Biology subjects

Ostrov, N.

Publications and source records attributed to Ostrov, N..

12 recordsLinked to original sources

bioRxiv2025

A scalable transposon mutagenesis system for non-model bacteria

Transposon mutagenesis enables genome-wide interrogation of gene function with a single self-contained genetic construct. However, its application to non-model bacteria remains limited because transposition efficiency depends on multiple host factors that are difficult to predict a priori including transposase activity, antibiotic resistance marker performance, and regulatory element compatibility. Here, we present a scalable system to identify functional transposon configurations in non-model bacteria through pooled library screening. We selected 18 promoters across multiple bacterial phyla to independently drive expression of the transposase and antibiotic resistance marker, generating 324 promoter combinatorial variants for each of six antibiotic resistance markers. We developed a high-throughput, automated workflow to deliver all 1,944 mariner-based transposon variants in a single experiment and applied this to 92 non-model bacteria spanning multiple phyla. From this, we identified functional transposons for 43 strains, with high-level mutagenesis (102-104 unique insertions) in 13 species, including seven with no previously described transposon mutagenesis. We then expanded to a dual-transposase system, mariner or Tn5, and devised a single transposon insertion sequencing method for high-throughput screening of 3,888 configurations. To demonstrate the practical utility of our screening approach, we used a top-performing variant to generate a genome-wide transposon mutant library for Comamonas testosteroni KF-1, a bacterium that metabolizes plastic- and lignin-derived polymers. We assayed this C. testosteroni mutant library to identify enzymatic pathways, transporter genes, and regulators essential for the metabolism of plastics-associated monomer terephthalate and lignin-associated monomer 4-hydroxybenzoate. Together, this work establishes a scalable approach to construct and identify genetic perturbation systems in non-model bacteria, expanding our ability to systematically probe gene function across the bacterial tree of life.

synthetic biology↗

bioRxiv2025

A genetic platform for a biocementation bacterium

Sporosarcina pasteurii is the most widely studied bacterium for microbially-induced calcium carbonate precipitation (MICP), a process of intense interest for materials and construction applications. Despite two decades of investigation, S. pasteurii has remained genetically intractable, limiting our mechanistic understanding of biomineralization pathways and constraining efforts to engineer scalable solutions. Here, we present the first genetic toolkit for S. pasteurii, including a stable replicating plasmid, a conjugation-based DNA delivery protocol, engineered inducible promoters, and methods for genome modification. Using homologous recombination, we precisely deleted 5.7 kb of the genome spanning two operons encoding urease activity and demonstrated complete loss of biocementation. We also screened a library of engineered transposon constructs for activity in S. pasteurii and generated a genome-wide mutant library with >15,000 unique insertion sites. Using this library, we identified putative genes affecting ureolytic growth, revealing previously inaccessible aspects of S. pasteurii genetics. This work establishes S. pasteurii as a genetically tractable platform for rational engineering of MICP and constitutes the first genetic modification capability within the Sporosarcina genus.

synthetic biology↗

bioRxiv2025

Active learning guides automated discovery of DNA delivery via electroporation for non-model microbes

Delivery of recombinant DNA is foundational for understanding and engineering a target organism. Electroporation can be applied to any cell type, yet identification of a working protocol for new organisms remains bespoke and laborious because no systematic framework exists, and appropriate instrumentation is lacking. Here, we describe an automated high-throughput platform which uses active learning to discover electroporation protocols for non-model microbes. We first devised a 24-condition electroporation screen, based on systematic evaluation and selection of key parameters, that can be applied to any microbe. Facilitated by a custom-built fully-programmable electroporator, we successfully identified electroporation protocols for eight non-model bacteria using this screen alone. We then combined this electroporation screen with our pooled POSSUM plasmid library to simultaneously evaluate 408 experimental conditions per organism and identified both a protocol and a replicating plasmid for five non-model Proteobacteria spanning three major classes (alpha-, beta-, and gamma-). We report the first electroporation protocols for Shewanella indica, Shewanella putrefaciens 200, Shewanella putrefaciens 95, Halomonas elongata, Piscinibacter sakaiensis, and Duganella zoogloeoides, as well as multiple alternative protocols for Shewanella amazonensis, Shewanella oneidensis, Azospirillum brasilense, Cupriavidus necator, Pseudomonas alcaliphila, and Escherichia coli. Finally, we developed an active learning pipeline to guide the selection of parameters based on gathered experimental data. Using our robotic platform, we iteratively tested 538 conditions over three iterations to improve electroporation for the emerging industrial chassis C. necator, achieving 8.6-fold higher transformation efficiency than state of the art. This work establishes a discovery platform for DNA delivery to diverse and recalcitrant microbes that can be extended broadly to non-model organisms in our biosphere.

microbiology↗

bioRxiv2025

Functional genomics in a microbe that degrades and metabolizes PET plastic

Piscinibacter sakaiensis (formerly Ideonella sakaiensis) was the first bacterial species known to both completely degrade and assimilate polyethylene terephthalate (PET). However, the absence of efficient genetic tools has limited direct engineering of this organism, forcing most efforts to rely on heterologous expression of PET-degrading enzymes in model hosts. Here, we establish foundational genetic tools to engineer P. sakaiensis. We identify a functional plasmid origin of replication, multiple new selectable markers, and a transposon system for the strain. We use these tools to construct a genome-wide, barcoded transposon mutant library for pooled high-throughput functional screens. We apply this mutant library to growth on PET and identify metabolic and physiological genes that impact PET biodegradation. We also use this library to reveal mutants with improved DNA uptake for genome engineering. Together, these advances provide a platform for functional genomics in P. sakaiensis and positions this naturally evolved plastic-degrading bacterium as an engineerable chassis for synthetic biology and sustainable materials research.

microbiology↗

bioRxiv2025

A reference set of functional plasmids for Vibrio natriegens

New microbial hosts with superior phenotypes, such as fast growth, are attractive for research and biotechnology, but often lack systematic evaluation of functional genetic parts. A reference set of working plasmids would increase reproducibility and encourage use of these hosts. Here, we use the POSSUM toolkit, a collection of 23 origins-of-replication and 6 antibiotic markers, to identify functional genetic parts for strains of Vibrio natriegens. We applied this to the wild-type strain ATCC 14048 and an engineered variant NBx CyClone, evaluating 414 combinations of origins of replication and antibiotic selection conditions. We show that both strains support five replicons (pNG2, pSa, pSC101ts, p15A, and RSF1010) with NBx CyClone supporting an extra replicon RK2. The assay can be performed in under a week and is compatible with multiple DNA delivery methods. This work demonstrates the feasibility of rapidly establishing reference information to accelerate the adoption of new microbial hosts.

synthetic biology↗

bioRxiv2024

Synthetic genomes unveil the effects of synonymous recoding

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1-4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement--recoding--is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organisms genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

synthetic biology↗

bioRxiv2024

Predicting microbial growth conditions from amino acid composition

The ability to grow a microbe in the laboratory enables reproducible study and engineering of its genetics. Unfortunately, the majority of microbes in the tree of life remain uncultivated because of the effort required to identify culturing conditions. Predictions of viable growth conditions to guide experimental testing would be highly desirable. While carbon and energy sources can be computationally predicted with annotated genes, it is harder to predict other requirements for growth such as oxygen, temperature, salinity, and pH. Here, we developed genome-based computational models capable of predicting oxygen tolerance (92% balanced accuracy), optimum temperature (R2=0.73), salinity (R2=0.81) and pH (R2=0.48) for novel taxonomic microbial families without requiring functional gene annotations. Using growth conditions and genome sequences of 15,596 bacteria and archaea, we found that amino acid frequencies are predictive of growth requirements. As little as two amino acids can predict oxygen tolerance with 88% balanced accuracy. Using cellular localization of proteins to compute amino acid frequencies improved prediction of pH (R2 increase of 0.36). Because these models do not rely on the presence or absence of specific genes, they can be applied to incomplete genomes, requiring as little as 10% completeness. We applied our models to predict growth requirements for all 85,205 species of sequenced bacteria and archaea and found that uncultivated species are enriched in thermophiles, anaerobes, and acidophiles. Finally, we applied our models to 3,349 environmental samples with metagenome-assembled genomes and showed that individual microbes within a community have differing growth requirements. This work guides identification of growth constraints for laboratory cultivation of diverse microbes.

microbiology↗

bioRxiv2024

Design and construction towards a pan-microbial toolkit

Establishing genetic tractability in non-model microbes requires identifying genetic parts that function in a target host. However, the paucity and purported narrow host range of available parts means that successful identification is governed by serendipity. Instead, a more comprehensive and scalable process would be desirable. Here, we describe the design principles for a pan-microbial genetic toolkit in which phylogenetically-diverse parts can be assembled and tested for function in microbes using high-throughput readouts. The architecture is based on Golden Gate Assembly, which simplifies the addition of parts and the construction of combinatorial libraries. We used this framework to develop two modules: first, the POSSUM (Plasmid Origins and Selection MarkerS for Undomesticated Microbes) module for identification of replicating plasmids in non-model microbes which includes 29 plasmid origin of replication sequences, 23 selection markers, and 30 unique DNA sequences for tracking by sequencing; second, the MACKEREL (Modular, NGS-trACKable ExpRession ELement) module, for identification of functional gene expression cassettes which includes 426 bacterial promoter-RBS sequences driving fluorescent reporter expression, trackable by flow cytometry. We demonstrate the use of these libraries to screen for functional promoter-RBS variants in 6 non-model microbes. Continued efforts to expand this pan-microbial toolbox will accelerate efforts to improve genetic tractability and guide research across the tree of life.

synthetic biology↗

bioRxiv2023

MicrobeMod: A computational toolkit for identifying prokaryotic methylation and restriction-modification with nanopore sequencing

Bacteria and archaea use restriction-modification (R-M) systems to distinguish self from foreign DNA by methylating their genomes with DNA methyltransferases with diverse sequence specificities, and these immunity systems often vary at the strain level. Identifying active methylation patterns and R-M systems can reveal barriers to the introduction of recombinant DNA or phage infection. Here, we present the computational MicrobeMod toolkit for identifying 5mC and 6mA methylation sequence motifs and R-M systems in bacterial genomes using nanopore sequencing of native DNA. We benchmark this approach on a set of reference E. coli strains expressing methyltransferases with known specificities. We then applied these analyses to 31 diverse bacterial and archaeal organisms to reveal the methylation patterns of strains with previously unexplored epigenetics, finding that prokaryotic 5-methylcytosine may be more common than previously reported. In summary, MicrobeMod can rapidly reveal new epigenetics within a prokaryotic genome sequenced with Oxford Nanopore R10.4.1 flow cells at sequencing depths as low as 10x and only requires native DNA. This toolkit can be used to advance fundamental knowledge of bacterial methylation and guide strategies to overcome R-M barriers of genetic tractability in non-model microbes.

microbiology↗

bioRxiv2023

A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria

Microbial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such as Escherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs for Halomonas alkaliphila, Halomonas neptunia, and Shewanella electrodiphila. Furthermore, we expand the list of functional ORIs for Duganella zoogloeoides, Pseudomonas alcaliphila, Shewanella oneidensis and Shewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.

synthetic biology↗

bioRxiv2018

Establishing a cell-free Vibrio natriegens expression system

The fast growing bacterium Vibrio natriegens is an emerging microbial host for biotechnology. Harnessing its productive cellular components may offer a compelling platform for rapid protein production and prototyping of metabolic pathways or genetic circuits. Here, we report the development of a V. natriegens cell-free expression system. We devised a simplified crude extract preparation protocol and achieved >260 g/mL of super-folder GFP in a small-scale batch reaction after three hours. Culturing conditions, including growth media and cell density, significantly affect translation kinetics and protein yield of extracts. We observed maximal protein yield at incubation temperatures of 26{degrees}C or 30{degrees}C, and show improved yield by tuning ions crucial for ribosomal stability. This work establishes an initial V. natriegens cell-free expression system, enables probing of V. natriegens biology, and will serve as a platform to accelerate metabolic engineering and synthetic biology applications.

synthetic biology↗

bioRxiv2017

Recombineering in Vibrio natriegens

Here, we show that {lambda}-Red homologs found in the Vibrio-associated SXT mobile element potentiate allelic exchange in V. natriegens by 10,000-fold. Specifically, we show SXT-Beta (s065), SXT-Exo (s066), and {lambda}-Gam proteins are sufficient to enable recombination of single- and double-stranded DNA with episomal and genomic loci. We characterize and optimize episomal oligonucleotide-mediated recombineering and demonstrate recombineering at genomic loci. We further show targeted genomic deletion of the extracellular nuclease gene dns using a double-stranded DNA cassette. Continued development of this recombination technology will advance high-throughput and large-scale genetic engineering efforts to domesticate V. natriegens and to investigate its rapid growth rate.

synthetic biology↗