Project description:One of the key innovations of the flowering plants is their female reproductive organ, the carpel. Here we show that a mechanism controlling carpel margin development in the model flowering plant Arabidopsis thaliana was recruited from light-regulated processes. This recruitment followed the loss from the basic Helix-Loop-Helix transcription factor SPATULA (SPT) of a domain previously responsible for its negative regulation by phytochrome. We propose that the loss of this domain was a prerequisite for the light-independent expression in female reproductive tissues of a genetic module that also promotes shade avoidance responses in vegetative organs. Striking evidence for this proposition is provided by the restoration of wild type carpel development to spt mutants by low red/far-red light ratios, simulating vegetation shade, which we show to occur via PHYB, PIF4 and PIF5. Our data illustrate the potential of modular evolutionary events to generate rapid morphological change, and thereby provide a molecular basis for neo-Darwinian theories that describe this non-gradualist phenomenon. Furthermore, the effects shown here of light quality perception on carpel development lead us to speculate on the potential role of light-regulated mechanisms in plant organs that, like the carpel, form within the shade of surrounding tissues. The SPATULA (SPT) coding sequence was fused to the 5M-bM-^@M-^Y-extremity of a sequence encoding the 75 C-terminal residues of the viral VP16 transcriptional activator and inserted into the pG0229-35S:GR plant transformation vector between the CaMV-35S promoter and sequences encoding the rat glucocorticoid receptor (GR), so as to conserve the entire reading frame. The resulting plasmid was transferred to Agrobacterium tumefaciens C58/pMP90 cells and used to transform A. thaliana Col-0 plants by standard methods. A homozygous, single-copy transformant was identified, from which two populations of ten T3 descendents were grown and treated by dipping of inflorescences for 2 min in cycloheximide (CHX; 10 M-BM-5g/ml) containing Silwet L-77 surfactant (0.01% v/v). This treatment reduced translation to approximately 5% of its native level in inflorescence tissues, as measured by the in vivo incorporation of [35S]-methionine into proteins. One hour later, inflorescence tissues of the two plant populations were dipped for 2 min in CHX solution, as described above, with and without dexamethasone (DEX; 10 M-BM-5M). After a further 2h, treated inflorescences, excluding open flowers, were harvested and pooled prior to RNA extraction for global gene expression analyses. Microarray analyses were performed using CATMA microarrays each containing 31776 gene-specific tags corresponding to 22089 A. thaliana genes. Three biological replicates were performed, based on three entirely separate induction experiments involving T3 plants from the same T2 parent. A transformed line containing a construction in which SPT had been replaced by an initiation codon and nuclear localization signal (NLS) was used to verify that the targets identified did not respond to the nuclear translocation of VP16-GR protein alone (35S:NLS-VP16-GR plants). In this NLS control experiment, two biological replicates were performed. One technical replicate with fluorochrome reversal was performed for each biological replicate. The labeling of cRNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products), the hybridization of these cRNAs to microarrays, and the subsequent scanning of microarrays, was performed as described by Lurin et al. (2004) Plant Cell 16, 2089-2103.

Project description:The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the M-bM-^@M-^Xdeath effectorM-bM-^@M-^Y. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm. Streptococcus mutans UA159 were grown with 2 uM CSP or without (uninduced control) to mid-log phase. Total RNA was extracted as described above. The cDNAs were prepared for hybridization using the PFGRC protocol. Microarray chips were scanned using a Gene Pix 4000B (Axon) and analyzed using the TM4 Microarray Software Suite (http://www.tm4.org/). Transcript levels were measured by cDNA hybridized to a fourfold redundant S. mutans microarray and averaged for three replicated hybridizations. Differential gene expression was based on a post-normalization cut-off of M-BM-1> twofold.

Project description:Otitis media, pneumonia, sinusitis and as well as severe diseases such as meningitis and bacteraemia are related to biofilm-like diseases, in which Streptococcus pneumoniae demonstrated differential and tissue specific gene expressions. In this study, we reported the differential gene expression profile of early in vitro biofilm and planktonic cell in c-DNA microarray analysis. The microarray analysis was performed on total RNA extracted from biofilms grown in 24-well microtiter plate and mid-log grown planktonic cells. To validate the results of microarray, real-time RT-PCR was performed on 13 differentially expressed genes and one constitutively expressed gene from six different functional groups. cDNA-microarray analyses indicated 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Among total differentially expressed gene, almost 50% were hypothetical genes. Of the 46 protein coding genes, 34 showed up-regulation and 16 showed down-regulation in biofilm. The functional annotation showed that many functional categories were differentially regulated in biofilm and planktonic cells, such as genes involve in purine, pyrimidine nucleotide metabolism, RNA/DNA metabolism, amino acid transport and metabolism, translation, transporter protein, carbohydrate transport and metabolism, cell wall biosynthesis, isoprenoid biosynthesis, transcription regulator and cellular process. Streptococcus pneumoniae R6 strain used in this is an unencapsulated and avirulent strain derived from encapsulated serotype 2 pathogenic strain D39. In vitro biofilm formation was carried out in 24-well, flat-bottom, polystyrene microtiter plate (BD falcon, MD, USA) in static model. S. pneumoniae grown up to mid-logarithmic phase in TSB medium was diluted 1:100 with fresh sterile TSB medium supplied with 1% glucose, inoculated 1.5 mL in 24-well microtiter plate and, incubated for 15 hours at 37M-BM-0C in 5% CO2. After incubation medium was discarded, and the plates were gently washed three times with 1.5 mL sterile, cold phosphate buffer saline (PBS). Adherent cell were scraped and immediately processed for RNA extraction. For planktonic cells RNA extraction, five ml of mid-logarithmic phase cell suspension was pelleted by centrifugation and wash three times with sterile PBS and immediately processed for RNA extraction. All experiments were performed in triplicate (3 independent biological replicates)

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Vibrio cholerae N16961 cpxA*

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