Case Study: mll BGC (Methylolanthanin) in Methylorubrum extorquens AM1

This case study demonstrates how to query the mll biosynthetic gene cluster across multiple genomics databases, identifying join points and data gaps.

Background

The mll (methylolanthanin) biosynthetic gene cluster produces a lanthanide-chelating metallophore in Methylorubrum extorquens AM1. This is a well-characterized system described in Zytnick et al. 2024 (PNAS).

Key identifiers: - Organism: Methylorubrum extorquens AM1 (formerly Methylobacterium extorquens) - NCBI Taxonomy ID: 272630 - RefSeq Assembly: GCF_000018845.1 - Megaplasmid: NC_012811.1 (contains mll cluster) - Gene locus tags: META1p4129-META1p4138

The mll Gene Cluster

Locus Tag	Gene	Product	Function
META1p4129	mluA	TonB-dependent receptor	Metallophore uptake
META1p4130	mluR	Anti-sigma factor	Regulation
META1p4131	mluI	ECF sigma factor	Regulation
META1p4132	mllA	IucA/IucC family	Siderophore biosynthesis
META1p4133	mllBC	Fused biosynthesis	Siderophore biosynthesis
META1p4134	mllDE	Fused biosynthesis	Siderophore biosynthesis
META1p4135	mllF	3,4-DHB synthesis	Siderophore biosynthesis
META1p4136	mllG	DUF2218	Regulation/transport
META1p4137	mllH	Acetyltransferase	Linker modification
META1p4138	mllJ	DUF4142 ferritin-like	Periplasmic export

---

Database Queries

1. KBase Pangenome Database

1.1 Find the genome

-- Query: Find M. extorquens genomes in KBase
SELECT genome_id, gtdb_species_clade_id, ncbi_biosample_id
FROM kbase_ke_pangenome.genome
WHERE gtdb_species_clade_id LIKE '%Methylobacterium_extorquens%'
LIMIT 10

Result:

{
  "result": [
    {
      "genome_id": "RS_GCF_000018845.1",
      "gtdb_species_clade_id": "s__Methylobacterium_extorquens--RS_GCF_900234795.1",
      "ncbi_biosample_id": "SAMN00000030"
    }
    // ... 26 total genomes
  ]
}

Join point identified: - genome_id: RS_GCF_000018845.1 → Strip RS_ prefix → GCF_000018845.1 (NCBI Assembly) - ncbi_biosample_id: SAMN00000030 → Direct link to NCBI BioSample

1.2 Check available genes

-- Query: What nucleotide accessions are in KBase for this genome?
SELECT DISTINCT SUBSTRING(gene_id, 1, 12) as accession_prefix, COUNT(*) as cnt
FROM kbase_ke_pangenome.gene
WHERE genome_id = 'RS_GCF_000018845.1'
GROUP BY SUBSTRING(gene_id, 1, 12)

Result:

NC_010172.1 - 4,983 genes (plasmid pMETA1 only)

⚠️ DATA GAP IDENTIFIED:

KBase pangenome is missing genes from: - NC_012808.1 - Main chromosome - NC_012811.1 - Megaplasmid (where mll cluster resides!)

1.3 Attempt to find mll genes

-- Query: Search for megaplasmid genes (expected to fail)
SELECT COUNT(*) as gene_count
FROM kbase_ke_pangenome.gene
WHERE genome_id = 'RS_GCF_000018845.1'
  AND gene_id LIKE 'NC_012811%'

Result: 0 genes found

Conclusion: Cannot query mll cluster in KBase pangenome.

1.4 Check EggNOG functional annotations

-- Query: Get functional annotations for available genes
SELECT query_name, Description, Preferred_name, COG_category
FROM kbase_ke_pangenome.eggnog_mapper_annotations
WHERE query_name LIKE 'NC_012808%'  -- chromosome
LIMIT 20

Result: Returns functional annotations with COG categories, gene names, etc. But megaplasmid genes are not present.

---

2. JGI GOLD Database

2.1 Find organism via NCBI Assembly

-- Query: Find M. extorquens AM1 in GOLD via assembly accession
SELECT
    assembly_accession,
    taxid,
    organism_name,
    biosample,
    refseq_category
FROM "gold-db-2 postgresql".gold.ncbi_assembly
WHERE assembly_accession = 'GCF_000018845.1'
   OR organism_name LIKE '%Methylorubrum extorquens AM1%'

Join point: - assembly_accession: GCF_000018845.1 ← matches KBase genome_id (after stripping RS_) - taxid: Links to ncbi_taxonomy table - biosample: Links to sample metadata

2.2 Get taxonomy lineage

-- Query: Full taxonomy for the organism
SELECT
    ncbi_tax_id,
    scientific_name,
    phylum,
    class,
    "order",
    family,
    genus,
    species
FROM "gold-db-2 postgresql".gold.ncbi_taxonomy
WHERE ncbi_tax_id = 272630  -- M. extorquens AM1

---

3. JGI IMG Core Database

3.1 Find taxon_oid for organism

-- Query: Get IMG taxon identifier
SELECT
    taxon_oid,
    taxon_display_name,
    ncbi_taxon_id,
    genome_type
FROM "img-db-2 postgresql".img_core_v400.taxon
WHERE ncbi_taxon_id = 272630
   OR taxon_display_name LIKE '%Methylorubrum extorquens AM1%'

Join point: - taxon_oid: IMG internal identifier → links to all IMG gene tables - ncbi_taxon_id: 272630 → links to GOLD/NCBI

3.2 Query biosynthetic gene clusters

-- Query: Find BCG regions for this organism
SELECT
    region_id,
    taxon_oid,
    start_coord,
    end_coord,
    bcg_type,
    scaffold_oid,
    bcg_method
FROM "img-db-2 postgresql".img_mysql_abc.bcg_region
WHERE taxon_oid = (
    SELECT taxon_oid FROM img_core_v400.taxon
    WHERE ncbi_taxon_id = 272630
)

3.3 Get genes in BCG regions

-- Query: Get genes within identified BCG regions
SELECT
    rg.region_id,
    rg.gene_oid,
    rg.bcg_gene_type,
    rg.gene_functions,
    g.locus_tag,
    g.gene_display_name
FROM "img-db-2 postgresql".img_mysql_abc.bcg_region_genes rg
JOIN "img-db-2 postgresql".img_core_v400.gene g
    ON rg.gene_oid = g.gene_oid
WHERE rg.region_id IN (
    -- BCG region IDs from previous query
)

3.4 Get scaffold/contig mapping

-- Query: Map scaffold_oid to NCBI accessions
SELECT
    scaffold_oid,
    scaffold_name,
    ext_accession,
    taxon_oid
FROM "img-db-2 postgresql".img_core_v400.scaffold
WHERE taxon_oid = (
    SELECT taxon_oid FROM img_core_v400.taxon
    WHERE ncbi_taxon_id = 272630
)

Join point: - scaffold_name or ext_accession may contain NC_012811 (megaplasmid accession) - Links IMG scaffold_oid → NCBI nucleotide accession

3.5 IMG Functional Annotations for mll Cluster

Using the taxon_oid (644736386) and locus tag pattern MexAM1_META1p*:

-- Query: Get gene product names for mll cluster
SELECT gene_oid, locus_tag, product_name
FROM "img-db-2 postgresql".img_core_v400.gene
WHERE taxon = 644736386
  AND locus_tag LIKE 'MexAM1_META1p413%'
  OR locus_tag = 'MexAM1_META1p4129'
ORDER BY locus_tag

Result - Product Names:

gene_oid	locus_tag	product_name
644814096	MexAM1_META1p4129	iron complex outermembrane recepter protein
644814097	MexAM1_META1p4130	anti-sigma-factor antagonist
644814098	MexAM1_META1p4131	RNA polymerase sigma factor
644814099	MexAM1_META1p4132	spermidine-citrate ligase
644814100	MexAM1_META1p4133	3,4-dihydroxybenzoyl-citryl-spermidine/N-citryl-spermidine--spermidine ligase
644814101	MexAM1_META1p4134	3,4-dihydroxybenzoyl-citryl-spermidine/N-citryl-spermidine--spermidine ligase
644814102	MexAM1_META1p4135	isochorismatase family protein
644814103	MexAM1_META1p4136	hypothetical protein
644814104	MexAM1_META1p4137	hypothetical protein
644814105	MexAM1_META1p4138	MMPL family transporter

Note: IMG uses locus tag format MexAM1_META1p4129 vs NCBI's META1p4129.

-- Query: Get COG annotations for mll cluster genes
SELECT g.locus_tag, gc.cog
FROM "img-db-2 postgresql".img_core_v400.gene g
JOIN "img-db-2 postgresql".img_core_v400.gene_cog_groups gc ON g.gene_oid = gc.gene_oid
WHERE g.taxon = 644736386
  AND (g.locus_tag LIKE 'MexAM1_META1p413%' OR g.locus_tag = 'MexAM1_META1p4129')
ORDER BY g.locus_tag

Result - COG Annotations:

locus_tag	cog	Description
MexAM1_META1p4129	COG4774	Outer membrane receptor for ferric coprogen and ferric-rhodotorulic acid
MexAM1_META1p4130	COG3712	Anti-sigma factor antagonist
MexAM1_META1p4131	COG1595	DNA-directed RNA polymerase sigma subunit
MexAM1_META1p4132	COG4264	IucA/IucC family siderophore biosynthesis
MexAM1_META1p4133	COG4264	IucA/IucC family siderophore biosynthesis
MexAM1_META1p4134	COG4264	IucA/IucC family siderophore biosynthesis
MexAM1_META1p4135	COG1535	Isochorismatase

-- Query: Get PFAM annotations for mll cluster genes
SELECT g.locus_tag, gp.pfam_family
FROM "img-db-2 postgresql".img_core_v400.gene g
JOIN "img-db-2 postgresql".img_core_v400.gene_pfam_families gp ON g.gene_oid = gp.gene_oid
WHERE g.taxon = 644736386
  AND (g.locus_tag LIKE 'MexAM1_META1p413%' OR g.locus_tag = 'MexAM1_META1p4129')
ORDER BY g.locus_tag, gp.pfam_family

Result - PFAM Annotations:

locus_tag	pfam_family	Description
MexAM1_META1p4129	pfam00593	TonB-dependent receptor
MexAM1_META1p4129	pfam07715	TonB-dependent receptor plug domain
MexAM1_META1p4132	pfam04183	IucA/IucC family (siderophore synthetase)
MexAM1_META1p4132	pfam06276	Ferric iron reductase FhuF-like transporter
MexAM1_META1p4133	pfam00501	AMP-binding enzyme
MexAM1_META1p4133	pfam04183	IucA/IucC family
MexAM1_META1p4133	pfam06276	Ferric iron reductase FhuF-like transporter
MexAM1_META1p4133	pfam13193	AMP-binding enzyme C-terminal domain
MexAM1_META1p4135	pfam01261	Xylose isomerase-like TIM barrel
MexAM1_META1p4138	pfam13628	Predicted permease

Key finding: The pfam04183 (IucA/IucC) domain is characteristic of siderophore biosynthesis enzymes. This domain is present in mllA (META1p4132) and mllBC (META1p4133), confirming their role in metallophore biosynthesis as described in Zytnick et al. 2024.

---

4. MIBiG Database

4.1 Search for organism BGCs

URL: https://mibig.secondarymetabolites.org/repository

Search: "Methylorubrum extorquens AM1"

Result: - BGC0001991 - Toblerol cluster (different BGC!) - ❌ mll cluster is NOT in MIBiG

BGC0001991 (Toblerol) details: - Accession: NC_012811.1 (same megaplasmid as mll) - Coordinates: 7,676–32,884 bp - Genes: MEXAM1_RS33125 through MEXAM1_RS25440

Note: The toblerol cluster uses different locus tag format (MEXAM1_RS) vs mll cluster (META1p).

---

5. NCBI Direct Access

5.1 Nucleotide database

# Fetch megaplasmid sequence info
curl "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?\
db=nuccore&id=NC_012811.1&rettype=gb&retmode=text" | head -100

5.2 Gene database

# Search for mll genes by locus tag
curl "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/esearch.fcgi?\
db=gene&term=META1p4129[locus_tag]+OR+META1p4132[locus_tag]"

---

Join Points Summary

┌─────────────────────────────────────────────────────────────────────┐
│                         IDENTIFIER MAPPING                          │
├─────────────────────────────────────────────────────────────────────┤
│                                                                     │
│  KBase Pangenome                    JGI GOLD                        │
│  ════════════════                   ════════                        │
│  genome_id: RS_GCF_000018845.1  ──► assembly_accession: GCF_000018845.1
│  ncbi_biosample_id: SAMN00000030 ─► biosample: SAMN00000030         │
│  gene_id: NC_010172.1_123       ──► (nucleotide accession)          │
│                                                                     │
│  JGI GOLD                           JGI IMG                         │
│  ════════                           ═══════                         │
│  taxid: 272630                  ──► ncbi_taxon_id: 272630           │
│  assembly_accession             ──► (via scaffold.ext_accession)    │
│                                                                     │
│  JGI IMG                            NCBI                            │
│  ═══════                            ════                            │
│  taxon_oid: 644736386           ──► ncbi_taxon_id: 272630           │
│  scaffold.ext_accession         ──► NC_012811.1 (megaplasmid)       │
│  gene.locus_tag: MexAM1_META1p* ──► META1p4129 (mll genes)          │
│  gene_oid: 644814096-644814105  ──► mll cluster genes               │
│                                                                     │
│  MIBiG                              NCBI                            │
│  ═════                              ════                            │
│  Accession: NC_012811.1         ──► Nucleotide accession            │
│  Locus tags: MEXAM1_RS*         ──► RefSeq locus tag format         │
│                                                                     │
└─────────────────────────────────────────────────────────────────────┘

---

Data Availability Matrix

Data Element	KBase	GOLD	IMG	MIBiG	NCBI
Genome assembly	✅	✅	✅	-	✅
Taxonomy	✅ (GTDB)	✅	✅	-	✅
Chromosome genes	❌	-	✅	-	✅
Megaplasmid genes	❌	-	✅	✅	✅
mll cluster	❌	-	✅	❌	✅
Toblerol cluster	❌	-	✅	✅	✅
COG annotations	⚠️ partial	-	✅	-	-
PFAM annotations	❌	-	✅	-	-
Functional annotations	⚠️ partial	-	✅	✅	✅
BioSample metadata	✅	✅	-	-	✅

---

Recommended Query Strategy

To get comprehensive mll cluster data:

Step 1: Start with NCBI (authoritative source)

# Get megaplasmid GenBank record with all gene annotations
efetch -db nuccore -id NC_012811.1 -format gbwithparts > NC_012811.gbk

Step 2: Query IMG for functional annotations

-- Get IMG taxon_oid first
SELECT taxon_oid FROM img_core_v400.taxon WHERE ncbi_taxon_id = 272630;

-- Then get all genes on megaplasmid scaffold
SELECT g.gene_oid, g.locus_tag, g.product_name, g.cog_id, g.pfam_id
FROM img_core_v400.gene g
JOIN img_core_v400.scaffold s ON g.scaffold = s.scaffold_oid
WHERE s.ext_accession = 'NC_012811.1'
  AND g.start_coord BETWEEN 4500000 AND 4520000  -- approximate mll region

Step 3: Cross-reference with KBase (for pangenome context)

-- Get species-level pangenome statistics
SELECT * FROM kbase_ke_pangenome.pangenome
WHERE gtdb_species_clade_id LIKE '%Methylobacterium_extorquens%'

-- Get ortholog clusters for comparison (note: mll genes not available)
SELECT gc.gene_cluster_id, gc.is_core, gc.is_auxiliary
FROM kbase_ke_pangenome.gene_cluster gc
WHERE gc.gtdb_species_clade_id LIKE '%Methylobacterium_extorquens%'

---

Lessons Learned

1. KBase pangenome has gaps: Not all replicons (chromosomes, plasmids, megaplasmids) are included for every genome. Always verify which sequences are present.

2. Locus tag formats vary across databases:

3. NCBI original: META1p4129
4. NCBI RefSeq: MEXAM1_RS25400 (used in MIBiG)
5. IMG format: MexAM1_META1p4129 (genome prefix + original tag)

6. All refer to same genes but require format-aware matching

7. MIBiG is curated but incomplete: Has toblerol cluster but not mll cluster, despite both being characterized in the literature.

8. IMG is most comprehensive for JGI organisms: Has complete functional annotations (COG, PFAM), BCG predictions, and scaffold-level mappings. The pfam04183 (IucA/IucC) domain is diagnostic for siderophore biosynthesis.

9. Assembly accession is best join key: GCF_000018845.1 works across KBase (with RS_ prefix), GOLD, and NCBI.

10. IMG taxon_oid enables rich queries: Once you have the taxon_oid (644736386 for M. extorquens AM1), you can efficiently join to gene, scaffold, and functional annotation tables.

---

References

• Zytnick AM, et al. (2024) "Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism" PNAS 121(32):e2322096121. PMC11317620

• Ueoka R, et al. (2018) "Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms" Angew Chem Int Ed PMID:29112783 MIBiG BGC0001991

• NCBI Reference Sequences:

• NC_012808.1 - Chromosome
• NC_012811.1 - Megaplasmid (contains mll)
• NC_010172.1 - Plasmid pMETA1