Project description:Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly understood that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during interactions with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. The interaction displays two distinct phases: first, there is a coexisting phase in which the alga grows exponentially and the bacterium grows as well. The interaction shifts to pathogenic when the virulence of Sulfitobacter D7 towards E. huxleyi is invoked upon exposure to high concentrations of algal dimethylsulfoniopropionate (DMSP), which occurs when the algae reach stationary growth or when DMSP is applied exogenously to algae in exponential growth. We aimed to unravel the response of Sulfitobacter D7 to the pathogenicity-inducing compound, DMSP, and to different algae-derived infochemicals that affect the lifestyle of the bacterium. We grew Sulfitobacter D7 in conditioned media (CM) derived from algal cultures at the different growth phases, exponential and stationary (Exp-CM and Stat-CM, respectively), in which DMSP concentration is low and high, respectively. This enabled us to separate between different phases of the interaction with E. huxleyi, i.e., Exp-CM representing the coexisting phase, and Stat-CM representing the pathogenic phase. An additional pathogenicity-inducing treatment was Exp-CM supplemented with 100 µM DMSP (herein Exp-CM+DMSP). This condition mimicked co-cultures to which we added DMSP exogenously and thus induced Sulfitobacter D7 pathogenicity, which lead to death of exponentially growing E. huxleyi. In order to identify bacterial genes that are specifically responsive to DMSP, and are not affected by other algae-derived factors, we grew Sulfitobacter D7 in defined minimal medium (MM), lacking algal metabolites, supplemented with 100 µM DMSP (herein MM+DMSP), and examined the transcriptional response. After 24 h of Sulfitobacter D7 growth in all 5 media, triplicates were taken for transcriptomic analysis. Altogether, this experimental design allowed to expand our understanding on the bacterial response to DMSP, algal infochemicals and which of these are essential for coexistence and pathogenicity.
Project description:P. tricornutum (Bacillariophyta, Pennatae, NEPCC640) was obtained from the Algal Center of the Institute of Oceanology of the Chinese Academy of Sciences. The cells were cultured in a modified f/2 medium (Guillard, 1975) at 20 +/- 1C, and illuminated with 120 umol photon m-2 s-1 under a 12:12 light: dark cycle. Flasks were shaken by the researchers twice a day at the fixed times. Experiments were conducted in triplicate in 3L sterilized and acid-washed Erlenmeyer flask containing 2L medium. The equipment used in this study is similar to the ones used in previous ocean acidification research (Fu et al., 2007; Hutchins et al., 2007; Wu et al., 2010). Prior to inoculation, the mediums were treated by different CO2 concentrations. The low CO2 medium was bubbled with ambient air of about 400 ppmv (low CO2, LC) and the high CO2 medium was bubbled with pre-mixed air-CO2 mixtures (1000 ppmv; high CO2, HC) from a plant growth CO2 chamber (HP400G-D, Ruihua Instrument & Equipment Ltd, Wuhan, China) with a variation of less than 5%. Semi-continuous cultures were used to maintain the pH stability during P. tricornutum growth in the present study, All the cultures were diluted to 1x104 cells mL-1 with fresh medium and pre-acclimated to the desired CO2 level every 24 h to maintain an exponential growth phase and minimize pH fluctuations of the cells. Cultures were harvested after 8 months of semi-continuous incubation. Significant differences between the carbonate systems in different cultures.
Project description:To study the effect of myoblast-derived paracrine factors on cardiomyocyte gene expression, microarray analysis was performed from isolated rat fetal cardiomyocyte cultures. These cultures were treated for 24 hours with fresh culture medium (control), skeletal myoblast-conditioned medium, or cardiac fibroblast-conditioned medium.
Project description:Wildtype and SIPK -silenced (RNAi) tobacco cell suspension cultures were grown in MS medium at 25 C in the dark. Cell suspension cultures were transferred to fresh growth medium 3 days prior to pooling each genotype and dividing the pool into 3 x 10ml volumes per genotype. A 30 minute pre-incubation was used with xanthine (for abiotic controls and treatments), but no pre-treatment was used for the biotic stressors. For each treatment and abiotic/biotic controls, the cells were harvested by vacuum filtration at the appropriate time points (controls sampled at 0 hrs) and frozen in N2(l) for RNA extraction. In order to discern the effect of silencing SIPK on the transciptome in defence response, biological replicates of WT and SIPK-Ri were randomly paired on 3 slides per treatment or control group. Series_weblink: http://www.tigr.org/tdb/potato Keywords = potato, Biotic stress Keywords: ordered
Project description:Algal biofuel production requires an input of synthetic nitrogen fertilizer. Fertilizer synthesized via the Haber-Bosch process produces CO2 as a waste by-product and represents a substantial financial and energy investment. Reliance on synthetic fertilizer attenuates the environmental significance and economic viability of algae production systems. To lower fertilizer input, the waste streams of algal production systems can be recycled to provide alternative sources of nitrogen such as amino acids to the algae. The halophytic green alga Dunaliella viridis can use ammonium (NH4+) derived from the abiotic degradation of amino acids, and previously, supplementation of NH4+ from glutamine (GLN) degradation was shown to support acceptable levels of growth and increased neutral lipid production compared to nitrate. To understand the effect of glutamine-released NH4+ on algae growth and physiology, metabolite levels, growth parameters, and transcript profiles of D. viridis cultures were observed in a time course after transition from media containing nitrate as a sole N source to medium containing GLN, glutamate (GLU), or a N-depleted medium. Growth parameters were similar between GLN (NH4+) and nitrate supplemented cultures, however, metabolite data showed that the GLN supplemented cultures (NH4+) more closely resembled cultures under nitrogen starvation (N-depleted and GLU supplementation). Neutral lipid accumulation was the same in nitrate and glutamine-derived NH4+ cultures. However, glutamine-derived NH4+ caused a transcriptional response in the immediate hours after inoculation of the culture. The strong initial response of cultures to NH4+ changed over the course of days to closely resemble that of nitrogen starvation. These observations suggest that release of NH4+ from glutamine was sufficient to maintain growth, but not high enough to trigger a cell transition to a nitrogen replete state. Comparative transcript profiling of the nitrogen-starved and nitrate-supplied cultures show an overall downregulation of fatty acid synthesis and a shift to starch synthesis and accumulation. The results indicate that a continuous, amino acid derived slow release of NH4+ to algae cultures could reduce the amount of synthetic nitrogen needed for growth, but optimization is needed to balance nitrogen starvation and cell division.
Project description:Conidia germination is critical for fungi to colonize various habitats. We sampled RNA expression at four stages of conidia germination, including fresh conidia (15min), polar growth (120min), doubling of long axis (240min), and first hyphal branching (360min) in Neurospora crassa. Cultures were made on two different media, including Bird medium supporting only asexual development and maple sap medium supporting both asexual and sexual development, and two biological replicates were collected for all data points. The growth was under a labratory condition of 25C and constant light.
Project description:Conidia germination is critical for fungi to colonize various habitats. We sampled RNA expression at four stages of conidia germination, including fresh conidia (15min), polar growth (120min), doubling of long axis (240min), and first hyphal branching (360min) in Neurospora crassa. Cultures were made on Bird medium, and two biological replicates were collected for all data points. The growth was under a labratory condition of 375C and constant light.