Biology subjects

Nierzwicki-Bauer, S.

Publications and source records attributed to Nierzwicki-Bauer, S..

4 recordsLinked to original sources

bioRxiv2026

Genetic access to an obligate cyanobacterial endosymbiont within the shoot meristem of fern hosts

Eukaryote-associated microbes are ubiquitous, but their essential roles in the development and ecology of their host is yet to be fully understood, partly because complex associations cannot be reconstituted and, in many instances, the genetic tools to elucidate those roles are not available. Here, we report the conjugative transfer of DNA into Nostoc azollae within two Azolla fern hosts. N. azollae is a filamentous, N2-fixing, heterocyst-forming cyanobacterium which is an obligate endosymbiont of the complex microbial community associated with the floating ferns of the genus Azolla. The cyanobiont provides fixed nitrogen to its host, supporting maximum growth rates without any N-fertilizer and making Azolla symbioses both ecologically and agriculturally important. Triparental mating protocols and fluorescent reporter detection were optimized for the cyanobiont isolated from the fern, allowing further demonstration of heterologous gene expression in N. azollae driven by several promoters, including some of a CRISPR-associated transposon (CAST) system. Azolla was then treated with a cytokinin hormone to render fern shoot apices amenable to in planta conjugation, permitting DNA transfer to, and stable gene expression in two distinct developmental stages of N. azollae within Azolla. These included (i) cells of filaments from the Shoot Apical Nostoc colony, the only cyanobacterial stem-cell population vertically transmitted across fern generations, and (ii) cells from differentiated filaments in early formed Azolla leaf cavities. Our approach represents a technically groundbreaking advance for the genetic engineering of cyanobacterial endosymbioses that may be useful for other symbiotic systems, opening a pathway to investigate these important biological entities.

microbiology↗

bioRxiv2025

Intercellular communication in the fern endosymbiotic cyanobacterium Nostoc azollae

The water fern Azolla spp. harbors as an endobiont the N2-fixing, filamentous, heterocyst-forming cyanobacterium Nostoc azollae. N. azollae provide the fern with fixed nitrogen permitting its growth in nitrogen-poor environments. In the filaments of heterocyst-forming cyanobacteria, an intercellular exchange of regulators and metabolites occur in which heterocysts provide vegetative cells with fixed nitrogen and vegetative cells provide heterocysts with reduced carbon. Intercellular molecular exchange takes place by diffusion through septal junctions and can be probed by fluorescence recovery after photobleaching (FRAP) analysis with fluorescent markers such as calcein and 5-carboxyfluorescein. The septal junctions traverse the septal peptidoglycan through nanopores that can be visualized in isolated septal peptidoglycan disks by electron microscopy. Here we obtained N. azollae material from Azolla plants, which contains the symbiotic cyanobacterium in a viable state and with different morphologies, including heterocyst-containing filaments. FRAP analysis showed effective transfer of the fluorescent markers between vegetative cells as well as from vegetative cells to heterocysts. Interestingly, communicating and noncommunicating vegetative cells and heterocysts could be distinguished showing conservation in the endobiont of a regulatory mechanism capable of opening and closing septal junctions. Peptidoglycan sacculi were also isolated and showed septal disks with arrays of nanopores that conform to those visualized in other heterocyst-forming cyanobacteria. However, a wider range of septal disk size was observed in N. azollae. In spite of its eroded genome, N. azollae maintains the intercellular communication system that is key for its growth as a multicellular organism. ImportanceThe water fern Azolla constitutes a unique symbiotic system in which cyanobacterial endobionts capable of fixation of atmospheric nitrogen provide the plant with the nitrogen needed for growth. This symbiosis is an important fertilizer for rice crops worldwide, thereby reducing the reliance on fossil fuel-derived nitrogen fertilizers. The symbiotic cyanobacterium, Nostoc azollae, is a heterocyst-forming strain in which a filament of cells is the organismic unit of growth. Here we show that the intercellular molecular exchange function necessary for the multicellular behavior of the organism is conserved in the endobiotic Nostoc azollae.

microbiology↗

bioRxiv2023

Crane fly semiochemical overrules coordinate cyanobiont differentiation in Azolla ferns

Semiochemicals from insects that restrict plant symbiont dinitrogen fixation had not been known. Here we report on a the glycosylated triketide {delta}-lactone only found in Nephrotoma cornicina crane flies, cornicinine, that causes chlorosis in the floating-fern symbioses from the genus Azolla. Cornicinine was chemically synthesized, as well as its aglycone and diastereoisomer. Only the glycosylated trans-A form was active: 500 nM cornicinine in the growth medium turned the dinitrogen-fixing cyanobacterial filaments from Nostoc azollae inside the host leaf cavities into akinete-like cells. Cornicinine further inhibited akinete germination in Azolla sporelings, precluding re-establishment of the symbiosis during sexual reproduction. It did not affect the plant Arabidopsis thaliana or several free-living cyanobacteria from the genera Anabaena or Nostoc. Chlorosis occurred in hosts on nitrogen with and devoid of cyanobiont. Cornicinine, therefore, targeted host mechanisms resulting in coordinate cyanobiont differentiation. Sequence profiling of messenger RNA from isolated leaf cavities confirmed high NH4-assimilation and proanthocyanidin biosynthesis in this trichome-rich tissue. Leaf-cavity transcripts in ferns grown on cornicinine reflected activation of Cullin-RING ubiquitin-ligase pathways, known to mediate metabolite signaling and plant elicitation consistent with the chlorosis phenotype. Transcripts accumulating when akinetes are induced, in leaf cavities of ferns on cornicinine and in megasporocarps, were consistent with increased JA-oxidase, sulfate transport and exosome formation. The work begins to uncover molecular mechanisms of cyanobiont differentiation in a seed-free plant symbiosis important for wetland ecology or circular crop-production today, that once caused massive CO2 draw-down during the Eocene geological past. SignificanceCoordinated differentiation of host and filamentous cyanobacteria underlies the development of ecologically important symbioses; this includes the floating ferns Azolla which share their wetland habitat with Nephrotoma cornicina craneflies containing the glycosylated triketide {delta}-lactone semiochemical, cornicinine. Cornicinine overrules cyanobiont differentiation thus inhibiting symbiosis N2-fixation and sexual reproduction; its mode of action resembles plant elicitation as suggested by transcriptional profiling of cells lining the cyanobiont cavities using a new release of the fern host genome.

plant biology↗

bioRxiv2022

Towards genome-engineering in complex cyanobacterial communities: RNA-guided transposition in Anabaena

In genome engineering, integration of incoming DNA has been dependent on enzymes produced by dividing cells which has been a bottle neck towards increasing DNA-insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here we developed Golden-Gate vectors to test CAST in filamentous cyanobacteria and show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing the CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide(sg)-RNA encoding the leading, but not the reverse complement strand of the target were effective with the protospacer associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci, therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.

synthetic biology↗