Article Figures & Data
Figures
- FIG 1
Phylogenetic trees. (A) 16S rRNA tree of representative species of the phyla Planctomycetes and Verrucomicrobia with sequenced genomes (as of June 2013) and five outgroup species. Bootstrap values are shown at nodes, and black hexagons indicate species with a BMC gene cluster. Each species' BMC gene cluster is shown schematically to the right of the species name, color-coded by gene annotation as follows: orange, transcriptional regulator; blue, enzyme; green, BMC-H shell protein; pink, BMC-P shell protein; gray, hypothetical protein. (B) Gene tree of the concatenation of aldehyde dehydrogenase (pduP) and phosphotransferase (pduL) homologs (Table 2; see also Table S3 in the supplemental material). Branches with less than 50% bootstrap support were collapsed and drawn in cartoon form. Branch color or symbol corresponds to phylum. Collapsed branches were similarly colored to represent phyla present within. Scale bar represents number of substitutions per site. Clades are annotated by the key enzymes present in the gene clusters (see Materials and Methods). PDU, vitamin B12-dependent propanediol utilization; PDU2, vitamin B12-independent (glycyl radical enzyme-utilizing) propanediol utilization; PV, Planctomycetes and Verrucomicrobia type; EUT, vitamin B12-dependent ethanolamine utilization; GRE, locus of unknown function containing a glycyl radical enzyme. Bootstrap values of branches separating major clades are shown.
- FIG 2
Homologous recombination to generate knockout mutants in P. limnophilus. (A) Schematic of the BMC gene cluster in P. limnophilus and the homologous recombination used to create the ΔpvmJ::IN(npt) mutant. Orange, transcriptional regulator; blue, enzyme; green, BMC-H shell protein; pink, BMC-P shell protein; gray, hypothetical gene; yellow, kanamycin resistance gene (npt). Orange arrows represent primers OE206 and OE192 (see Table S2 in the supplemental material) used for PCR genotyping. OE206 is outside the homology region, showing that recombination happened at the desired locus. (B) PCR amplification using primers OE206 and OE192 showing that amplicons are different sizes for the wild-type (wt) and knockout strains.
- FIG 3
Growth curves of Δplim_1104, ΔpvmJ, and ΔpvmN strains grown on l-fucose (A) and l-rhamnose (B). “No sugar” data in panel B are reproduced from panel A. (C) Spiking experiment in which the ΔpvmN strain was grown on 5 mM d-glucose for 2 days, at which point 5 mM l-fucose was added to half of the cultures. Solid squares represent nonspiked cultures, and open squares represent cultures with l-fucose added. In all graphs, each data point is an average of three independently grown cultures (n = 3); each error bar represents 1 standard deviation.
- FIG 4
BMCs are expressed during growth on fucose and rhamnose. (A) Growth curves using l-fucose or l-rhamnose of the ΔpvmDE and Δplim_1104 strains grown with or without sugars. Data for the Δplim_1104 strain are reproduced from Fig. 3A and B. Each data point is an average of three independently grown cultures (n = 3); each error bar represents 1 standard deviation. (B to D) Electron microscopy of wild-type P. limnophilus showing BMCs (indicated by white arrowheads) when grown for 9 days on l-fucose (B) and l-rhamnose (C) but not d-glucose (D).
- FIG 5
Growth curves of Δplim_1104, ΔpvmDE, and ΔpvmN strains grown on fucoidan from Laminaria spp. “No sugar” data are reproduced from Fig. 3A and 4A. Each data point is an average of three independently grown cultures (n = 3); each error bar represents 1 standard deviation.
- FIG 6
Proposed model of l-fucose metabolism in P. limnophilus. Two potential pathways to reach lactaldehyde are shown. Enzymes encoded by the pvm gene cluster are shown in blue, and shell proteins are shown in green (BMC-H) or pink (BMC-P). Cofactors are depicted in gray. Enzymes that potentially catalyze reactions not directly involved in the BMC chemistry are shown in black. “n.r.” stands for no result, meaning that there was no significant BLAST hit using characterized enzymes as a query. DHAP, dihydroxyacetone phosphate.
Tables
- TABLE 1
Strains used in this study
Strain name Genotype Term used in text Reference or source Mü 290T Wild type Wild type 31 OE3 Δplim_1104::npt Δplim_1104, control strain This work OE4 Δplim_1751::IN(npt) ΔpvmJ::IN(npt) This work OE22 Δlim_1751::npt ΔpvmJ This work OE23 Δ(plim_1756-plim_1755)::npt ΔpvmDE, shell-less mutant This work OE24 Δplim_1747::npt ΔpvmN This work - TABLE 2
Protein compositions of functionally distinct metabolosomes
BMC type No. and name of shell protein gene(s) Gene(s) for aldehyde-generating enzymes Gene for core metabolosome enzymes BMC-H BMC-T BMC-P 1,2-Propanediol oxidoreductase Aldolase Propionaldehyde generating Acetaldehyde generating Aldehyde dehydrogenase Phosphotransferase Acyl kinase Alcohol dehydrogenase PDU (S. enterica) 3; pduA, -J, -K, -U 2; pduB, -T 1; pduN None None pduCDEa (B12-dependent diol dehydratase) None pduPa pduLa pduWa pduQa EUT (S. enterica) 3; eutK, -M, -S 1; eutL 1; eutN None None None eutBCa (ethanolamine ammonia lyase) eutEa eutDa acka eutGa PDU2 (C. phytofermentans) 4; Cphy_1176, Cphy_1180, Cphy_1181, Cphy_1182 1; Cphy_1186 1; Cphy_1184 Cphy_1185 Cphy_1177 Cphy_1174 (B12-independent diol dehydratase) None Cphy_1178 Cphy_1183 Cphy_1327 Cphy_1179 PV (P. limnophilus) 2; pvmD, -E 0 3; pvmH, -K, -M None pvmN None None pvmJ pvmB pvmG pvmO ↵a Experimentally verified function. Accession numbers for genes are provided in Tables S1 and S4 in the supplemental material.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Individual gene trees for PduL homologs and PduP homologs (Fig. S1), results of pairwise comparisons of BMC-related enzymes from P. limnophilus and C. phytofermentans (Fig. S2), description of mutants used in this study (Fig. S3), analysis of Biolog phenotypic microarray data (Fig. S4), raw Biolog phenotypic microarray data (Fig. S5), growth profile comparison (Fig. S6), new nomenclature of BMC-related genes of the PVM BMC gene cluster (Table S1), list of oligonucleotides used in this study (Table S2), genomes and accession numbers of genes used in phylogenetic analysis for Fig. 1B and Fig. S1A and S1B (Table S3), and accession numbers and references for genes in Table 2 (Table S4).
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- Supplemental file 1 -
