Project description:Recent studies have shown that Pelagibacter oxidize a wide range of one carbon (C1) and methylated compounds that are ubiquitous in the oceans. However, the metabolic pathways used to oxidize and assimilate these compounds are complex and have been only partly described. To understand the metabolism of these compounds in Pelagibacter and to identify candidate genes involved in these pathways, we used microarray to study changes in gene expression in response to five different compounds (trimethylamine N-oxide (TMAO), methylamine, dimethylsulfoniopropionate (DMSP), methanol, and glycine betaine (GBT)) in Pelagibacter strain HTCC1062.
Project description:Recent studies have shown that Pelagibacter oxidize a wide range of one carbon (C1) and methylated compounds that are ubiquitous in the oceans. However, the metabolic pathways used to oxidize and assimilate these compounds are complex and have been only partly described. To understand the metabolism of these compounds in Pelagibacter and to identify candidate genes involved in these pathways, we used microarray to study changes in gene expression in response to five different compounds (trimethylamine N-oxide (TMAO), methylamine, dimethylsulfoniopropionate (DMSP), methanol, and glycine betaine (GBT)) in Pelagibacter strain HTCC1062. This project will examine the transcriptional response of the marine microorganism Pelagibacter HTCC1062 to five methylated compounds. To do this, 18 flasks were innoculated with cells - 3 flasks without any methylated compounds and rest of 15 treated with different methylated compounds respectively (TMAO (trtmA), methylamine (trtmB), DMSP (trtmC), methanol (trtmD) and glycine betaine (trtmE)). Cells were all harvested at the same timepoint in the exponential phase.
Project description:Bacteria respond to stimuli in the environment using transcriptional control, but this may not be the case for most marine bacteria having small, streamlined genomes. Candidatus Pelagibacter ubique, a cultivated representative of the SAR11 clade, which is the most abundant clade in the oceans 4, has a small, streamlined genome and possesses an unusually small number of transcriptional regulators. This observation leads to the hypothesis that transcriptional control is low in Pelagibacter and limits its response to environmental conditions. However, the extent of transcriptional control in Pelagibacter is unknown. Here we show that transcriptional control is extremely low in Pelagibacter and another oligotroph (SAR92) compared to two marine copiotrophic bacterial taxa, Polaribacter MED152 and Ruegeria pomeroyi. We found that ~0.1% of protein-encoding genes in Pelagibacter are under transcriptional control compared to >10% of genes in other marine bacteria. Regardless of the growth condition, the same genes were highly expressed while most genes were always expressed at very low levels. Quantitative RNA sequencing revealed that abundances of most Pelagibacter transcripts were <0.01 copies per cell whereas transcript abundances were 1 to 10 copies per cell in some other bacteria. Our results demonstrate that Pelagibacter can change growth without shifts in transcript levels, suggesting that transcriptional control plays a minimal role in the adaptive strategy for one of the most successful organisms in the biosphere.
Project description:Thiamine is often undetectable in ocean surface waters where Pelagibacter cells are numerically abundant. Despite this, Pelagibacter cells are missing de novo thiamine synthesis pathways. We show that an eogenous source of the thiamine precursor HMP is required for thiamine synthesis in Pelagibacter and that this precursor is abundant in the Sargasso sea.
Project description:Bacteria respond to stimuli in the environment using transcriptional control, but this may not be the case for most marine bacteria having small, streamlined genomes. Candidatus Pelagibacter ubique, a cultivated representative of the SAR11 clade, which is the most abundant clade in the oceans 4, has a small, streamlined genome and possesses an unusually small number of transcriptional regulators. This observation leads to the hypothesis that transcriptional control is low in Pelagibacter and limits its response to environmental conditions. However, the extent of transcriptional control in Pelagibacter is unknown. Here we show that transcriptional control is extremely low in Pelagibacter and another oligotroph (SAR92) compared to two marine copiotrophic bacterial taxa, Polaribacter MED152 and Ruegeria pomeroyi. We found that ~0.1% of protein-encoding genes in Pelagibacter are under transcriptional control compared to >10% of genes in other marine bacteria. Regardless of the growth condition, the same genes were highly expressed while most genes were always expressed at very low levels. Quantitative RNA sequencing revealed that abundances of most Pelagibacter transcripts were <0.01 copies per cell whereas transcript abundances were 1 to 10 copies per cell in some other bacteria. Our results demonstrate that Pelagibacter can change growth without shifts in transcript levels, suggesting that transcriptional control plays a minimal role in the adaptive strategy for one of the most successful organisms in the biosphere. Bacteria were grown in batch culture and sampled twice during the initial, rapid phase of exponential growth and twice during the phase of slower growth that followed.
Project description:Thiamine is often undetectable in ocean surface waters where Pelagibacter cells are numerically abundant. Despite this, Pelagibacter cells are missing de novo thiamine synthesis pathways. We show that an eogenous source of the thiamine precursor HMP is required for thiamine synthesis in Pelagibacter and that this precursor is abundant in the Sargasso sea. Batch cultures of P. ubique were grown in a defined arificial seawater media. Three cultures were given no thiamine amendment, and three other cultures received an excess concentration of thiamine. Cultures were harvested for microarray analyses just prior to and after thiamine limitation for the purpose of observing differences in gene expression related to thiamine limitation.
Project description:Candidatus pelagibacter ubique HTCC1062 requires 4-amino-5-hydroxymethyl-2-methylpyrimidine, an abundant thiamine precursor in the sea
Project description:Chemoheterotrophic marine bacteria of the SAR11 clade are Earth's most abundant organisms. Following the first cultivation of a SAR11 bacterium, 'Candidatus Pelagibacter ubique' strain HTCC1062 (Ca. P. ubique) in 2002, unusual nutritional requirements were identified for reduced sulfur compounds and glycine or serine. These requirements were linked to genome streamlining resulting from selection for efficient resource utilization in nutrient-limited ocean habitats. Here we report the first successful cultivation of Ca. P. ubique on a defined artificial seawater medium (AMS1), and an additional requirement for pyruvate or pyruvate precursors. Optimal growth was observed with the collective addition of inorganic macro- and micronutrients, vitamins, methionine, glycine and pyruvate. Methionine served as the sole sulfur source but methionine and glycine were not sufficient to support growth. Optimal cell yields were obtained when the stoichiometry between glycine and pyruvate was 1:4, and incomplete cell division was observed in cultures starved for pyruvate. Glucose and oxaloacetate could fully replace pyruvate, but not acetate, taurine or a variety of tricarboxylic acid cycle intermediates. Moreover, both glycine betaine and serine could substitute for glycine. Interestingly, glycolate partially restored growth in the absence of glycine. We propose that this is the result of the use of glycolate, a product of phytoplankton metabolism, as both a carbon source for respiration and as a precursor to glycine. These findings are important because they provide support for the hypothesis that some micro-organisms are challenging to cultivate because of unusual nutrient requirements caused by streamlining selection and gene loss. Our findings also illustrate unusual metabolic rearrangements that adapt these cells to extreme oligotrophy, and underscore the challenge of reconstructing metabolism from genome sequences in organisms that have non-canonical metabolic pathways.