehensive mutation-spectrum analysis package that utilises routinely used methodologies and visualisation tools. To demonstrate the use and capabilities of iMARS, we have analysed the distribution, types and sequence context of spontaneous base substitutions derived from the cII gene mutation assay in transgenic animals. Analysis of spontaneous mutation spectra revealed variation both within and between the transgenic rodent test systems Big Blue Mouse, MutaMouse and Big Blue Rat. The most common spontaneous base substitutions were G:C-->A:T transitions and G:C-->T:A transversions. All Big Blue Mouse spectra were significantly different from each other by distribution and nearly all by mutation type, whereas the converse was true for the other test systems. Twenty-eight mutation hotspots were observed across all spectra generally occurring in CG, GA/TC, GG and GC dinucleotides. A mutation hotspot at nucleotide 212 occurred at a higher frequency in MutaMouse and Big Blue Rat. In addition, CG dinucleotides were the most mutable in all spectra except two Big Blue Mouse spectra. Thus, spontaneous base-substitution spectra showed more variation in distribution, type and sequence context in Big Blue Mouse relative to spectra derived from MutaMouse and Big Blue Rat. The results of our analysis provide a baseline reference for mutation studies utilising the cII gene in transgenic rodent models. The potential differences in spontaneous base-substitution spectra should be considered when making comparisons between these test systems. The ease at which iMARS has allowed us to carry out an exhaustive investigation to assess mutation distribution, mutation type, strand bias, target sequences and motifs, as well as predict mutation hotspots provides us with a valuable tool in helping to distinguish true chemically induced hotspots from background mutations and gives a true reflection of mutation frequency.     

Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum: characterization of two di- and tripeptide transporters (HcPTR2A and B)

Constraints on plant growth imposed by low availability of nitrogen are a characteristic feature of ecosystems dominated by ectomycorrhizal plants. Ectomycorrhizal fungi play a key role in the N nutrition of plants, allowing their host plants to access decomposition products of dead plant and animal materials. Ectomycorrhizal plants are thus able to compensate for the low availability of inorganic N in forest ecosystems. The capacity to take up peptides, as well as the transport mechanisms involved, were analysed in the ectomycorrhizal fungus Hebeloma cylindrosporum. The present study demonstrated that H. cylindrosporum mycelium was able to take up di- and tripeptides and use them as sole N source. Two peptide transporters (HcPTR2A and B) were isolated by yeast functional complementation using an H. cylindrosporum cDNA library, and were shown to mediate dipeptide uptake. Uptake capacities and expression regulation of both genes were analysed, indicating that HcPTR2A was involved in the high-efficiency peptide uptake under conditions of limited N availability, whereas HcPTR2B was expressed constitutively.     

Volatiles modulate the development of plant pathogenic rust fungi

Rust fungi are obligate biotrophic pathogens that differentiate a series of specialized cells to establish infection. One of these cells, the haustorium, which serves to absorb nutrients from living host cells, normally develops only in planta. Here, we show that the rust fungus Uromyces fabae (Pers.) Schroet. stimulates volatile emission of its host, broad bean (Vicia faba L.). Volatiles were identified and shown to be perceived by the fungus in in vitro assays that excluded the host. Three of them, nonanal, decanal, and hexenyl acetate promoted the development of haustoria on artificial membranes. In contrast, the terpenoid farnesyl acetate suppressed this differentiation. In assays using whole plants, farnesyl acetate reduced rust disease not only on broad bean but also on several cereals and legumes including soybean. This natural substance was effective against all rusts tested when directly applied to the host. This demonstrated that farnesyl acetate may serve as a powerful novel tool to combat rust fungi including Phakopsora pachyrhizi that currently threatens the production of soybeans world-wide.     

Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development

In multicellular organisms, organogenesis requires tight control and coordination of cell proliferation, cell expansion, and cell differentiation. We have identified Arabidopsis (Arabidopsis thaliana) nucleosome assembly protein 1 (AtNAP1;1) as a component of a regulatory mechanism that connects cell proliferation to cell growth and expansion during Arabidopsis leaf development. Molecular, biochemical, and kinetic studies of AtNAP1;1 gain- or loss-of-function mutants indicate that AtNAP1;1 promotes cell proliferation or cell expansion in a developmental context and as a function of the farnesylation status of the protein. AtNAP1;1 was farnesylated and localized to the nucleus during the cell proliferation phase of leaf development when it promotes cell division. Later in leaf development, nonfarnesylated AtNAP1;1 accumulates in the cytoplasm when it promotes cell expansion. Ectopic expression of nonfarnesylated AtNAP1;1, which localized to the cytoplasm, disrupts this developmental program by promoting unscheduled cell expansion during the proliferation phase.     

Virus-bacterium interactions in water and sediment of West African inland aquatic systems

The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virus-bacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water.     

The Bifidobacterium longum NCIMB 702259T ctr gene codes for a novel cholate transporter

Preexposure of Bifidobacterium longum NCIMB 702259T to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The B. longum ctr gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant Escherichia coli KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [14C]cholate.     

The Tra domain of the lactococcal CluA surface protein is a unique domain that contributes to sex factor DNA transfer

CluA is a cell surface-presented protein that causes cell aggregation and is essential for a high-efficiency conjugation process in Lactococcus lactis. We know from previous work that in addition to promoting cell-to-cell contact, CluA is involved in sex factor DNA transfer. To define the CluA domains involved in aggregation and in transfer, we first performed random mutagenesis of the cluA gene using a modified mini-Tn7 element which generated five amino acid insertions located throughout the encoded protein. Thirty independent cluA insertion mutants expressing modified CluA proteins at the cell surface were isolated and characterized further. The level of aggregation of each mutant was determined. The cell binding capacity of CluA was affected strongly when the protein had a mutation in its N-terminal region, which defined an aggregation domain extending from amino acid 153 to amino acid 483. Of the cluA mutants that still exhibited aggregation, eight showed an attenuated ability to conjugate, and six mutations were located in a 300-amino-acid C-terminal region of the protein defining a transfer domain (Tra). This result was confirmed by a phenotypic analysis of an additional five mutants obtained using site-directed mutagenesis in which charged amino acids of the Tra domain were replaced by alanine residues. Two distinct functional domains of the CluA protein were defined in this work; the first domain is involved in cell binding specificity, and the Tra domain is probably involved in the formation of the DNA transport machinery. This is the first report of a protein involved in conjugation that actively contributes to DNA transfer and mediates contact between donor and recipient strains.