One carbon metabolism in SAR11 pelagic marine bacteria

Abstract

The SAR11 Alphaproteobacteria are the most abundant heterotrophs in the oceans and are believed to play a major role in mineralizing marine dissolved organic carbon. Their genomes are among the smallest known for free-living heterotrophic cells, raising questions about how they successfully utilize complex organic matter with a limited metabolic repertoire. Here we show that conserved genes in SAR11 subgroup Ia (Candidatus Pelagibacter ubique) genomes encode pathways for the oxidation of a variety of one-carbon compounds and methyl functional groups from methylated compounds. These pathways were predicted to produce energy by tetrahydrofolate (THF)-mediated oxidation, but not to support the net assimilation of biomass from C1 compounds. Measurements of cellular ATP content and the oxidation of (14)C-labeled compounds to (14)CO(2) indicated that methanol, formaldehyde, methylamine, and methyl groups from glycine betaine (GBT), trimethylamine (TMA), trimethylamine N-oxide (TMAO), and dimethylsulfoniopropionate (DMSP) were oxidized by axenic cultures of the SAR11 strain Ca. P. ubique HTCC1062. Analyses of metagenomic data showed that genes for C1 metabolism occur at a high frequency in natural SAR11 populations. In short term incubations, natural communities of Sargasso Sea microbial plankton expressed a potential for the oxidation of (14)C-labeled formate, formaldehyde, methanol and TMAO that was similar to cultured SAR11 cells and, like cultured SAR11 cells, incorporated a much larger percentage of pyruvate and glucose (27-35%) than of C1 compounds (2-6%) into biomass. Collectively, these genomic, cellular and environmental data show a surprising capacity for demethylation and C1 oxidation in SAR11 cultures and in natural microbial communities dominated by SAR11, and support the conclusion that C1 oxidation might be a significant conduit by which dissolved organic carbon is recycled to CO(2) in the upper ocean.

Figure 1. Demethylation and C1 oxidation regions of the strain HTCC1062 genome.

(A) formate dehydrogenase; (B) methanol metabolism; (C) methylamine oxidation; (D) glycine betaine oxidation; (E) aminomethyltransferases (Asterisk). fdhF, formate dehydrogenase, alpha subunit; fdsB, NAD-dependent formate dehydrogenase, beta subunit; fdhD , formate dehydrogenase, chain D; mobA , molybdopterin-guanine dinucleotide biosynthesis protein A; moeA, molybdopterin biosynthesis protein; fhs , formate-THF ligase; SAR11_1286, putative glutamine amidotransferase; Fe-ADH, iron-containing alcohol dehydrogenase; ssdH, aldehyde dehydrogenase family; SAR11_1289, short chain dehydrogenase; soxB, sarcosine oxidase; soxD & soxD₂, sarcosine oxidase delta chain; soxA & soxA₂, sarcosine oxidase alpha chain; soxG & soxG₂, sarcosine oxidase gamma subunit; soxB ₂, sarcosine oxidase beta subunit; glxBCD, glutamate synthase; glnT, Glutamine synthetase III (putative gamma-glutamylmethylamide synthetase); bhmT, betaine-homocysteine methyltransferase; sardh, sarcosine dehydrogenase; dmgdh, dimethylglycine dehydrogenase; gcvT , glycine system cleavage T-protein; gcvH , glycine cleavage H-protein; gcvP , glycine cleavage P-protein; dmdA , dimethylsulfoniopropionate-dependent demethylase; mhpC, hydrolase, alpha/beta hydrolase fold family; fadD, CoA activator for DMSP beta oxidation; mmgC , acyl-CoA dehydrogenase for DMSP beta oxidation; metF, methylene-THF reductase; opuAB , glycine betaine transport system permease protein; opuAA , glycine betaine transport ATP-binding protein; opuAC , substrate-binding region of ABC-type glycine betaine transport system; SAR11_1265 & SAR11_1303, gcvT-like aminomethyltransferase protein; SAR11_1304, monomeric sarcosine oxidase. Colors correspond to pathways in Figure 2.

Figure 2. Proposed C1 and methylated compound oxidation pathways in SAR11 Group Ia.

(A) THF-linked oxidation pathway; (B) methanol oxidation pathway; (C) glycine betaine demethylation and oxidation; (D) methylamine oxidation pathways; (E) TMAO degradation pathway; (F) glycine cleavage pathway; (G) DMSP demethylation. Note: ? - unidentified metabolic processes/enzymes; * - spontaneous reaction; † - two paralogous operons.

Figure 3. Phylogenetic tree of Fe-ADH proteins.

Coloration is according to 16S rRNA gene phylogeny, as shown in the boxed legend. Bootstrap values were omitted for clarity; nodes with less than 60% support were collapsed. Arrows indicate Fe-ADH proteins for which methanol dehydrogenase activity has been demonstrated experimentally. Scale bar  = 0.4 changes per position.

Figure 4. Phylogeny of SAR11 AMT proteins.

Four paralogous AMTs in HTCC1062 were placed into three functional subgroups: DmdA-like, GcvT, and an AMT of unknown function. All four AMTs were also identified in HTCC1002 and HTCC7211 genomes. This phylogenetic tree was generated using the neighbor-joining method. Bootstrap values are based on 100 iterations.

Figure 5. ¹⁴C-labeled compound utilization by HTCC1062 in culture.

HTCC1062 Cells from log phase were collected and resuspended in artificial seawater media (ASW). Radioisotope assays were conducted at room temperature (22°C) in ASW amended with (A) 1 µM ¹⁴C-[methyl]-GBT; (B) 5 µM ¹⁴C-TMA; (C) 20 µM ¹⁴C-methanol; or (D) 100 nM ¹⁴C-formaldehyde. Where not visible, error bars are smaller than the size of the symbols.

Figure 6. Utilization of ¹⁴C-labeled C1 and methylated compounds by bacterioplankton in the western Sargasso Sea.

The oxidation and incorporation rates were calculated from the initial linear part of each curve. Rate of ¹⁴C-compound oxidation to ¹⁴CO₂ (▪); rate of ¹⁴C-compounds incorporation into biomass (□).