| Aspect | Details | Quantitative evidence / conditions | Primary source (date; URL/DOI) |
|---|---|---|---|
| Verified target identity | **mllG** in *Methylorubrum extorquens* AM1 corresponds to **META1p4136 / MexAM1_META1p4136** within the **mll (methylolanthanin) locus META1p4129–META1p4138**. Literature identified this gene specifically as **mllG** and annotated it as a **DUF2218-containing protein**. Importantly, the literature does **not** support the UniProt reaction annotation “2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase” for this AM1 protein; instead, available evidence places it in a lanthanophore biosynthetic/uptake locus with a likely **transport or regulatory** role. (pqac-00000000, pqac-00000001) | mll locus reported as strongly induced under poorly soluble lanthanide conditions; average upregulation of the cluster was reported as **~32-fold** under growth with **Nd2O3**. (pqac-00000001) | Zytnick et al., **2022**, bioRxiv, “Discovery and characterization of the first known biological lanthanide chelator,” https://doi.org/10.1101/2022.01.19.476857 |
| Cluster context | The **mll locus** spans **META1p4129–META1p4138** and encodes methylolanthanin-associated functions. The locus includes predicted **uptake/regulatory genes** (**mluA/m/u?** assignments reported for META1p4129–4131: TonB-dependent outer membrane receptor, anti-sigma factor, sigma factor), followed by **biosynthetic genes** homologous to petrobactin/rhodopetrobactin loci (**META1p4132–4135: mllA, mllBC, mllDE, mllF**), then **mllG = META1p4136 (DUF2218)**, plus **mllH = META1p4137** (acetyltransferase) and **mllJ = META1p4138** (ferritin-like DUF4142 protein, putatively exported to the periplasm). (pqac-00000001, pqac-00000006) | Cluster identified from transcriptomic response to poorly soluble lanthanide source and linked to a secreted lanthanide chelator. (pqac-00000001, pqac-00000003) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857; Phi, **2024** dissertation, https://doi.org/10.5282/edoc.33507 |
| Gene-by-gene proposed functions in the locus | **META1p4129–4131**: predicted **transport/regulation** for metallophore uptake and expression control; **META1p4132–4135**: predicted **methylolanthanin biosynthesis** based on homology to citrate-based siderophore pathways; **META1p4136/mllG**: **DUF2218** family protein, also present in related rhodopetrobactin loci; homology to *Vibrio cholerae* **VCA0233** near iron-uptake/xenosiderophore genes suggests **regulation or transport rather than direct biosynthesis**; **META1p4137/mllH**: acetyltransferase; **META1p4138/mllJ**: ferritin-like DUF4142 protein, proposed periplasmic accessory role. (pqac-00000000, pqac-00000001) | No direct enzymatic assay for **mllG** reported in the available sources; no substrate specificity or aldolase reaction was experimentally shown for mllG. (pqac-00000000, pqac-00000001) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857 |
| Evidence specifically about mllG | **mllG/META1p4136** is explicitly named in the mll cluster and annotated as **DUF2218**. Available literature frames DUF2218 in this context as likely involved in **transport/regulation**, not as a characterized catalytic aldolase. Thus, for this specific protein, the gene symbol is **not ambiguous in the methylolanthanin literature**, but its **molecular function remains incompletely defined**. (pqac-00000000, pqac-00000001) | Evidence is inferential: locus membership, conservation in related metallophore loci, and homology/context to VCA0233-like proteins. No kinetics, purified-protein activity, or localization experiment for mllG alone was reported. (pqac-00000000) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857 |
| Methylolanthanin product and pathway role | The **mll cluster** encodes production of **methylolanthanin (MLL)**, described as the **first known biological lanthanide chelator/lanthanophore**, structurally related to citrate-based siderophores and containing a **4-hydroxybenzoate** moiety. MLL is secreted and participates in **lanthanide acquisition**, especially when lanthanides are poorly bioavailable. (pqac-00000002, pqac-00000003, pqac-00000004, pqac-00000006) | MLL was observed to bind **La3+, Nd3+, and Lu3+** by mass spectrometry. (pqac-00000002, pqac-00000003) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857; Phi, **2024**, https://doi.org/10.5282/edoc.33507 |
| Expression induction by insoluble lanthanide source | Transcriptomic studies showed the **mll locus** is among the most highly induced gene clusters when cells are grown with **poorly soluble Nd2O3** rather than soluble lanthanide sources, consistent with a role in improving access to mineral/insoluble lanthanides. (pqac-00000001, pqac-00000003) | Average induction reported as **~32-fold** for the cluster under **Nd2O3** growth conditions; the dissertation notes broad transcriptional remodeling involving **nearly 1,500 genes** across lanthanide-source comparisons. (pqac-00000001, pqac-00000003) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857; Phi, **2024**, https://doi.org/10.5282/edoc.33507 |
| Growth phenotype: overexpression | Overexpression of the **mll biosynthetic cluster** improved growth when lanthanides were poorly bioavailable, supporting a role for the MLL system in lanthanide scavenging. (pqac-00000001, pqac-00000002, pqac-00000007) | In a **ΔmxaF/pAZ1** overexpression background grown with **Nd2O3**, growth rate was **0.026 h^-1**, compared with **0.037 h^-1** on **NdCl3**; overexpression partially rescued poor-growth conditions imposed by insoluble Nd source. (pqac-00000002) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857 |
| Growth phenotype: deletion / nonessentiality under tested lab conditions | Deletion of the **mll** cluster impaired lanthanide accumulation but did **not abolish growth** under the tested laboratory conditions, indicating MLL enhances but is not absolutely essential for lanthanide-dependent growth in those settings. (pqac-00000002, pqac-00000005) | **ΔmxaFΔmll** showed growth similar to **ΔmxaF** in some tested conditions, but lanthanide bioaccumulation decreased by about **30%** in one analysis. (pqac-00000005) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857 |
| Nd accumulation phenotype | The mll system contributes to intracellular lanthanide accumulation/bioaccumulation. Deletion reduces, and overexpression increases, intracellular **Nd** levels. (pqac-00000002, pqac-00000004, pqac-00000007) | Deletion caused a reported **1.8-fold decrease** in intracellular Nd accumulation on **NdCl3**; overexpression increased intracellular Nd by about **3.5-fold on average**. Separate summary text describes deletion as causing a **~30% decrease** in bioaccumulation. (pqac-00000002, pqac-00000005) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857 |
| Rescue by exogenous methylolanthanin | Purified **methylolanthanin** added exogenously can rescue or enhance growth, showing that the secreted small molecule itself is functionally active in lanthanide acquisition. (pqac-00000002) | Addition of **50 nM MLL** to cultures grown with **2 µM NdCl3** significantly increased growth yield (**p = 0.036 and 0.037** in reported comparisons). (pqac-00000002) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857 |
| Localization and cellular site of action | The **lanthanide-chelating product MLL** is extracellular/secreted, while uptake is tied to **TonB-dependent outer membrane** transport and downstream ABC-type transport. More broadly, known lanthanide-dependent methanol oxidation enzymes in *M. extorquens* AM1 are **periplasmic**, placing MLL-mediated acquisition upstream of periplasmic lanthanide use. For **mllG** specifically, no direct localization experiment was reported. (pqac-00000001, pqac-00000003, pqac-00000004) | Supported by cluster architecture and linked transport systems rather than direct mllG localization assays. (pqac-00000001, pqac-00000004) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857; Phi, **2024**, https://doi.org/10.5282/edoc.33507 |
| Bottom-line annotation for C5B1I8 | For **UniProt C5B1I8 / mllG / META1p4136**, the strongest current evidence supports annotation as an **uncharacterized DUF2218 family protein in the methylolanthanin lanthanophore locus**, probably contributing to **transport/regulation/accessory steps in lanthanide acquisition**, rather than a confidently established **2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase**. (pqac-00000000, pqac-00000001) | Major knowledge gap: no direct enzymology, no confirmed substrate, no specific subcellular localization for mllG alone in the available evidence. (pqac-00000000) | Zytnick et al., **2022**, https://doi.org/10.1101/2022.01.19.476857 |


*Table: This table summarizes verified identity, cluster context, proposed gene functions, and the main experimental evidence linking the mll locus to methylolanthanin-mediated lanthanide acquisition in Methylorubrum extorquens AM1. It is useful for distinguishing gene-level evidence for mllG from broader cluster-level functional data.*