Project description:Small non coding RNA molecules (sncRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sncRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. Enterococcus faecalis. We characterized 11 sncRNAs by tiling microarray analysis, 5’ and 3’ RACE-PCR, and Northern blot analysis. Six sncRNAs were specifically expressed at exponential phase, two sncRNAs were observed at stationary phase, and three were detected during both phases. This is the first experimental genome-wide identification of sncRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen. Identification of sncRNA candidates transcribed by E. faecalis V583 was undertaken with two samples of cells harvested in mid-log growth phase and stationary phase after 24h of incubation at 37°C in M17 glucose media.
Project description:A long-term-survival (LTS) phase in Listeria monocytogenes was recently discovered. Cells in this phase are coccoid in shape, survive for at least 30 d without any decrease in viable cell numbers, and are very resistant to heat and high pressure. However, how cells of L. monocytogenes transition to this long-term-survival phase is little understood. Therefore, a whole-genome expression analysis was conducted to study the transcription profile of L. monocytogenes as it enters the LTS phase. Transcription profiles at log, stationary and death phases were analyzed since differential gene expressions at these phases may contribute to the eventual transition to the LTS phase. Specifically, cells of L. monocytogenes F2365 at log, stationary, death and LTS phases were obtained by incubating cultures in TSBYE at 35°C for 13 h, 17 h, 24 h and 168-336 h, respectively. Also, to study cells transitioning from the LTS phase back to the log phase, 1 ml of the LTS-phase culture at 336 h was reinoculated into 100 ml of fresh TSBYE with incubation at 35°C for 8 h. Total RNAs of all samples were extracted, reverse transcribed into cDNAs and then hybridized to the L. monocytogenes expression microarray (Roche NimbleGen). During the transition from log phase to stationary phase, differential changes in gene expression involved genes associated with cell envelope, cell division, stress response, energy metabolism, protein synthesis and material transport. During the transition from stationary to death phase, differential changes were observed in genes related to cell envelope, detoxification, pathogenesis, energy metabolism, protein synthesis and material transport. When cultures transitioned from death phase to 168-h LTS phase, significant downregulation of genes associated with amino acid and protein biosynthesis, as well as stress responses, were observed (P < 0.05), while multiple genes related to cell envelope, energy production and material transportation were significantly upregulated (P < 0.05). High similarity of transcription profiles (r = 0.93) within LTS phase was observed when comparing transcriptomes at 168 h and 336 h. RNA quality measurement revealed a high level of degradation of ribosomal RNA during the LTS phase. The transcription profile at 8-h (log-phase) after re-inoculation of LTS cells also resembled that at 13 h (r = 0.94). We hypothesize that the upregulation of some compatible solute transporters during the LTS phase may result in accumulation of these solutes, which may lower intracellular water activity and thus enhance resistance of L. monocytogenes to heat and high pressure. Dormancy may be induced at the LTS phase which is suggested by the downregulation of genes associated with transcription and translation. Once fresh nutrients are provided, LTS cells may quickly exit dormancy and become metabolically active as they transition to the log phase. Based on the annotated genome of L. monocytogenes F2365 (GenBank accession# NC_002973) (Donaldson et al., 2009), a gene expression microarray was designed to target 2821 protein-coding genes (including putative protein-coding genes). Each of the 2821 genes was targeted by 12 unique 60-mer oligonucleotide probes. Each unique probe was printed in duplicates on the microarray. The microarrays were in a format of 4 × 72 k (4 microarrays per slide, with each microarray containing 72,000 probes) and provided by Roche NimbleGen (Roche NimbleGen, WI).
Project description:Expression profiling of C. albicans homozygous sfl1 and sfl2 mutants expressing (sfl1-CaEXP-SFL1-HA3, sfl2-CaEXP-SFL2-HA3) or not (sfl1-CaEXP, sfl2-CaEXP, vector only control) SFL1-HA3 and SFL2-HA3, respectively, under hyphae-inducing conditions (Lee's medium at 37 degrees C).
Project description:G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 30 °C in chemostats, as previously described (for more information see Esteve-Núñez, A., M. M. Rothermich, M. Sharma, and D. R. Lovley. 2004. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol.:in press.), with acetate (5 mM) as the electron donor and fumarate (27.5 mM) as the electron acceptor. For growth in the absence of fixed nitrogen, the ammonium chloride (4.7 mM) was omitted from the medium and fumarate served as the electron acceptor. Therefore, cultures with ammonium chloride were limited by acetate while cultures without ammonium chloride were limited by nitrogen. Cultures were maintained at a dilution rate of 0.05 h-1 for 5 culture vessel volumes to ensure that cells were at steady-state prior to harvesting. Cells were harvested by centrifugation at 4 °C and the cell pellet was flash-frozen in liquid nitrogen and then stored at -80 °C prior to RNA extraction. Cells grown in the absence of ammonium had acetylene reduction rates of 0.012 nmol/hr compared to 0.003 nmol/hr in ammonium-grown cells, providing evidence that these cells were fixing nitrogen.. The steady-state concentration of cells in the nitrogen-fixing chemostats (0.178 mg/mL + 0.011; mean + standard deviation, n=3) was significantly lower than chemostats provided with ammonium (0.45 mg/mL + 0.007). Three cultures of acetate limited growth with fumarate as the electron acceptor and three cultures of nitrogen limited growth with fumarate as the electron acceptor were grown. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment.
Project description:G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 23 °C in batch culture. Both control and experimental populations were grown with 10mM acetate as the electron donor while Fe(III) as 55 mM ferric citrate served as the electron acceptor for the control populations and graphite anodes as the electron acceptor for the experimental populations. Controls populations were harvested at a concentration of 30 mM Fe(II). Fuel cell power output was monitored by measuring the voltage across a known resistance in the fuel cell and experimental populations were harvested when current production reached a steady state of 0.6 milliamperes. Cells were harvested at log phase by centrifugation at 4 °C and the cell pellet was flash-frozen in liquid nitrogen and then stored at -80 °C prior to RNA extraction. Three cultures of control and experimental populations were collected. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment. Additional information regarding the fuel cell experimental set up and culture conditions for this experiment can be found in both the current reference and the following reference: Bond DR and Lovley DR. Electricity production by Geobacter sulfurreducens attached to electrodes. 2003. Applied and Environmental Microbiology 69: 1548-1555.
Project description:We investigated how Candida albicans adapts its transcriptional pattern to a sudden availability of glucose in the extracellular medium, having previously grown on a non-fermentable carbon source.
Project description:G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 23 �Â�°C in batch culture. Both control and experimental populations were grown with 10mM acetate as the electron donor while Fe(III) as 55 mM ferric citrate served as the electron acceptor for the control populations and graphite anodes as the electron acceptor for the experimental populations. Controls populations were harvested at a concentration of 30 mM Fe(II). Fuel cell power output was monitored by measuring the voltage across a known resistance in the fuel cell and experimental populations were harvested when current production reached a steady state of 0.6 milliamperes. Cells were harvested at log phase by centrifugation at 4 �Â�°C and the cell pellet was flash-frozen in liquid nitrogen and then stored at -80 �Â�°C prior to RNA extraction. Three cultures of control and experimental populations were collected. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment. Additional information regarding the fuel cell experimental set up and culture conditions for this experiment can be found in both the current reference and the following reference: Bond DR and Lovley DR. Electricity production by Geobacter sulfurreducens attached to electrodes. 2003. Applied and Environmental Microbiology 69: 1548-1555.