Biology subjects

Reisch, C. R.

Publications and source records attributed to Reisch, C. R..

5 recordsLinked to original sources

bioRxiv2023

Characterizing constitutive promoters across the Proteobacteria

Although research on promoters has spanned decades, the precise prediction of promoter activity from DNA sequence remains a challenge even in model organisms. Recent literature has identified important differences in the core sequence of {sigma}70 promoters across classes of Proteobacteria as well as a lack of transferability when promoters are moved from host to host. Currently, there is a need for synthetic constitutive promoters spanning a range of expression levels in species outside of Escherichia coli. Additionally, characterization data defining behavior of the same promoter across multiple species would be extremely valuable to the field. Here, we analyzed promoter activity in three classes of Proteobacteria, which enabled us to better understand the sequence elements correlated with a strong promoter in different hosts. In doing so, we identified and characterized constitutive promoters spanning a range of expression in these species for community use and described the portability of a subset of these promoters as they were moved between hosts. These promoter libraries have broad applications as predictable genetic tools to control gene expression in diverse species (1-3). This work adds to the toolkit for gene expression in non-model bacteria and is a step towards the larger goal of accurate promoter prediction in a given host from a de novo sequence.

synthetic biology↗

bioRxiv2023

Comparative Genomics and Directed Evolution Reveal Genetic Determinants of Extreme UVC Radiation Tolerance in Bacteria Recovered from the Stratosphere

Aerosolized microbes surviving transport to and in the stratosphere endure extremes of low temperature, atmospheric pressure, and relative humidity, and high shortwave ultraviolet radiation flux. However, the genetic determinants for traits enabling resistance to the combination of stresses experienced by microbes in the high atmosphere have not been systematically investigated. In this study, we examined Proteobacteria and Actinobacteria isolated from the stratosphere (18 to 29 km ASL) and that demonstrated high tolerance to desiccation (15-25% RH) and UVC radiation (UVCR; {lambda}= 254 nm). Closely related reference strains were more sensitive to UVCR than the stratospheric isolates, indicating that extreme resistance is not universally distributed in these phylogenetically related bacteria. Comparative genomic analyses revealed DNA repair and antioxidant defense genes in the isolates that are not possessed by the related reference strains, including genes encoding photolyase, DNA nucleases and helicases, and catalases. Directed evolution by repeated exposure to increasing doses of UVCR improved the LD90 in a sensitive reference strain by [~]3.5-fold. The mutations acquired in Curtobacterium flaccumfaciens pv. flaccumfaciens strain DSM 20129 incrementally increased its UVCR resistance, with the accumulation of 20 point mutations in protein coding genes increasing tolerance to a level approaching that of stratospheric isolate Curtobacterium sp. L6-1. The genetic basis for the increased UVCR tolerance phenotypes observed is discussed, with a specific emphasis on the role of genes involved in DNA repair and detoxification of reactive oxygen species. ImportanceUltraviolet radiation is omnipresent in sunlight and has important biological effects on organisms. The stratosphere is the only location on Earth where microbes receive natural exposure to highly mutagenic wavelengths (<280 nm) of ultraviolet radiation. Genetic studies of bacteria from an environment that selects for extreme ultraviolet radiation resistant phenotypes has expanded what is known from studies of model species (e.g., E. coli) and identified potentially novel protection and repair strategies. In addition to deepening understanding of ultraviolet radiation photobiology in atmospheric microbes and bacteria in general, these advancements are also highly relevant to astrobiology and space biology. The cold, dry, hypobaric, and high radiation environment of the stratosphere provides an earthly analog for thin extraterrestrial atmospheres (e.g., Mars) and is ideal for bioprospecting extremophile phenotypes that enable engineering of genetic stability and functionality in bio-based space life-support systems or any application where long-term persistence is desirable (e.g., biocontrol).

molecular biology↗

bioRxiv2022

Broad-host-range mutagenesis with CRISPR-associated transposase

Transposons have been instrumental tools in microbiology enabling random mutagenesis, with transposons like Tn5 and Mariner, and site-specific DNA integrations with Tn7. However, programmable targeting of transposons was impossible until CRISPR-associated transposase (CasTn) systems were described. Like other CRISPR-derived systems, CasTn can be programmed with a short DNA encoded sequence that is transcribed into a guide-RNA. Here we describe a broad-host-range CasTn system and demonstrate its function in bacteria from three classes of the Proteobacteria. The CasTn genes are expressed from a broad-host-range replicative plasmid, while the guide-RNA and transposon are provided on a high-copy pUC plasmid that is suicidal in most bacteria outside of E. coli. Using our CasTn system, single-gene disruptions were performed with on-target efficiencies approaching 100% in the Beta- and Gammaproteobacteria, Burkholderia thailandensis, and Pseudomonas putida, respectively. The results were more modest in the Alphaproteobacterium Agrobacterium fabrum, with a peak efficiency of 45%, though for routine single-gene disruptions, this efficiency is adequate. In B. thailandensis, the system allowed simultaneous co-integration of transposons at two different target sites. The CasTn system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in P. putida. Given the iterative capabilities and large payload size, this system will be helpful for genome engineering experiments across several fields of research. SignificanceThe genetic modification of bacteria to disrupt native genes and integrate recombinant genes is necessary for basic and applied research. Traditional methods for targeted disruptions and insertions are often cumbersome and inefficient, limiting experiments' scale and throughput. This work developed a system for targeted transposon mutagenesis that is easy to use, iterative, and efficient. We demonstrate that the system functions across three different classes of the Proteobacteria in species widely used in research and biotechnology. Moreover, the framework of the system and accompanying plasmids that we developed will facilitate porting the system to other bacteria. Our system provides a fast and efficient protocol to genetically modify these bacteria by inserting desired genetic cargo into specific genomic targets.

synthetic biology↗

bioRxiv2021

GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes

BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens. FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes. ConclusionsGuidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process smaller eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

bioinformatics↗

bioRxiv2020

Potential For Applying Continuous Directed Evolution To Plant Enzymes

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in todays agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cells growth rate to the target genes function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

synthetic biology↗