Supplementary Note 1 Regulation of cofactor biosynthesis. The accurate prediction of genes involved in de novo vitamin biosynthesis and salvage was facilitated by identifying genes downstream to to binding sites of regulators that control biotin or NAD metabolism and to riboswitches that bind thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), or adenosylcobalamin (B12).
The biotin-responsive repressor domain of BirA was present only in the five Gammaproteobacterial members and in each case it was predicted to control biosynthetic genes (Supplementary Data S2). However, while they all encode the YigM transporter for biotin uptake, its BirA-dependent regulation is only predicted in the two Halomonas spp. Two different transcriptional regulators were identified for NAD metabolism genes. In cyanobacteria, the NtrR repressor co-regulated the biosynthetic enzyme, NadA, the pyridine salvage enzyme PncB, and the two central NAD synthesis enzymes, NadD and NadE. In Oceanicaulis bin04, the predicted transcription factor, NadQ, co-regulates the NAD synthetaseNadE and all three de novo NAD biosynthesis enzymes, NadABC.
The Halomonassp. HL-93 genome encodes the only FMN riboswitch detected and is located upstream to a second gene (ribH2) encoding 6,7-dimethyl-8-ribityllumazine synthase. The ribH2 gene, occurs in the locus encoding enzymes and the transporter responsible for erythritol degradation. Growth on erythritol as a carbon source leads to exclusive assimilation of carbohydrate via erythrose-4-phosphate(Barbier et al., 2014) which can then easily be converted to the FAD precursor, ribulose-5-phosphate (Supplementary Fig. 2) as needed.
The B12 riboswitch was detected in all members of the consortia except Aliidiomarina HL-53, which lacks any B12 biosynthesis or salvage genes. In all cases it controls expression of genes encoding Btu transporter components and, in five of the Rhobacteraceae, a dedicated TonB-type energization system (TonB-ExbB-ExbD) for the TonB-dependent B12 receptor. Expression of corrin ring biosynthetic genes (cobGJH) is under control of the B12 riboswitch in only S. fredricksonii HL-109 suggesting that they are less likely to produce excess cofactor and consequently excrete this resource. The riboswitch in Marinobacter HL-58 controls biosynthetic genes (cobA and cobB), which likely reflects its ability to salvage an early precursor in B12 biosynthesis. In R. calidilacus HL-91, the B12-dependent MetH methionine synthase is under riboswitch control while in the Marinobacter spp. the ribonucleotide synthesis enzyme, NrdAB (an alternative to the B12-dependent reductase, NrdJ) is regulated, which is consistent with their ability to synthesize alternative B12-independent enzymes, MetE and NrdJ, respectively.
The TPP riboswitch is present in all members and controls the HMP biosynthetic gene, thiC, in all prototrophs. The riboswitch in all except one of the prototrophs with salvage capability (Marinobacter HL-58) controls biosynthetic enzymes and/or transporters used in salvage. In the auxotrophs, all predicted operons encoding thiamine precursor salvage enzymes (ThiMED, TenA) and predicted transporters for thiamine (ThiBPQ, Omr1, PnuT), HMP (ThiXYZ, YkoFEDC), and thiazole (TRAP) are co-regulated by TPP riboswitches.
Supplementary Note 2. Extended Description of Thiamine salvage. Nine members are predicted to salvage thiamine based on identified transporters and the presence of a thiamine kinase. Four auxotrophs encode ThiD, ThiM, and ThiE and, therefore, should be able to salvage HET and HMP. Salvage of both precursors is the only option for generating TPP by S. fredricksonii HL-109 and thus Wolf’s vitamin solution should not support growth of this organism. The presence of thiamine-monophosphate kinase (ThiL) in some auxotrophs is unexpected. For example, Bacterioidetes bin01 salvages thiamine and phosphorylates it with ThiN and thus it unclear why ThiL is needed unless salvage systems for HET and HMP are present in sequencing gaps. Conversely all three auxotrophic Rhodobacteraceae lack ThiL yet require it for salvage of HET and HMP. The only thiamine-salvaging prototroph, M. excellens HL-55, lacks a known thiamine kinase. These findings suggest that existence of novel salvage enzymes in both the auxotrophs and prototrophs.
Eight types of TPP precursor transporters were identified, five of which are dedicated to thiamine uptake. ThiV2 is a new candidate thiamine transporter found in bacteria belonging to the FCB group and distantly related to the candidate HMP transporter, ThiV (Carini et al., 2014). The predicted role of thiV2 in HMP transport is supported by its frequent occurrence downstream of the TPP riboswitch and in the genomic neighborhood of thiN and sometimes omr1, both of which are involved in thiamine salvage. The only candidate HET transporter identified is also novel and was first found in S. fredricksonii HL-109 where thiD was found to overlap a downstream gene encoding the substrate-binding component of a TRAP-type transporter. Further analysis revealed orthologs in both Halomonas spp., as well as other microbial genomes, allowing us to identify the other two components of this transport system in HL-109 and to recognize that genes in this system frequently occur in the thiMED neighborhood. Since S. fredricksonii HL-109 encodes the ThiXYZ HMP transporter but lacks a candidate HET transporter, we propose that this TRAP transporter family is used in HET salvage. We were unable to identify HMP and/or HET transporters in Rhodobacteraceae HL-91, bin07, and bin09 but suspect that they exist due to the presence of ThiDEM in these auxotrophs.
Supplementary Notes References
Barbier T, Collard F, Zuniga-Ripa A, Moriyon I, Godard T, Becker J et al. (2014). Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella. Proc Natl Acad Sci U S A111: 17815-17820.
Carini P, Campbell EO, Morre J, Sanudo-Wilhelmy SA, Thrash JC, Bennett SE et al. (2014). Discovery of a SAR11 growth requirement for thiamin's pyrimidine precursor and its distribution in the Sargasso Sea. ISME J8: 1727-1738.
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