Unique glycine-activated riboswitch linked to glycine-serine auxotrophy in SAR11 - PubMed (original) (raw)

Unique glycine-activated riboswitch linked to glycine-serine auxotrophy in SAR11

H James Tripp et al. Environ Microbiol. 2009 Jan.

Abstract

The genome sequence of the marine bacterium 'Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that 'Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 microM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 x 10(6) cells ml(-1) to 8.16 x 10(6) cells ml(-1) (R(2) = 0.992). Serine was capable of substituting for glycine at 1.5 microM. 'Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that 'Cand. P. ubique' is therefore a glycine-serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.

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Figures

Figure 1

Figure 1

Molar growth yield curve for glycine. The maximum cell density achieved for batch cultures grown in the dark at 16° C is plotted against addition of glycine in the presence of excess nutrients (avg., N=2 for each data point, R2=0.992, error bars show complete range). Carbon was supplied as 10 μM glucose and 50 μM acetate. Other nutrients were 10 μM NH4Cl, 1 μM KH2PO4, 53.6 μM FeCl3 and a mix of vitamins (Davis and Guillard, 1958).

Figure 2

Figure 2

Secondary structure of SAR11 riboswitches and alignment to environmental data. Aptamers are labeled with Roman Numerals I and II, and base pairing stems are labeled P1, P2, and P3 with subsections labeled “a” and “b.” Base pairs with an asterisk were shown to be accessible to spontaneous cleavage, as would be expected if they are not part of a base pair. Base pairs shaded in grey show 100% conservation in upstream regions of metagenomic data. gcvT, glycine cleavage protein T; glcB, malate synthase.

Figure 3

Figure 3

Structural probing and KD measurements for gcvT and glcB riboswitches. A. Spontaneous cleavage products of the glcB associated riboswitch in the presence of 1 mM of ligand candidates and the absence of ligand. Selected RNase T1 cleavage products (cleaves 3′ of G residues) are identified on the left. Arrows on the right indicate bands used to quantify extent of RNA cleavage. B. Plot of percentage RNA cleaved (normalized between no ligand and saturating ligand conditions) versus the logarithm of the molar concentration of glycine used to determine KD and h constants for the glcB associated riboswitch. C. Identical to B for the gcvT associated riboswitch.

Fig 4

Fig 4

Maximum cell yields for glycine substitutes (avg., N=2 for each data point, error bars show complete range). Response to 1.5 μM additions of compounds. Gly, glycine; Bet, betaine; Ser, serine; Glc, glycolate; Glx, glyoxylate; Thr, threonine; No Gly, no glycine; No Glu, no glucose; No ace, no acetate. Excess nutrients were supplied as in Fig. 1.

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