Sequencing Pseudomonas putida, Predicting Pyoverdine Structure

bio
I sequenced the genome of the pyoverdine-producing Pseudomonas from a previous post, and had some fun digging into the genes to figure out what molecule exactly it makes
Published

January 22, 2026

In my previous post I showed a bacteria isolated from the water around the roots of a Jade plant on our windowsill, which secreted something that fluoresced a lovely blue under UV light. My best guess was that it was some species of Pseudomonas, and the blue was from pyoverdine. But how can we know for sure? Well, I put it off because money, but since I like working with this bacteria and want to do more with it, I figured I’d bite the bullet and pay the ~$120 to get my answers :) It turns out that the closest match is P. putida, and that the pyoverdine variant that this makes is probably different to anything documented in the literature. Exciting stuff! Update: found a likely match in the literature - see end of post :)

Sequencing with Plasmidsaurus

I went with Plasmidsaurus’ ‘Standard Bacterial Genome Sequencing with Extraction’ service. I sent the sample (~15mg cells suspended in Zymo DNA Shield) off on Tuesday morning and by Wednesday evening the results were ready.

Species ID (Mash)

  • Best match: Pseudomonas putida NBRC 14164
  • Identity: 95.6% (1946/5000 shared hashes)

Genome Quality (CheckM)

  • Completeness: 99.88% — excellent!
  • Contamination: 2.15% — very low
  • Lineage marker: Pseudomonas

Assembly Stats

  • Genome size: 6.54 Mb (typical for Pseudomonas)
  • Total reads: 66,830 (398 Mb total)
  • Estimated coverage: ~58x
  • Longest read: 88.4 kb
  • Read N50: 9.7 kb

They give you lots of data, including annotating the genome for you with bakta. Great stuff! We can see right away that our guess was right - this is a Pseudomonas species (P. putida is the closest match) and are ready to dig in further to see what we can learn.

Pyoverdine Investigation

Pyoverdines vary but all come with three key parts: a dihydroxyquinoline core, a peptide chain, and a side chain. The peptide chain especially varies strain-to-strain - see here for some examples.

Step one was looking for the pyoverdine Biosynthetic Gene Cluster (BGC). A Deep Research run gave some promising genes to start the hunt with. Some hits let us narrow in on a key region between 4.71 and 4.76M bp, with a number of promising-looking genes. A tool called antiSMASH likewise identified a region (4,696,705–4,796,912), labeled as ‘NRP-metallophore’ - this is our BGC alright :)

By looking at the A-domain predictions for this region, we can start on a predicted peptide sequence: Glu-D-Tyr-Dab─Asp─Ala─Asp─D-OHOrn─X─OHOrn (where Glu-D-Tyr-Dab forms the conserved pyoverdine chromophore after cyclization). That X is an unknown, which we’ll have to work on later - my best guess is Gly.

The next step is to figure out which side chain is present. (Some strains make some of both). The side-chain type is determined by which gene is present:

  • pvdN (PLP-dependent aminotransferase) → succinamide side chain
  • ptaA (periplasmic transaminase) → α-ketoglutarate side chain

We BLAST the genome against known reference sequences, finding an 83% match to PvdN (NP_251095.1) but only a 23% match for PtaA (AAY94407.1) -> strain produces succinamide side chain.

This info gives us enough info to reasonably guess at the structure of the pyoverdine precursor (ferribactin):

🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠🧠

From Genome to Siderophore: Characterizing a Novel Pyoverdine from Pseudomonas putida

The Starting Point

We began with a high-quality assembled genome from a Pseudomonas putida strain isolated from a houseplant root system (sequenced by Plasmidsaurus). The assembly was excellent: 99.88% complete, only 2.15% contamination, and a 6.54 Mb genome at ~58× coverage. Species identity was confirmed via Mash (95.6% to P. putida NBRC 14164).

Finding the Pyoverdine Biosynthetic Gene Cluster

We searched the Bakta annotations for pyoverdine-related genes (pvd genes, NRPS, siderophore terms) and identified a cluster spanning ~4.7–4.8 Mb containing:

  • pvdT — pyoverdine exporter
  • Two large NRPS genes (~14.7 kb and ~6.4 kb) — the peptide assembly machinery
  • PvdA-like monooxygenase — hydroxylates ornithine
  • Formylglycine-generating enzyme (likely pvdF) — formyltransferase
  • argD — makes diaminobutyrate (Dab), essential for the chromophore
  • TonB-dependent receptor — likely the ferripyoverdine receptor (fpvA)

Notably, pvdA was located separately at ~3.99 Mb — this split arrangement is common in pyoverdine BGCs. (Note from Johno: There’s a PvdA-like hydroxylase in the main cluster too, so this might not be a case of a split arrangement after all? TBD if one or both are used, and how they’re regulated.)

antiSMASH Analysis

Running the genome through antiSMASH confirmed Region 10 as an NRP-metallophore cluster (siderophore NRPS). The top KnownClusterBlast hit was Pf-5 pyoverdine from Pseudomonas protegens, but with only ~49% NRPS identity — indicating a related but distinct peptide.

Predicting the Peptide Sequence

antiSMASH provided A-domain substrate predictions for each NRPS module:

Module Substrate Configuration
1 Glu L
2 Tyr D (E-domain present)
3 Dab L
4 Asp L
5 Ala L
6 Asp L
7 OH-Orn D (E-domain present)
8 ? —
9 OH-Orn L

Resolving the Unknown Residue

We explored two hypotheses:

  1. Lysine — has a terminal -NH₂ on its side chain
  2. Glycine — essentially just an α-amino group with no real side chain

After deeper analysis (including literature review of other P. putida pyoverdines), glycine emerged as the more likely candidate: - The “NH₂” prediction could mean “minimal/no side chain” - Glycine is common in mid-chain positions of P. putida pyoverdines (KT2440, GB-1 both contain Gly) - Prediction tools are known to have difficulty with Gly vs Ala

(Note fro Johno: Opus was sure an unrelated minowa NH2 mention was related to this and predicted lysine as a result, I tried to determine the unknown residue but had no luck, Gly is a pretty good guess AFAICT but will need analysis to confirm).

Determining the Side Chain: pvdN vs ptaA

Pyoverdine side chains come in two main types: - Succinamide — produced by strains with pvdN - α-Ketoglutarate — produced by strains with ptaA

We BLASTed known pvdN (P. aeruginosa PAO1, NP_251095.1) and ptaA sequences against the genome:

  • pvdN: 83% identity match → gene LDMLEE_03614 at ~3.99 Mb (near pvdA)
  • ptaA: No significant match

Conclusion: Succinamide side chain

The Final Predicted Structure

CHROMOPHORE PRECURSORS          VARIABLE PEPTIDE
        ↓                              ↓
┌───────────────────┐  ┌─────────────────────────────────────┐
 L-Glu → D-Tyr → L-Dab → L-Asp → L-Ala → L-Asp → D-OHOrn → Gly → L-OHOrn
                  ↓                                    ↓            ↓
           cyclizes to form                      Fe³⁺ binding  Fe³⁺ binding
        dihydroxyquinoline                       (hydroxamate) (hydroxamate)
           chromophore

Key features: - 9-residue peptide (longer than typical P. putida pyoverdines) - Two hydroxyornithine residues — provides two hydroxamate groups for Fe³⁺ chelation - Succinamide side chain on the chromophore - Novel sequence — no match in SIDERITE database (best similarity only 0.35)

SMILES (with stereochemistry)

NN[C@@H](CCC(=O)O)C(=O)N[C@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCN)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@H](CCCNO)C(=O)NCC(=O)N[C@@H](CCCNO)C(=O)O

(Note from Johno: Should probably be a single N at the start there)

What Still Needs Confirmation

This is a bioinformatic prediction. To definitively confirm the structure:

  1. LC-MS/MS — will reveal the exact mass and fragment pattern, confirming each residue (especially Gly at position 8)
  2. The hydroxamates — could be N-formylated (if pvdF is active), which would add +28 Da per residue
  3. Cyclization — the C-terminal OHOrn likely cyclizes with the chromophore, which MS/MS should reveal

Why This Matters

This appears to be a genuinely novel pyoverdine variant — it doesn’t match any characterized P. putida pyoverdines in the literature or databases. Discovering the diversity of siderophores in environmental isolates helps us understand:

  • Microbial competition for iron in the rhizosphere
  • The evolution of NRPS systems
  • Potential for novel iron chelators with biotechnology applications

END AI SLOP

Here’s a notebook running over the steps, with some light annotation. (I’ve since been fiddling a bit more, LMK if you’re wanting to dive into this deeply)

Future Plans

It’s one thing to poke at a genome and predict a structure, it’s another entirely to verify it. I’m hoping to work with a local university to run LC-MS on this to answer a few remaining questions and nail down the final structure exactly. Will require dusting off and improving my organic chemistry and learning a lot more! Stay tuned for that in some future blog post hopefully :)

Also, it’s probably obvious but worth stating explicityl: I AM OUT OF MY DEPTH HERE AND EVERYTHING IN THIS POST SHOULD BE TAKEN WITH A GRAIN OF SALT, ESPECIALLY WHILE IT IS MARKED ‘DRAFT’ :)

For the curious, raw data is on Google Drive, I’m open to questions @johnowhitaker. This solveit dialog has the key code in a nice rendered form.

Update: A Potential Match

In A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies BioMetals, 2013 I found the following: P. putida BTP1/90-40 Asp–Ala–Asp–AOHOrn–Ser–cOHOrn (citing Jacques et al. (1995)). This is pretty much spot on for ours, and would mean the mystery X is Ser (serine), not Gly as guessed. It also answers a mystery - I’d been searching fruitlessly for pvdF in my genome to figure out if OHOrn gets formylated, this would indicate it doesn’t in this case.

This is a great find! Let’s compare:

Literature (P. putida BTP1/90-40): Asp–Ala–Asp–AOHOrn–Ser–cOHOrn

Our prediction: Asp–Ala–Asp–D-OHOrn–X–OHOrn

That’s nearly identical! The key insight: X = Ser (serine), not Gly as guessed.

The nomenclature:

AOHOrn = “amide-linked” hydroxyornithine (connected via α-amino group) cOHOrn = “cyclic” hydroxyornithine (C-terminal, forms lactam ring with its own side chain) This is a known pyoverdine type! Your strain likely produces a pyoverdine very similar or identical to the BTP1/90-40 type.

Why the A-domain gave “X”: Ser can be tricky to predict – its small hydroxymethyl side chain sometimes doesn’t give strong specificity signatures. Much more plausible than Gly given this literature match.

Updated peptide structure:

Copied! Chromophore─Glu─D-Tyr─Dab─Asp─Ala─Asp─D-OHOrn─Ser─cOHOrn This also answers the pvdF question – if the literature strain has OHOrn (not fOHOrn/formyl-hydroxyornithine), then absence of pvdF is consistent.

PS: Molecule viewer test

Here’s the predicted ferrobactin precursor molecule, copied from the output of this code:

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