Carbon catabolite repression in <Emphasis Type="Italic">Thermoanaerobacterium saccharolyticum</Emphasis>

Background

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.

Results

We report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.

Conclusion

Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

The CHD3 remodeler PICKLE associates with genes enriched for trimethylation of histone H3 lysine 27

In Arabidopsis (Arabidopsis thaliana), the ATP-dependent chromatin remodeler PICKLE (PKL) determines expression of genes associated with developmental identity. PKL promotes the epigenetic mark trimethylation of histone H3 lysine 27 (H3K27me3) that facilitates repression of tissue-specific genes in plants. It has previously been proposed that PKL acts indirectly to promote H3K27me3 by promoting expression of the POLYCOMB REPRESSIVE COMPLEX2 complex that generates H3K27me3. We undertook expression and chromatin immunoprecipitation analyses to further characterize the contribution of PKL to gene expression and developmental identity. Our expression data support a critical and specific role for PKL in expression of H3K27me3-enriched loci but do not support a role for PKL in expression of POLYCOMB REPRESSIVE COMPLEX2. Moreover, our chromatin immunoprecipitation data reveal that PKL protein is present at the promoter region of multiple H3K27me3-enriched loci, indicating that PKL directly acts on these loci. In particular, we find that PKL is present at LEAFY COTYLEDON1 and LEAFY COTYLEDON2 during germination, which is when PKL acts to repress these master regulators of embryonic identity. Surprisingly, we also find that PKL is present at the promoters of actively transcribed genes that are ubiquitously expressed such as ACTIN7 and POLYUBIQUITIN10 that do not exhibit PKL-dependent expression. Taken together, our data contravene the previous model of PKL action and instead support a direct role for PKL in determining levels of H3K27me3 at repressed loci. Our data also raise the possibility that PKL facilitates a common chromatin remodeling process that is not restricted to H3K27me3-enriched regions.     

IFN-gamma-producing gamma delta T cells help control murine West Nile virus infection

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, thereby partially mimicking human disease. Using this model, we have demonstrated that mice deficient in gammadelta T cells are more susceptible to WN virus infection. TCRdelta(-/-) mice have elevated viral loads and greater dissemination of the pathogen to the CNS. In wild-type mice, gammadelta T cells expanded significantly during WN virus infection, produced IFN-gamma in ex vivo assays, and enhanced perforin expression by splenic T cells. Adoptive transfer of gammadelta T cells to TCRdelta(-/-) mice reduced the susceptibility of these mice to WN virus, and this effect was primarily due to IFN-gamma-producing gammadelta T cells. These data demonstrate a distinct role for gammadelta T cells in the control of and prevention of mortality from murine WN virus infection.     

Dielectrophoresis of Cells

Dielectrophoresis, the motion produced by the action of nonuniform electric field upon a neutral object, is shown to be a simple and useful technique for the study of cellular organisms. In the present study of yeast (Saccharomyces cerevisiae) using a simple pin-pin electrode system of platinum and high-frequency alternating fields, one observes that the collectability of cells at the electrode tip, i.e. at the region of highest field strength, depends upon physical parameters such as field strength, field uniformity, frequency, cell concentration, suspension conductivity, and time of collection. The yield of cells collected is also observed to depend upon biological factors such as colony age, thermal treatment of the cells, and chemical poisons, but not upon irradiation with ultraviolet light. Several interesting side effect phenomena coincident with nonuniform electric field conditions were observed, including stirring (related to “jet” effects at localized electrode sites), discontinuous repulsions, and cellular rotation which was found to be frequency dependent.     

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane‐rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast‐targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici‐populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora‐specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N‐terminal signal‐dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant‐specific sorting signals to traffic within plant cells.     

T cell responses to the RTS,S/AS01(E) and RTS,S/AS02(D) malaria candidate vaccines administered according to different schedules to Ghanaian children

Background

The Plasmodium falciparum pre-erythrocytic stage candidate vaccine RTS,S is being developed for protection of young children against malaria in sub-Saharan Africa. RTS,S formulated with the liposome based adjuvant AS01_E or the oil-in-water based adjuvant AS02_D induces P. falciparum circumsporozoite (CSP) antigen-specific antibody and T cell responses which have been associated with protection in the experimental malaria challenge model in adults.

Methods

This study was designed to evaluate the safety and immunogenicity induced over a 19 month period by three vaccination schedules (0,1-, 0,1,2- and 0,1,7-month) of RTS,S/AS01_E and RTS,S/AS02_D in children aged 5–17 months in two research centers in Ghana. Control Rabies vaccine using the 0,1,2-month schedule was used in one of two study sites.

Results

Whole blood antigen stimulation followed by intra-cellular cytokine staining showed RTS,S/AS01_E induced CSP specific CD4 T cells producing IL-2, TNF-α, and IFN-γ. Higher T cell responses were induced by a 0,1,7-month immunization schedule as compared with a 0,1- or 0,1,2-month schedule. RTS,S/AS01_E induced higher CD4 T cell responses as compared to RTS,S/AS02_D when given on a 0,1,7-month schedule.

Conclusions

These findings support further Phase III evaluation of RTS,S/AS01_E. The role of immune effectors and immunization schedules on vaccine protection are currently under evaluation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00360230","term_id":"NCT00360230"}}NCT00360230     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.     

Parental contribution and coefficient of coancestry among maize inbreds: pedigree, RFLP, and SSR data

The genetic relationship between inbreds i and j can be estimated from pedigree or from molecular marker data. The objectives of this study were to: (1) determine whether pedigree, restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) data give similar estimates of parental contribution and coefficient of coancestry (f _ij ) among a set of maize (Zea mays L.) inbreds, and (2) compare the usefulness of RFLP and SSR markers for estimating genetic relationship. We studied 13 maize inbreds with known pedigrees. The inbreds were genotyped using 124 RFLP and 195 SSR markers. For each type of marker, parental contributions were estimated from marker similarity among an inbred and both of its parents, and were subsequently used to estimate f _ij . Estimates of parental contribution differed significantly (α<0.05) between pedigree data and either type of marker, but not between the marker systems. The RFLP estimates of parental contribution failed to sum to 1.0, reflecting a higher frequency of non-parental bands with RFLP than with SSR markers. The f _ij estimated from pedigree, RFLP, and SSR data were highly correlated (r=0.87–0.97), although significant differences were found among the three sets of f _ij estimates. We concluded that pedigree and marker data often lead to different estimates of parental contribution and f _ij , and that SSR markers are superior to RFLP markers for estimating genetic relationship. A relevant question is whether or not the inbreds previously genotyped with an older marker system (e.g., RFLP) need to be re-analyzed with a newer marker system (e.g., SSR) for the purpose of estimating genetic relationship. Such re-analysis seems unnecessary if data for the same type of marker are available for a given inbred and both of its parents. Received: 2 June 1999 / Accepted: 30 July 1999     

Pseudomonas fluorescens' view of the periodic table

Growth in a biofilm modulates microbial metal susceptibility, sometimes increasing the ability of microorganisms to withstand toxic metal species by several orders of magnitude. In this study, a high-throughput metal toxicity screen was initiated with the aim of correlating biological toxicity data in planktonic and biofilm cells to the physiochemical properties of metal ions. To this end, Pseudomonas fluorescens ATCC 13525 was grown in the Calgary Biofilm Device (CBD) and biofilms and planktonic cells of this microorganism were exposed to gradient arrays of different metal ions. These arrays included 44 different metals with representative compounds that spanned every group of the periodic table (except for the halogens and noble gases). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) values were obtained after exposing the biofilms to metal ions for 4 h. Using these values, metal ion toxicity was correlated to the following ion-specific physicochemical parameters: standard reduction-oxidation potential, electronegativity, the solubility product of the corresponding metal–sulfide complex, the Pearson softness index, electron density and the covalent index. When the ions were grouped according to outer shell electron structure, we found that heavy metal ions gave the strongest correlations to these parameters and were more toxic on average than the other classes of the ions. Correlations were different for biofilms than for planktonic cells, indicating that chemical mechanisms of metal ion toxicity differ between the two modes of growth. We suggest that biofilms can specifically counter the toxic effects of certain physicochemical parameters, which may contribute to the increased ability of biofilms to withstand metal toxicity.