This study functionally characterise the import system of cyanochelin B, a photolytic β-hydroxy aspartate siderophore produced by several filamentous cyanobacteria, and reports the first functional heterologous expression of a cyanobacterial TonB-dependent transporter and establishes Synechocystis as a promising platform for cyanobacterial xenosiderophore-uptake studies.
Abstract
Siderophore transport is central to microbial competition, because it determines access to iron, frequently a limiting nutrient. While siderophore-mediated iron uptake via TonB-dependent transporters (TBDTs) has been extensively studied in heterotrophic bacteria, little is known about the functionality and specificity of TBDTs in cyanobacteria. In the present study we functionally characterise the import system of cyanochelin B, a photolytic β-hydroxy aspartate siderophore produced by several filamentous cyanobacteria, including Leptolyngbya sp. NIES-3755. We have identified a cyanochelin B putative transport cassete localized in the vicinity of the cyanochelin biosynthetic gene cluster in Leptolyngbya genome. By expressing the import genes heterologously in a model unicellular cyanobacterium Synechocystis sp. PCC 6803, we established that the transport cassette reconstitutes cyanochelin B-dependent growth, consistent with cyanochelin-mediated iron acquisition. Systematic gene dissection showed that the TBDT (CctA) and the substrate-binding protein (CctB), responsible for binding the siderophore in the periplasm, are alone sufficient for cyanochelin import into Synechocystis cells, with the permeases, ATPase and a cassette-associated ferredoxin supplied in trans by the host. CctA carries an N-terminal AMIN domain, a fusion found only in cyanobacterial TBDTs. The cassette accepts the structurally similar cyanochelin A but not cyanochelin C, enterobactin or pyoverdine, indicating limited promiscuity. The phylogenetic placement of cyanochelin receptors within a broader clade containing citrate-hydroxamate-type siderophore receptors suggests an evolutionary link between transport systems for chemically distinct cyanobacterial siderophores. Our study reports the first functional heterologous expression of a cyanobacterial TonB-dependent transporter and establishes Synechocystis as a promising platform for cyanobacterial xenosiderophore-uptake studies.
Cyanobacteria are key prokaryotic primary producers in diverse ecosystems, yet the role of cyanobacterial siderophores in shaping their associated microbiomes remains unexplored. Our study demonstrates the benefits provided to the heterotrophic co-habitants of filamentous cyanobacteria in terrestrial microbial biofilms, focusing on the recently discovered widespread siderophores cyanochelins. To address the acceptance of cyanochelin B (CychB) across multiple bacterial classes, we first investigated its role in providing iron to a model siderophore producer P. aeruginosa PAO1 and selected Pseudomonas natural isolates, which were found to utilize CychB under iron limiting conditions while downregulating endogenous siderophore production. In response to CychB, PAO1 expresses a siderophore internalization cluster, which is localized in multiple Pseudomonas natural isolates. Using metagenome analysis, we characterized the bacterial community recruited along with CychB producing Phormidesmis cyanobacteria under long-term iron starvation. Potential CychB acceptor bacteria associated with the CychB producer were predominantly lacking endogenous siderophore machineries. Using siderophore selective pressure, we isolated a genuine CychB acceptor, gram-negative bacterium Methyloversatilis sp. S146 and demonstrated that its genome hosts an iron processing cluster overexpressed after CychB feeding, recognizing Methyloversatilis as a candidate for further mechanistic investigation of iron acquisition–driven microbial interactions. Our results indicate that CychB supports a specific subset of co-habiting heterotrophic bacteria during iron starvation, further emphasizing the role of cyanobacteria as key drivers of nutrient flows within globally important microbial soil crust ecosystems, supporting microbial life in nutrient-limited environments. These findings provide a mechanistic foundation to elucidate the role of cyanochelins as a public good in these communities.
B. P. Falcao, Martinez Yerena Jose Alberto, T. Galica et al.· bioRxiv· 0 citations
Iron is a key micronutrient that constrains microbial growth and productivity in many aquatic and terrestrial environments due to its limited bioavailability. Microorganisms evolved sophisticated acquisition strategies, including the production of siderophores, high-affinity iron-chelating molecules that facilitate iron solubilisation and uptake. Cyanobacteria, photosynthetic prokaryotes and major contributors to global primary production, also depend on iron as a cofactor to their core metabolic enzymes. However, very few cyanobacterial siderophores were described so far, and cyanobacteria remain an underxplored source of possibly novel siderophores. Here we report a novel cyanobacterial siderophore, cyanochelin C, that employs two β-hydroxyaspartate residues for iron chelation. We provide extensive nuclear magnetic resonance (NMR) and mass spectrometry (MS) evidence on the molecular structure and identify the corresponding biosynthetic gene cluster (BGC). Bioinformatic analysis of the BGC further revealed the presence of an acylase CcsQ clustering with a broader cyanobacteria-specific family of acylases associated with predicted siderophore-encoding BGCs. Discovery of cyanochelin C and its deacylation by CcsQ expands the known structural diversity of cyanobacterial siderophores and improves the understanding of important enzymatic reactions.
Viviana Di Matteo, L. Štenclová, B. P. Falcao et al.· bioRxiv· 0 citations
With 21 outer-membrane transporters dedicated to iron acquisition, Pseudomonas aeruginosa possesses an extensive toolkit for securing this essential nutrient, but activates the expression only of those relevant to its immediate environment. Eighteen of these pathways rely on siderophores, two produced by P. aeruginosa and the others acquired from neighboring microorganisms through a siderophore-piracy strategy. Using fluorescent reporters and mathematical modeling, we quantified how catechol-type siderophores regulate transcription of their matched transporters, PfeA, FvbA, PirA, and PiuA. pfeA and fvbA showed switch-like induction by enterobactin and vibriobactin, starting at around 100 nM and plateauing at 1 µM. In contrast, N-(2,3-dihydroxybenzoyl)-L-serine, an enterobactin degradation product, selectively induced piuA transcription only in strains lacking endogenous siderophores, via a gradual response spanning 250 nM to 100 µM. pirA transcription was strongly induced by host catecholamines, with activation thresholds of 1 µM for epinephrine, 5 µM for norepinephrine, and 10 µM for L-DOPA, and plateaus between 30 and 150 µM. Iron repression uncovered distinct regulatory regimes: pfeA, fvbA, and pirA display biphasic Fur-dependent repression, with transcription decreasing beginning at 4 to 16 nM Fe and fully repressed by 32 µM, whereas piuA transcription began to decrease at 8 µM iron and was not fully repressed even at 2 mM. Together, these results reveal distinct transcriptional logics that allow P. aeruginosa to sense and decode a wide array of environmental catechols, integrate this information with iron availability, and thereby deploy an iron-uptake strategy finely tuned to competitive and host-associated niches.
Florian Volck, Anne-Eloïse Revillot-Schmidt, Thibaut Hubert et al.· Proceedings of the National...· 0 citations
Plants acquire iron using two canonical mechanisms: reduction-based uptake (Strategy I) and chelation-based uptake (Strategy II). Although Strategy II is characteristic of grasses, genes associated with chelated-metal transport occur broadly across plant lineages, obscuring how this pathway evolved. Here, we reconstruct the evolutionary history of Yellow Stripe-Like (YSL) transporters and Nicotianamine Synthase (NAS) across Archaeplastida and their closest non-plant homologs. Phylogenomic, distributional, and structural analyses reveal pronounced temporal uncoupling between these gene families. YSL most likely originated early in Viridiplantae and represents the deepest evolutionary module of the chelated-metal transport system. In contrast, NAS appeared much later through independent horizontal gene transfer events from fungi into euphyllophytes and specific moss lineages. This timing indicates that YSL-mediated transport initially functioned independently of nicotianamine, implying ancestral use of alternative siderophores. A pronounced expansion of YSL genes in Poaceae coincides with the emergence of canonical Strategy II, suggesting that this pathway arose by exaptation of pre-existing chelated-metal transport mechanisms.
Matheus L. C. B. de Campos, W. Rodrigues, Helena F. Gomes et al.· bioRxiv· 0 citations
The widespread oceanic cyanobacterial Prochlorococcus genus is a major contributor to global carbon fixation, yet mechanisms enabling this lineage to elevate intracellular inorganic carbon as a substrate for photosynthesis remain unresolved. Cyanobacterial CO2-concentrating mechanisms typically rely on membrane-bound bicarbonate (HCO3-) transporters SbtA1, SbtA2, BicA and BCT1, and CO2-to-HCO3- conversion uptake systems (CO2 pumps; NDH-I3 and NDH-I4), to elevate a cellular HCO3- pool for use by Rubisco-containing carboxysomes. Evidence suggests Prochlorococcus harbours carboxysomes with a low-CO2-specificity Rubisco, implying a functional CCM dependent on active HCO3- uptake. However, canonical CO2 pumps are absent, leaving distant HCO3- transporter homologues, BicA2 and SbtA2, as prime candidates for HCO3- transport in this group. Yet these have not been functionally characterised. Here we demonstrate that BicA2 from P. marinus CCMP1375 mediates Na+-dependent HCO3- uptake in E. coli, whereas BicA2 from P. marinus CCMP1986 is inactive in its native form but acquired transport function through a single amino acid substitution during adaptive laboratory evolution. These findings confirm BicA2 as a low-affinity, Na+-dependent bicarbonate transporter with variable flux, revealing a previously uncharacterized CCM component in Prochlorococcus. This mechanistic insight reshapes our understanding of carbon acquisition strategies in the most abundant photosynthetic organism on Earth and highlights evolutionary plasticity in transporter function with implications for global biogeochemical cycles.
Loraine M. Rourke, C. Byrt, G. D. Price et al.· The ISME Journal· 0 citations