Nutrient requirements for growth of the extreme oligotroph 'Candidatus Pelagibacter ubique' HTCC1062 on a defined medium - PubMed (original) (raw)
Nutrient requirements for growth of the extreme oligotroph 'Candidatus Pelagibacter ubique' HTCC1062 on a defined medium
Paul Carini et al. ISME J. 2013 Mar.
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.
Figures
Figure 1
Simplified illustration of central metabolism in Ca. P. ubique. Black lines: reactions predicted to occur in Ca. P. ubique based on genome content. Red lines: reactions predicted to occur in E. coli, but missing from Ca. P. ubique. Blue lines: putative glucose oxidation pathway (see Schwalbach et al., 2010). Gene names in green are discussed further in the article. Bolded compounds were previously identified as growth substrates for Ca. P. ubique. ald, alanine dehydrogenase; glcB, malate synthase; APS, adenosine 5′-phosphosulfate; G3-P, glyceraldehyde-3-phosphate; 3-HP, 3-hydroxypyruvate; PAPS: 3′-phosphoadenylyl sulfate, PEP: phosphoenolpyruvate.
Figure 2
Growth of Ca. P. ubique in AMS1 with organic carbon additions. Black: cells grown without additions of glycine, methionine or pyruvate. Red: cells amended with methionine (10 μℳ), glycine (50 μℳ) and pyruvate (50 μℳ). Blue: cells amended with methionine (10 μℳ) only. Green: cells amended with glycine (50 μℳ) and pyruvate (50 μℳ) only. Points are the average density of triplicate cultures. Error bars indicate±1.0 s.d. (_n_=3). When error bars are not visible, they are smaller than the size of the symbols.
Figure 3
Maximum cell yields of Ca. P. ubique in response to pyruvate and glycine additions. (a) Pyruvate titration in AMS1 supplemented with glycine (50 μℳ) and methionine (10 μℳ). Using the formula of the regression line, the maximum cell density achievable from pyruvate carryover with source inoculum (156 pM) was calculated to be 400 cells ml−1 (filled star). (b) Glycine titration into AMS1 supplemented with pyruvate (50 μℳ) and methionine (10 μℳ). Using the formula of the regression line, the maximum cell density achievable from glycine carryover with source inoculum (500 pM) was calculated to be 5000 cells ml−1 (filled star). Filled circles are the average maximum cell densities of triplicate batch cultures. Error bars indicate±1.0 s.d. (_n_=3). When error bars are not visible, they are smaller than the size of the symbols.
Figure 4
DNA content and morphology of SYBR Green I-stained stationary-phase cells from pyruvate-deplete and -replete batch cultures. Red dashed line in a and b represents the minimum threshold of fluorescence detection. Black dashed lines in a and b represent relative DNA fluorescence values of 300–325 per event and 475–500 per event, as indicated with black arrows. (a) Relative DNA fluorescence of cells from pyruvate-replete (50 μℳ) stationary-phase cultures, and (b) pyruvate-deplete (0.5 μℳ) stationary-phase cultures. (c) Fluorescent microscopy image of cells from (a). Arrowheads point to single cells. (d) Fluorescent microscopy image of cells from (b). Arrowheads point to cell doublets.
Figure 5
Glycolate assimilation gene organizations in E. coli and Ca. P. ubique. Reaction arrows are colored by the genes predicted to catalyze the reaction. Green stem-loop images represent the glycine-activated riboswitch (Tripp et al., 2009). Gene annotations are as described in NCBI, however, we predict the reaction catalyzed by AspC to be as described in the figure. accA, acetyl-CoA carboxylase; aspC, probable aspartate transaminase; glcC, DNA-binding transcriptional dual regulator, glycolate-binding; glcD, glycolate oxidase subunit, FAD-linked; glcE, glycolate oxidase, FAD-binding subunit; glcF, glycolate oxidase, iron-sulfur subunit; G, glcG; Putative glc operon gene, function unknown; glcB, malate synthase G; recA, recombinase A; yghK, glycolate transporter; ldh; probable 2-hydroxyacid dehydrogenase; 0274, SAR11_0274; major facilitator superfamily transporter, possible sugar-phosphate transporter; TCA, tricarboxylic acid.
References
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