Structural modelling uncovers diverse predicted transcriptional and post-transcriptional modulators among type III effectors of symbiotic Rhizobia

A conserved partner-switching system controls terminal differentiation in multicellular cyanobacteria

ABSTRACT Canonical partner-switching systems (PSSs) regulate sigma factor activity through reversible phosphorylation, but their established roles have been largely limited to stress responses and sporulation in Firmicutes. Whether this regulatory mechanism also controls developmental cell fate decisions in other bacterial phyla has remained unknown. Here, we identify a canonical PSS that governs heterocyst differentiation in the multicellular cyanobacterium Anabaena sp. PCC 7120. This system comprises the anti-sigma factor All2284 (NfsS) and the anti-anti-sigma factor All2283 (NfsR). Structural predictions and biochemical assays showed that NfsS phosphorylates NfsR on a conserved serine residue, whereas bacterial two-hybrid and co-purification assays demonstrated that NfsS binds the developmental sigma factors SigC and SigE. Deletion of nfsR abolished heterocyst formation and diazotrophic growth, and transcriptomic analysis revealed broad failure to induce late heterocyst genes, including nitrogen fixation functions such as nifHDK and fdxH. Phylum-wide comparative genomics further showed that PSS genes and putative functional clusters are strongly enriched in filamentous and heterocyst-forming taxa, indicating an association between the expansion of these signaling modules and the emergence of multicellularity and developmental specialization. Together, these findings establish a PSS as a direct regulator of terminal cell differentiation in a gram-negative bacterium and reveal partner switching as a conserved regulatory principle linking environmental signaling to developmental fate in a major bacterial phylum. IMPORTANCE While partner-switching systems are classically associated with stress responses and sporulation control in Firmicutes, whether this regulatory logic governs developmental decisions in other bacterial phyla has remained unknown. Here, we establish that a related partner-switching mechanism operates in a distinct bacterial lineage, the cyanobacteria, where it controls a major developmental transition involving terminal cell differentiation. Specifically, we show that a phosphorylation-dependent checkpoint involving the anti-sigma factor NfsS and the anti-anti-sigma factor NfsR directly regulates heterocyst formation. Disruption of this switch abolishes cell differentiation and diazotrophic growth, revealing that this system is an obligate gatekeeper for terminal differentiation. Conceptually, these findings substantially extend the known functional repertoire of partner-switching circuits: rather than controlling stress adaptation or spore dormancy, this module has been co-opted to govern a complex, multicellular developmental program in an organism that underpins global carbon and nitrogen cycles. This work, therefore, establishes a new paradigm for phosphorylation-based control of developmental sigma factors and provides a tractable model for dissecting how conserved signaling modules are rewired to drive lineage-specific innovations across the bacterial domain. While partner-switching systems are classically associated with stress responses and sporulation control in Firmicutes, whether this regulatory logic governs developmental decisions in other bacterial phyla has remained unknown. Here, we establish that a related partner-switching mechanism operates in a distinct bacterial lineage, the cyanobacteria, where it controls a major developmental transition involving terminal cell differentiation. Specifically, we show that a phosphorylation-dependent checkpoint involving the anti-sigma factor NfsS and the anti-anti-sigma factor NfsR directly regulates heterocyst formation. Disruption of this switch abolishes cell differentiation and diazotrophic growth, revealing that this system is an obligate gatekeeper for terminal differentiation. Conceptually, these findings substantially extend the known functional repertoire of partner-switching circuits: rather than controlling stress adaptation or spore dormancy, this module has been co-opted to govern a complex, multicellular developmental program in an organism that underpins global carbon and nitrogen cycles. This work, therefore, establishes a new paradigm for phosphorylation-based control of developmental sigma factors and provides a tractable model for dissecting how conserved signaling modules are rewired to drive lineage-specific innovations across the bacterial domain.

A rhizobial extracellular vesicle-conveyed auxin transporter mediates phytohormone mobilization and optimizes rhizobium-legume symbiosis

Bacterial extracellular vesicles (bEVs) are emerging as key players in interkingdom communication, yet their role in delivering functional proteins to host cells during symbiosis remains unexplored. This study shows that Sinorhizobium fredii HH103 packages a PIN-like auxin transporter, AuxT, into bEVs that traffic within the peribacteroid space of soybean nodules. AuxT is chromosomally encoded and constitutively expressed, genetically uncoupled from the flavonoid-inducible auxin biosynthesis machinery located on the symbiotic plasmid. Structural prediction reveals that AuxT adopts an eight-transmembrane-helix architecture with striking homology to plant PIN auxin exporters, despite negligible sequence identity. Molecular docking demonstrates that AuxT binds indole-3-acetic acid within a central cavity, with dimerization inducing ligand-specific conformational changes consistent with transport activity. The auxT mutant exhibits significant symbiotic defects including reduced shoot biomass, nodule number, and nodule mass that are fully restored by complementation. Critically, AuxT-enriched bEVs contain elevated auxin levels, and nodules colonized by the complemented strain accumulate more auxin specifically within the bEVs peribacteroid space, while bacteroids themselves show no auxin retention. We postulate that bEV-associated AuxT mediates localized auxin export into the symbiosome, modulating the host hormonal environment to optimize symbiotic development. This work reveals a previously unrecognized mechanism of interdomain hormonal modulation, where a bacterium uses a structurally convergent transporter and vesicular delivery to actively shape host physiology and to improve the symbiotic performance.

Functional analysis of natural variation in the RNA-binding protein CsrA across the bacterial domain predicts regulatory activity

Abstract Bacteria employ sophisticated post-transcriptional regulatory mechanisms to adapt to environmental changes. Carbon storage regulator A (CsrA), a highly conserved RNA-binding protein, serves as a critical post-transcriptional regulator by typically recognizing GGA-containing hairpin loops in target mRNAs and repressing translation. However, how this conserved regulator evolved diverse species-specific regulatory networks remains unclear. We developed Swarm-seq, a high-throughput platform assessing CsrA homologs across the bacterial domain for regulating flagella-dependent swarming in Bacillus subtilis. Testing over five-hundred codon-optimized csrA homologs revealed functional divergence, partitioning CsrAs into two broad classes. Class I (CsrAHp, CsrASm, RsmNPa) strongly inhibited swarming, while Class II (CsrAEc, RsmAPa) failed despite sequence conservation. This differential activity occurred despite canonical GGA motifs in flagellin (hag) transcript, suggesting evolutionary plasticity in RNA-binding specificity beyond motif recognition. Leveraging this dataset, we trained machine learning algorithms to predict CsrA functionality, experimentally validating Bdellovibrio bacteriovorus CsrA (CsrABb) and Pseudomonas putida RsmA (RsmAPp) as Class I. Our findings establish Swarm-seq as a powerful platform for characterizing CsrA homologs from genetically intractable or unculturable bacteria and demonstrate the potential for machine learning-guided discovery of functional regulatory proteins, providing insights into post-transcriptional regulatory network evolution.

Computational Identification and Structural Analysis of Ustilago maydis Effector Proteins.

The phytoplasma effector, Phyllogen, structurally mimics host plant MADS transcription factors.

Phytoplasmas are obligate plant pathogens that include species causing severe developmental abnormalities in diverse plant families. Phytoplasma infections may result in dwarfism, witches' broom, virescence and phyllody, the conversion of reproductive organs into leaf-like structures. Phyllody is due to the activity of phytoplasma effector proteins, called phyllogens, that interact with the host MADS transcription factor (MTF) network. This interaction was believed to be specific for members of the AP1/SEP/AGL6 superclade of tetrameric MTFs, key regulators of floral meristem identity and organogenesis. Here, we determined the molecular and atomic basis for the interaction between the phyllogen​ from Onions Yellow phytoplasma (PHYLOY) and the floral MTFs, APETALA1, SEPALLATA and AGAMOUS using biochemical, structural and genetic experiments. The DNA-binding patterns of homo and heteromeric MTFs with PHYLOY are shown using electrophoretic mobility shift assays, revealing broader interaction patterns between PHYLOY and MTFs that depend on highly conserved hydrophobic residues. Point mutations of these residues allowed gain or loss of interaction between MTFs and PHYLOY. Expressing PHYLOY under the control of the SEP3 and AP1 promoter in Arabidopsis further demonstrated whorl specific phenotypes, recapitulating the in vitro interaction and DNA-binding patterns. These findings reveal how PHYLOY targets specific MTFs through structural mimicry, resulting in impaired MTF tetramer formation and homeotic conversion of floral organs due to loss of active MTF tetramers.

B-Box (BBX) proteins and transcriptional regulation: dynamics, signaling and functions in plants.

This review synthesizes recent progress in how BBX activity is modulated through chromatin remodeling, alternative splicing and E3-ligase-mediated protein stability, among other mechanisms, and proposes that understanding BBX function requires a shift from identifying isolated target genes to decoding the combinatorial logic of their interactions.