Biology subjects

Lindblad, P.

Publications and source records attributed to Lindblad, P..

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

Alka(e)nes contribute to membrane lipid homeostasis and resilience of photosynthesis to high light in cyanobacteria

Alka(e)nes are produced by many living organisms and exhibit diverse physiological roles, reflecting a high functional versatility. Alka(e)nes serve as water proof wax in plants, communicating pheromones for insects, and microbial signaling molecules in some bacteria. Although alka(e)nes have been found in cyanobacteria and algal chloroplasts, a possible role in photosynthesis and chloroplast function remains elusive. In this study, we investigated the consequences of the absence of alka(e)nes on membrane lipid remodeling and photosynthesis using the cyanobacteria Synechocystis PCC6803 as a model organism. By following the dynamics of membrane lipids and the photosynthetic performance in strains defected and altered in alka(e)ne biosynthesis, we show that a profound remodeling of the membrane lipidome and carotenoid content occur in the absence of alka(e)nes, including a decrease in the membrane carotenoid content, a decrease in some digalactosyldiacylglycerol (DGDG) species and a parallel increase in monogalactosyldiacylglycerol (MGDG) species. Under high light, this effect is accompanied in alka(e)ne deficient strains by a higher susceptibility of photosynthesis and growth, the effect being reversed by expressing an algal photoenzyme producing alka(e)nes from fatty acids. We conclude that alka(e)nes play a crucial role in maintaining lipid homeostasis of photosynthetic membranes, thereby contributing to the proper functioning of photosynthesis, particularly under elevated light intensities. Significance statementWe used cyanobacteria as a model organism to explore the role of alka(e)nes related to photosynthesis. Our findings reveal that the absence of alka(e)nes induces alterations in the composition of membrane lipids and carotenoid content, resulting in an increased susceptibility of photosynthesis. By introducing a fatty acid photodecarboxylase to produce alkanes, we could reverse these effects, highlighting the critical role of alka(e)nes in maintaining lipid balance in photosynthetic membranes and ensuring efficient photosynthesis. Uncovering the physiological role of alka(e)nes provides insights to a better understanding of the widespread presence of genes encoding alka(e)nes-synthesizing enzymes in cyanobacteria and microalgae, organisms of major ecological and evolutionary importance in the global CO2 assimilation.

microbiology↗

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↗