Biology subjects

Springstein, B. L.

Publications and source records attributed to Springstein, B. L..

6 recordsLinked to original sources

bioRxiv2025

Evolutionary repurposing of a DNA segregation machinery into a cytoskeletal system controlling cyanobacterial cell shape

Bacteria, like eukaryotes, use conserved cytoskeletal systems for intracellular organization. The plasmid-encoded ParMRC system forms actin-like filaments that segregate low-copy plasmids. In multicellular cyanobacteria like Anabaena sp., we found that a chromosomally-encoded ParMR has evolved into a novel cytoskeleton-termed CorMR-with a function in cell shape control rather than DNA segregation. Using live-cell imaging, in vitro reconstitution and Cryo-EM, we demonstrate that CorM forms dynamically unstable, antiparallel double-stranded filaments, which are recruited to the membrane by CorR via an amphipathic helix conserved in multicellular cyanobacteria. CorMR filaments are regulated by MinC, which excludes them from the poles and division plane. Comparative genomics reveal that the repurposing of ParMR and Min systems co-evolved with cyanobacterial multicellularity, highlighting the evolutionary plasticity of cytoskeletal systems in bacteria.

microbiology↗

bioRxiv2023

Measuring prion propagation in single bacteria elucidates mechanism of loss

Prions are self-propagating protein aggregates formed by specific proteins that can adopt alternative folds. Prions were discovered as the cause of the fatal transmissible spongiform encephalopathies in mammals, but prions can also constitute non-toxic protein-based elements of inheritance in fungi and other species. Prion propagation has recently been shown to occur in bacteria for more than a hundred cell divisions, yet a fraction of cells in these lineages lost the prion through an unknown mechanism. Here, we investigate prion propagation in single bacterial cells as they divide using microfluidics and fluorescence microscopy. We show that the propagation occurs in two distinct modes with distinct stability and inheritance characteristics. We find that the prion is lost through random partitioning of aggregates to one of the two daughter cells at division. Extending our findings to prion domains from two orthologous proteins, we observe similar propagation and loss properties. Our findings also provide support for the suggestion that bacterial prions can form more than one self-propagating state. We implement a stochastic version of the molecular model of prion propagation from yeast and mammals that recapitulates all the observed single-cell properties. This model highlights challenges for prion propagation that are unique to prokaryotes and illustrates the conservation of fundamental characteristics of prion propagation across domains of life.

microbiology↗

bioRxiv2021

A bacteria-based assay to study SARS-CoV-2 protein-protein interactions

Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of COVID-19, an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of SARS-CoV-2-encoded proteins. Here we describe the use of a convenient bacteria-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identify sixteen distinct intraviral protein-protein interactions (PPIs), involving sixteen proteins. We find that many of the identified proteins interact with more than one partner. We further show how our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm and we use this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examine how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins. ImportanceUnderstanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here we describe the use of a versatile bacteria-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current pandemic. We identify sixteen distinct intraviral protein-protein interactions, involving sixteen proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacteria-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically its receptor binding domain) and its cognate human cell surface receptor ACE2 and we investigate the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.

molecular biology↗

bioRxiv2020

Natural competence in Chlorogloeopsis fritschii PCC 6912 and other ramified cyanobacteria

Lateral DNA transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA acquisition via transformation involves the uptake of DNA from the environment. The ability of recipient cells to actively transport DNA into the cytoplasm - termed natural competence - depends on the presence of type IV pili and competence proteins. Natural competence has been described in cyanobacteria for several organisms including unicellular and filamentous species. However, the presence of natural competence in ramified cyanobacteria, which are considered the peak of cyanobacterial morphological complexity, remains unknown. Here we show that ramified cyanobacteria harbour the genes essential for natural competence and experimentally demonstrate natural competence in the ramified cyanobacterium Chlorogloeopsis fritschii PCC 6912 (hereafter Chlorogloeopsis). Searching for homologs to known natural competence genes in ramified cyanobacteria showed that these genes are conserved in the majority of tested isolates. Experimental validation of natural competence using several alternative protocols demonstrates that Chlorogloeopsis could be naturally transformed with a replicative plasmid. Our results show that natural competence is a common trait in ramified cyanobacteria and that natural transformation is likely to play an important role in cyanobacteria evolution. ImportanceCyanobacteria are crucial players in the global biogeochemical cycles where they contribute to CO2- and N2-fixation. Their main ecological significance is the oxygen-producing photosynthetic apparatus that contributes to contemporary food chains. Ramified cyanobacteria form true-branching and multiseriate cell filament structures that represent a peak of prokaryotic multicellularity. Species in that group inhabit fresh and marine water habitats, thermal springs, arid environments, as well as endolithic and epiphytic habitats. Here we show that ramified cyanobacteria harbor the mechanisms required for DNA acquisition via natural transformation. The prevalence of mechanisms for natural uptake of DNA has implications for the role of DNA acquisition in the evolution of cyanobacteria. Furthermore, presence of mechanisms for natural transformation in ramified cyanobacteria opens up new possibilities for genetic modification of ramified cyanobacteria.

evolutionary biology↗

bioRxiv2019

Identification and characterization of novel filament-forming proteins in cyanobacteria

Filament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a {Delta}syc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.

microbiology↗

bioRxiv2019

A cytoskeletal network maintains filament shape in the multicellular cyanobacterium Anabaena sp. PCC 7120

Polymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits - e.g. MreB and FtsZ in bacteria - or heteropolymers that are composed of two subunits, e.g. keratin and /{beta} tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament forming cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) that assemble into a heteropolymer and function in the maintenance of the Anabaena multicellular shape (termed trichome). The two CCRPs - Alr4504 and Alr4505 (named ZicK and ZacK) - are strictly interdependent for the assembly of protein filaments in vivo and polymerize nucleotide-independently in vitro, similar to known intermediate filament (IF) proteins. A {Delta}zicK{Delta}zacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linear Anabaena trichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize the Anabaena trichome and are likely essential for the manifestation of the multicellular shape in Anabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.

microbiology↗