Giovannoni, Stephen J

Abstract 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.

Abstract Background Metagenomic sequence data are proving to be a vast resource for the discovery of biological components. Yet analysis of this data to identify functional RNAs lags behind efforts to characterize protein diversity. The genome of 'Candidatus Pelagibacter ubique' HTCC 1062 is the closest match for approximately 20% of marine metagenomic sequence reads. It is also small, contains little non-coding DNA, and has strikingly low GC content. Results To aid the discovery of RNA motifs within the marine metagenome we exploited the genomic properties of 'Cand. P. ubique' by targeting our search to long intergenic regions (IGRs) with relatively high GC content. Analysis of known RNAs (rRNA, tRNA, riboswitches etc.) shows that structured RNAs are significantly enriched in such IGRs. To identify additional candidate structured RNAs, we examined other IGRs with similar characteristics from 'Cand. P. ubique' using comparative genomics approaches in conjunction with marine metagenomic data. Employing this strategy, we discovered four candidate structured RNAs including a new riboswitch class as well as three additional likely cis-regulatory elements that precede genes encoding ribosomal proteins S2 and S12, and the cytoplasmic protein component of the signal recognition particle. We also describe four additional potential RNA motifs with few or no examples occurring outside the metagenomic data. Conclusion This work begins the process of identifying functional RNA motifs present in the metagenomic data and illustrates how existing completed genomes may be used to aid in this task.