Biology subjects

Benzerara, K.

Publications and source records attributed to Benzerara, K..

7 recordsLinked to original sources

bioRxiv2026

Targeted genome editing of the non-model cyanobacterium Cyanothece PCC 7425 via CRISPR/Cas12a

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI

microbiology↗

bioRxiv2026

Redox distribution of Asgard archaea and co-occurring taxa in microbial mats from an early Proterozoic ecosystem analog

Increasing evidence supports that eukaryotes originated from the symbiosis of an Asgard archaeon, the alphaproteobacterial ancestor of mitochondria, and possibly additional bacterial contributions. Eukaryogenesis likely occurred in redox-transition environments such as microbial mats or shallow sediments [~]2 billion years ago, when atmospheric oxygen was far lower than today, and oceans were euxinic. We investigated Asgardarchaeota-enriched microbial mats from the low-oxygen, sulfidic Catherine volcano lake (Afar region, Ethiopia), mimicking early Proterozoic conditions. 16S rRNA gene metabarcoding, metagenomics, and metagenome-assembled genome (MAG) analyses across redox-stratified layers of in situ and mesocosm-maintained mats revealed that Asgardarchaeota thrived in the sulfate-reduction zone, mainly co-occurring with Desulfurobacterota and Myxococcota, among others. Lokiarchaeia and Thorarchaeia were more abundant in deeper, anoxic layers. Within Heimdallarchaeia, Heimdallarchaeales were enriched in upper layers, correlating with oxygen-tolerant hydrogenase and sulfate-reduction genes, whereas Hodarchaeales were more relatively abundant in anoxic layers, correlating with methanogenesis. Although reactive-oxygen-species defense mechanisms were widespread in Asgardarchaeota MAGs, they lacked genes for aerobic respiration. These results support the hypothesis that Asgard archaea engaged primarily in syntrophic interactions with sulfate-reducers under conditions prevailing on the early Earth at the onset of eukaryogenesis.

microbiology↗

bioRxiv2025

Intracellular amorphous calcium carbonate biomineralization in methanotrophic gammaproteobacteria was acquired by horizontal gene transfer from cyanobacteria

Some bacteria genetically control the biomineralization of intracellular amorphous calcium carbonates (iACC) with potential implications for microbial physiology, evolution, bioremediation and biogeochemical cycling. Until now, this capacity has been documented in Cyanobacteria, the giant gammaproteobacterium Achromatium and a few magnetotactic Pseudomonadota and Nitrospirota. Here, we report the discovery of iACC biomineralization in members of the Methylococcaceae, a family of aerobic methanotrophic Gammaproteobacteria. A homolog of the ccyA gene, previously considered a diagnostic marker for iACC formation in Cyanobacteria, was identified in several Methylococcaceae genomes, based on a search of the conserved C-terminal (GlyZip)3 domain of the encoded calcyanin protein, with a sequence coverage higher than 60% and an E-value lower than 1e-20. Moreover, two cultivated strains, Methylococcus geothermalis and Methylococcus mesophilus, whose genomes contained the ccyA gene, were consistently shown to form iACC granules. The ccyA genes of Methylococcaceae and Microcystis share higher sequence similarity (47%) than with other Cyanobacteria (around 30%) within their common (GlyZip)3 domain, suggesting horizontal gene transfer (HGT) from an ancestral Microcystis-like cyanobacterium to Methylococcaceae. This finding extends the known taxonomic distribution of ccyA and suggests that the capability to biomineralize iACC was acquired by HGT, possibly in environments such as those close to the oxyclines of lakes, where Cyanobacteria and Methylococcaceae commonly co-exist. The discovery of iACC in methane-oxidizing Methylococcaceae highlights a previously unrecognized coupling between calcium carbonate biomineralization and methane cycling in aquatic environments, suggesting that iACC formation may play an overlooked role in microbial carbon storage and local geochemical regulation.

microbiology↗

bioRxiv2024

Diel changes in the expression of a marker gene and candidate genes for intracellular amorphous CaCO3 biomineralization in Microcystis

Phylogenetically diverse cyanobacteria biomineralize intracellular amorphous calcium carbonate (iACC) inclusions. This includes several genotypes of the Microcystis genus, a potentially toxic, bloom-forming cyanobacterium found worldwide in freshwater ecosystems. While we ignore the biological function of iACC and the molecular mechanisms driving their formation, this process may impact local geochemical cycles and/or be used for bioremediation strategies. Recently, a marker gene of this biomineralization pathway, named ccyA, was discovered. However, the function of the calcyanin protein encoded by ccyA remains unknown. Here, based on an RNA- Seq approach, we assess the expression of the ccyA gene in Microcystis aeruginosa PCC 7806 during a 24 h day/night cycle. The ccyA gene shows a clear day/night expression pattern with maximum transcript abundances during the second half of the night. This is consistent with the assumption that iACC biomineralization is related with photosynthesis and may therefore follow a day/night cycle as well. Moreover, several genes directly co-localized upstream and downstream of ccyA, on the same DNA strand show a similar expression pattern, including a cax gene encoding a calcium/proton exchanger and a gene encoding a protein with a domain also present in the N-terminal region of calcyanins in many iACC-forming cyanobacteria. This suggests that they all could be part of an operon, and may play a concerted role in iACC formation. Last, several other genes involved in carbon concentrating mechanisms and calcium transport show an expression pattern similar to that of ccyA. Overall, this study provides a list of candidate genes that may be involved in the biomineralization of iACC by cyanobacteria and whose role could be, in the future, analyzed by biochemistry and genetics approaches.

microbiology↗

bioRxiv2022

Description of Gloeomargarita ahousahtiae sp. nov., a thermophilic member of the order Gloeomargaritales with intracellular carbonate inclusions

A unicellular cyanobacterium, strain VI4D9, was isolated from thermophilic microbial mats thriving in a hot spring of the Ahousaht territory of Vancouver Island, Canada. The cells were elongated rods (5.1 m in length and 1.2 m in width on average). Their UV-visible absorption spectra revealed that they contain chlorophyll a, phycocyanin, and carotenoids. Transmission electron microscopy showed the presence of thylakoids concentrated on one side of the cells. The strain grew within a temperature range of 37-50{degrees}C, with an optimum at 45{degrees}C. Its genome had a size of 3,049,282 bp and a DNA G+C content of 51.8 mol%. The cells contained numerous intracellular spherical granules easily visible under scanning electron microscopy. Energy-dispersive x-ray spectroscopy revealed that these granules were made of Ca-, Ba- and Sr-containing carbonates. A phylogenetic 16S rRNA gene tree robustly placed this strain as sister to several environmental sequences and the described species Gloeomargarita lithophora, also characterized by the possession of intracellular carbonate inclusions. We consider strain VI4D9 to represent a new Gloeomargarita species based on its marked phenotypic differences with G. lithophora, notably, its thermophilic nature and different thylakoid organization. We propose the name Gloeomargarita ahousahtiae sp. nov. for this newly isolated thermophilic cyanobacterium. The type strain is VI4D9 (Culture Collection of Algae and Protozoa strain 1472/1; Laboratorio de Algas Continentales Mexico strain LAC 140). G. ahousahtiae is the second species described within the recently discovered order Gloeomargaritales.

microbiology↗

bioRxiv2021

A new gene family diagnostic for intracellular biomineralization of amorphous Ca-carbonates by cyanobacteria

Cyanobacteria have massively contributed to carbonate deposit formation over the geological history. They are traditionally thought to biomineralize CaCO3 extracellularly as an indirect byproduct of photosynthesis. However, the recent discovery of freshwater cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges this view. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and evolutionary history remain elusive. Here, using comparative genomics, we identify a new gene (ccyA) and protein (calcyanin) family specifically associated with cyanobacterial iACC biomineralization. Calcyanin is composed of a conserved C-terminal domain, which likely adopts an original fold, and a variable N-terminal domain whose structure allows differentiating 4 major types among the 35 known calcyanin homologues. Calcyanin lacks detectable full-length homologs with known function. Yet, genetic and comparative genomic analyses suggest a possible involvement in Ca homeostasis, making this gene family a particularly interesting target for future functional studies. Whatever its function, this new gene family appears as a gene diagnostic of intracellular calcification in cyanobacteria. By searching for ccyA in publicly available genomes, we identified 13 additional cyanobacterial strains forming iACC. This significantly extends our knowledge about the phylogenetic and environmental distribution of cyanobacterial iACC biomineralization, especially with the detection of multicellular genera as well as a marine species. Phylogenetic analyses indicate that iACC biomineralization is ancient, with independent losses in various lineages and some HGT cases that resulted in the broad but patchy distribution of calcyanin across modern cyanobacteria. Overall, iACC biomineralization emerges as a new case of genetically controlled biomineralization in bacteria. Significance statementFew freshwater species of Cyanobacteria have been known to mineralize amorphous CaCO3 (ACC) intracellularly. Despite the geochemical interest of this biomineralization, its evolutionary history and molecular mechanism remain poorly known. Here, we report the discovery of a new gene family that has no homolog with known function, which proves to be a good diagnostic marker of this process. It allowed to find cyanobacteria in several phyla and environments such as seawater, where ACC biomineralization had not been reported before. Moreover, this gene is ancient and was independently lost in various lineages with some later horizontal transfers, resulting in a broad and patchy phylogenetic distribution in modern cyanobacteria.

evolutionary biology↗

bioRxiv2019

Hyperdiverse archaea near life limits at the polyextreme geothermal Dallol area

Microbial life has adapted to various individual extreme conditions; yet, organisms simultaneously adapted to very low pH, high salt and high temperature are unknown. We combined environmental 16S/18S rRNA-gene metabarcoding, cultural approaches, fluorescence-activated cell sorting, scanning electron microscopy and chemical analyses to study samples along such unique polyextreme gradients in the Dallol-Danakil area (Ethiopia). We identify two physicochemical barriers to life in the presence of surface liquid water defined by: i) high chaotropicity-low water activity in Mg2+/Ca2+-dominated brines and ii) hyperacidity-salt combinations (pH~0/ NaCl-dominated salt-saturation) When detected, life was dominated by highly diverse ultrasmall archaea widely distributed across phyla with and without previously known halophilic members. We hypothesize that high cytoplasmic K+-level was an original archaeal adaptation to hyperthermophily, subsequently exapted during multiple transitions to extreme halophily. We detect active silica encrustment/fossilization of cells but also abiotic biomorphs of varied chemistry. Our work helps circumscribing habitability and calls for cautionary interpretations of morphological biosignatures on Earth and beyond.

microbiology↗