Fig 1.
Processes and components of lipoate assembly pathways.
(a) Main known steps of established lipoate assembly pathways. Enzymes and steps not occurring in E. coli but described for other organisms are printed in gray. LipM and LipL have been demonstrated in Firmicutes, B. subtilis [24], Staphylococcus aureus [25], and Listeria monocytogenes [26], as well as in Tenericutes, Mycoplasma hyopneumoniae [15,16]. (b) Predicted novel lipoate assembly pathway. The pathway is substantiated by experiments reported here as well as by published work on proteins from the 3 model organisms depicted in c [2,9] and by genetic and biochemical work on LipS1 and LipS2 from the archaeon Thermococcus kodakarensis [27]. Lipoate:protein ligases from sulfur oxidizers were originally reported not to contain a carboxy-terminal LplB domain based on superposition of the structure modeled for Thioalkalivibrio sp. K90mix by using the automated mode of SWISS_Model on E. coli Lpl(AB). We challenged this view and indeed, modeling by Alphafold [28] as well as sequence alignments yielded clear proof for the presence of the LplB domain (S2 Fig). (c) Genetic arrangement of 3 novel systems for lipoate assembly in Proteobacteria. Colors correspond to the biochemical roles as depicted in b. For Ts. sibirica locus tags are given according to JGI-IMG. LipT is an FAD-binding NAD(P)H-dependent oxidoreductase possibly delivering electrons for the LipS1/S2-catalyzed sulfur insertion step. The genes lipY and lipX encode a putative fatty acid transporter and a putative glutamine amidotransferase, respectively. ACP, acyl carrier protein; GcvH, glycine cleavage system protein H; LbpA, lipoate-binding protein; LD, lipoyl domains of the 2-oxoacid dehydrogenases.
Fig 2.
Biochemical and genetic evidence for a novel lipoate assembly pathway in bacteria.
(a) LbpAs from Thioalkalivibrio sp. K90mix and Ts. sibirica were produced in E. coli BL21(DE3) ΔiscR, a strain designed for improved synthesis of iron-sulfur proteins [32], either with or without a helper plasmid (pACYC-Tklpm) carrying genes lipS1-slpl(AB)-lipT-lipS2 from Thioalkalivibrio sp. K90mix (shown in b) under control of the constitutive pACYC184 tet promoter. Holo-LbpAs migrate faster in native PAGE due to loss of the positive lysine charge upon modification. In the heterologous host, TsLbpA proteins are—albeit not fully—modified by the assembly proteins stemming from a different species, i.e., Thioalkalivibrio. (c) Thiosulfate (triangles) and sulfite (boxes) concentrations for 4 different H. denitrificans strains during growth on methanol (24.4 mM) as a carbon source in the presence of 2 mM thiosulfate. (d) Growth of H. denitrificans strains. Symbols and lines in c and d correspond to H. denitrificans strains as follows: filled black symbols, solid lines: H. denitrificans ΔtsdA; symbols filled gray, solid lines: H. denitrificans ΔtsdA lbpA2-His; open symbols, dotted lines: H. denitrificans ΔtsdA ΔlbpA2; open symbols, solid lines: H. denitrificans ΔtsdA lbpA2-His Δslpl(AB). For all measurements, standard deviations based on 3 technical replicates are indicated, but too small to be visible for determination of biomass and sulfite. (e) SDS-PAGE of HdLbpA2-His enriched from H. denitrificans ΔtsdA lbpA2-His (left lane, 2.5 μg protein) and ΔtsdA Δslpl(AB) lbpA2-His (right lane, 1.5 μg protein). (f) Native gel mobility shift assay for HdLbpA2-His enriched from H. denitrificans ΔtsdA lbpA2-His (left lane, 2.5 μg protein) and ΔtsdA Δslpl(AB) lbpA2-His (right lane, 1.3 μg protein). HdLbpA2-His proteins were visualized after Western blotting using an Anti-His peroxidase conjugate. The data underlying panels c and d is provided as S1_data.xlsx.
Fig 3.
Taxonomic distribution of the lipoate synthesis systems, lipoate scavenging, and lipoate requiring proteins.
Venn diagrams show the abundance and overlap of lipoate:protein ligases (Lpl), octanoyl transferase (LipB, LipM), and lipoate synthases (LipA, LipS1/S2) in the bacteria (a) and the archaea (b). Panels c and d visualize the taxonomic distribution of these enzymes, the sulfur-oxidizing sHdr system (S) and lipoate-binding domains (LD). For each bacterial (c) and archaeal phylum (d), the percentage of genomes possessing these proteins is indicated by dots of different sizes and colors. Note that the proportion was normalized to the size of the phylum and does not show absolute counts or overall phylum size. The data underlying parts a and b are provided in S2 and S3 Tables, respectively. S4 Table supplies the data underlying parts c and d.
Fig 4.
Rooted phylogenetic tree for the complete lipoate:protein ligase/octanoyltransferase dataset.
The tree was rooted with the structurally related biotin ligase BirA as an outgroup. Red or blue dots placed on each leaf identify the source organisms as archaea or bacteria, respectively. The ligase/transferase type is color-coded in the next circle. In the outermost rings, the presence of other lipoate synthesis enzymes occurring in the same genome is labeled. The data underlying this figure is provided in Supplementary S3 Data.
Fig 5.
Phylogeny for clade 3 lipoate:protein ligases without LipM and cpLpl(BA).
LipMs do not have LplB domains and their sequences are consequently shorter. If a sequence is incomplete, parts of the information used to calculate the phylogenetic tree are missing. This can lead to erroneous estimates of the relationships between sequences and can bias the result and weakens statistical significance of the calculation. In addition, Lpl(BA) clearly shows an individual evolution and may also cause weakening of statistical support. The data underlying this figure is provided in S3 Data.
Fig 6.
Phylogenetic trees for LipS1/S2.
(a) To investigate the evolution of LipS1 and LipS2, their sequences were concatenated, as both units are usually found in synteny, are catalytically active together and should therefore be under the same evolutionary pressure [29]. Incomplete sequences and concatenated sequences from genomes lacking either LipS1 or LipS2 were removed from the analysis. The lower panels show schematic representation of phylogenetic trees generated using only archaeal sequences (red, b) or bacterial sequences (blue, c). Bacterial clades represented by single sequences were left out to increase readability. The data underlying this figure is provided in S3 Data.