Biology subjects

Russo, D. A.

Publications and source records attributed to Russo, D. A..

9 recordsLinked to original sources

bioRxiv2026

Activation-independent capture of free fatty acids at the bacterial cell envelope by BrtB

Fatty acids (FAs) are key metabolites in living organisms, shaping membrane architecture and helping cellular acclimatization to changing conditions. Bacteria synthesize FA de novo but can also reclaim them from membrane lipids or uptake them from the environment, usually converting free fatty acids (FFAs) into activated FAs that can then be further metabolized. In cyanobacteria, it is the acyl-acyl carrier protein synthetase (Aas) that activates exogenous FFAs. Yet, the cyanobacterial enzyme BrtB was recently shown to esterify, in vitro, FFAs directly onto abundant chlorinated glycolipids (bartolosides), generating bartoloside fatty acid esters (B-FAs). Whether this chemistry operates in vivo, where it occurs, and what its implications are for cell physiology has remained unclear. Here we show that in the cyanobacterium Synechocystis salina LEGE 06099, BrtB captures FFAs at the cell envelope without prior activation to generate B-FAs. We found that supplemented FFAs were converted into B-FAs within minutes. Strikingly, this response occurred with minimal changes in gene expression and little alteration of the extracellular proteome, consistent with a pathway already in place. Additionally, we observed that B-FAs can further be hydrolyzed into hydroxybartolosides - the levels of the latter metabolites increase in response to FA supply, suggesting a transient sequestration of FFA. Our results demonstrate that activation of FFA is not the only route to their cellular incorporation and identify a specialized-metabolite pathway that captures exogenous FFAs at the cyanobacterial cell envelope.

microbiology↗

bioRxiv2025

Establishing betaxanthin pigment biosynthesis in cyanobacteria

Betalains are water-soluble pigments with two major classes: red-violet betacyanins and yellow-orange betaxanthins. These pigments are increasingly being sought after as natural replacements for synthetic pigments in the food industry. Traditionally, betalains are extracted from cultivated plants but, due to low native pigments, the process is inherently inefficient. Now, as consumer demand increases, scalable and sustainable production routes are needed. To address this challenge, we engineered a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of the cytochrome P450 CYP76AD1 and the L-DOPA 4,5-dioxygenase DODA1 from Beta vulgaris (beet). Introduction of the two-enzyme betaxanthin pathway in Synechocystis sp. PCC 6803 did not result in detectable betaxanthins. Subsequent metabolic adjustments to the shikimate pathway, using a feedback resistant AroGfbr from E. coli, led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of 0.11 mg L-1 phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO2-enriched air) increased titers almost 150 times and led to the production of phenylalanine-betaxanthin with a final titer of 15.3 mg L-1. Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.

synthetic biology↗

bioRxiv2024

Competition and interdependence define multifaceted interactions of symbiotic Nostoc sp. and Agrobacterium sp. under inorganic carbon limitation

Cyanobacteria of the Nostoc genus are capable of forming symbiotic relationships with plants, thus transitioning to a heterotrophic lifestyle in return for providing bioavailable nitrogen to the host. The diazotrophic photoautotrophs also serve as a hub for specialized heterotrophic bacterial communities whose physiological contributions are poorly understood. By comparing the axenic strain Nostoc punctiforme PCC 73102 and the related strains Nostoc sp. KVJ2 and KVJ3, which still maintain their heterotrophic microbiome, we were able to demonstrate an almost obligate dependence of the cyanobacteria on the heterotrophic partners under carbon-limiting conditions. Detailed analysis of the intimate bilateral relationship between N. punctiforme and the isolate Agrobacterium tumefaciens Het4 using shotgun proteomics and microscopy uncovered a complex partnership characterized, among other traits, by competition for iron and facilitation for carbon. Although competitive interactions with A. tumefaciens Het4 compromise nitrogen fixation and stimulate the degradation of cyanophycin, mutualistic dependency prevails under inorganic carbon limitation. Both the absence of the high affinity bicarbonate uptake transporter SbtA and the prevalent extracarboxysomal localization of the carbon-fixing enzyme RubisCO, as detected by immunofluorescence microscopy, suggest that a weak carbon concentrating mechanism in N. punctiforme enforces a dependence on heterotrophic bacteria. Further, immunofluorescence, electron microscopic and proteomic analyses reveal a pronounced extracellular recycling of proteins under N- and C-limiting conditions. Our study shows that the pivotal influence of heterotrophic bacteria on symbiotic Nostoc strains should be considered when analyzing these cyanobacteria, especially in the free-living state. This work also sheds new light on how Nostoc benefits from the organic carbon provided by plant hosts.

microbiology↗

bioRxiv2024

EXCRETE enables deep proteomics of the cyanobacterial extracellular environment

Extracellular proteins play a significant role in shaping microbial communities which, in turn, can impact ecosystem function, human health, and biotechnological processes. Yet, for many ubiquitous microbes, there is limited knowledge regarding the identity and function of secreted proteins. Here, we introduce EXCRETE (enhanced exoproteome characterization by mass spectrometry), a workflow that enables comprehensive description of microbial exoproteomes from minimal starting material. Using cyanobacteria as a case study, we benchmark EXCRETE and show a significant increase over current methods in the identification of extracellular proteins. Subsequently, we show that EXCRETE can be miniaturized and adapted to a 96-well high-throughput format. Application of EXCRETE to cyanobacteria from different habitats (Synechocystis sp. PCC 6803, Synechococcus sp. PCC 11901, and Nostoc punctiforme PCC 73102), and in different cultivation conditions, identified up to 85% of all predicted secreted proteins. Finally, functional analysis reveals that cell envelope maintenance and nutrient acquisition are central functions of the cyanobacterial secretome. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across freshwater, marine, and terrestrial species.

biochemistry↗

bioRxiv2023

Self-assembly of nanofilaments in cyanobacteria for protein co-localization

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts but, so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that overexpression of a modified bacterial microcompartment shell protein, PduA*, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly only had a minor impact on cellular metabolism. Finally, we targeted the fluorescent reporter mCitrine to the nanofilaments using an encapsulation peptide that natively interacts with PduA. To our knowledge, this is the first study to apply bacterial microcompartment based nanotechnology in cyanobacteria. The establishment of nanofilaments in cyanobacterial cells is an important step towards cellular organization of heterologous pathways and the establishment of cyanobacteria as next generation hosts.

synthetic biology↗

bioRxiv2022

Cell surface composition and ionic strength mediate fast sedimentation in the cyanobacterium Synechococcus elongatus PCC 7942

Cyanobacteria are photosynthetic prokaryotes of high ecological and biotechnological relevance that have been cultivated in laboratories around the world for more than 70 years. Prolonged laboratory culturing has led to multiple microevolutionary events and the appearance of a large number of "domesticated" substrains among model cyanobacteria. Despite its widespread occurrence, strain domestication is still largely ignored. In this work we describe Synechococcus elongatus PCC 7942-KU, a novel domesticated substrain of the model cyanobacterium Synechococcus elongatus PCC 7942, which presents a fast-sedimenting phenotype. Under higher ionic strengths the sedimentation rate increases leading to complete sedimentation in just 12 h. Through whole genome sequencing and gene deletion, we demonstrate that the Group 3 alternative sigma factor F (SigF) plays a key role in cell sedimentation. In addition, sedimentation analysis of an unpiliated mutant and differences in surface hydrophobicity suggest that mutations in SigF lead to significant changes of cell surface structures and, consequentially, to the appearance of a fast-sedimenting phenotype. This work sheds light on the determinants of the planktonic to benthic transitions and provides genetic targets to generate fast-sedimenting strains that could unlock cost-effective cyanobacterial harvesting at scale.

microbiology↗

bioRxiv2022

Droplet-based microfluidics as a media optimization tool for cyanobacteria

The urgent need to increase sustainability in biotechnology has led to an increased interest in photosynthetic production platforms. Cyanobacteria are particularly attractive for their fast photoautotrophic growth and genetic accessibility. However, the lack of systematic strain optimization is holding back progress towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidics platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial cultivation. We successfully grew three different unicellular, biotechnologically relevant cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus sp. UTEX 3154. Highly-resolved 1D screening of nitrate, phosphate, carbonate, and salt content show that nitrate and/or phosphate can be limiting in standard cultivation media. Finally, we show that 2D screening results from our microfluidic setup translate well to a laboratory scale high-density cultivation setup. This study demonstrates that droplet-based microfluidics by micro segmented-flow are a powerful tool for high-throughput optimization of cyanobacterial cultivation.

microbiology↗

bioRxiv2021

Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further. ImportanceProtein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

microbiology↗

bioRxiv2019

Detection and characterization of a novel copper-dependent intermediate in a lytic polysaccharide monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose and the enzyme has large practical application in the process of refining biomass. The LPMO catalytic mechanism still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t[1/2] = 6 - 8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-Cu(I) reacts with H2O2. UV-vis absorption spectroscopy, electron paramagnetic resonance (EPR), and stopped-flow spectroscopy indicate that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). We propose that the reaction with H2O2 first forms a highly reactive short-lived Cu(III)-intermediate which is subsequently transformed into the observed long-lived copper-dependent intermediate. Since sub-equimolar amount of H2O2 to LPMO boosts oxidation of phosphoric acid swollen cellulose (PASC) suggests that the long-lived copper-dependent intermediate is part of the catalytic mechanism for LPMOs. The proposed mechanism offers new perspectives in the oxidative reaction mechanism of copper enzymes and hence for the biomass oxidation and the reactivity of copper in biological systems.

biochemistry↗