Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria (Burkholderia thailandensis and Pseudomonas putida, respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.

Recombineering is an important tool in gene editing, enabling fast, precise and highly specific in vivo modification of microbial genomes. Oligonucleotide-mediated recombineering via the in vivo production of single-stranded DNA can overcome the limitations of traditional recombineering methods that rely on the exogenous delivery of editing templates. By modifying a previously reported plasmid-based system for fully in vivo single-stranded DNA recombineering, we demonstrate iterative editing of independent loci by utilizing a temperature-sensitive origin of replication for easy curing of the editing plasmid from recombinant cells. Optimization of the promoters driving the expression of the system’s functional components, combined with targeted counterselection against unedited cells with Cas9 nuclease, enabled editing efficiencies of 90–100%. The addition of a dominant-negative mutL allele to the system allowed single-nucleotide edits that were otherwise unachievable due to mismatch repair. Finally, we tested alternative recombinases and found that efficiency significantly increased for some targets. Requiring only a single cloning step for retargeting, our system provides an easy-to-use method for rapid, efficient construction of desired mutants. Graphical Abstract

Microbes that survive transport to and in the stratosphere endure extremes of low temperature, atmospheric pressure, and relative humidity, as well as high fluxes in ultraviolet radiation (UVR). The high atmosphere thus provides an ideal environment to explore the genetic and physiological determinants conveying high tolerance to desiccation and UVR. In this study, we examined Curtobacterium aetherium L6-1, an actinobacterium obtained from stratospheric aerosol sampling that displays high resistance to desiccation and UVR. We found that its phylogenetic relatives are resistant to desiccation, but only C. aetherium displayed a high tolerance to UVR. Comparative genome analysis and directed evolution experiments implicated genes encoding photolyase, DNA nucleases and helicases, and catalases as responsible for UVR resistance in C. aetherium. Differential gene expression analysis revealed the upregulation of DNA repair and stress response mechanisms when cells were exposed to UVR, while genes encoding sugar transporters, sugar metabolism enzymes, and antioxidants were induced upon desiccation. Based on changes in gene expression as a function of water content, C. aetherium can modulate its metabolism through transcriptional regulation at very low moisture levels (X_w < 0.25 g H₂O per gram dry weight). Uncovering the genetic underpinnings of desiccation and UVR resistance in C. aetherium provides new insights into how bacterial DNA repair and antioxidant mechanisms function to exhibit traits at the extreme ends of phenotypic distributions.

Although the high-altitude limit for microbial survival in the Earth–atmosphere system has remained a scientific curiosity and topic of study, the ecological significance of long-distance microbial dispersal in the atmosphere has been perceived to have marginal relevance. Here, we report the characterization of novel plant pathogenic species of Curtobacterium that were isolated from samples collected at altitudes ranging from 1.5 to 29 km above sea level. Whole genome-based phylogenies of three strains, paired with plant challenge assays, indicate that each is a previously unrecognized species and causes disease on beans comparable to Curtobacterium flaccumfaciens . Isolates from the stratosphere (strain L6-1) and agricultural millet (G77) were identified to be the same species and designated as Curtobacterium aetherium sp. nov. C. aetherium displays high levels of tolerance to desiccation and UV radiation, which are stresses that increase in intensity with altitude. Back trajectory air mass analysis implied that the phytopathogens may have had an intercontinental source, but regional origins in the continental US cannot be excluded. The environmentally robust phytopathogens we have documented in the upper atmosphere provide new perspective on the role that high-altitude transport may play in microbial dispersal, gene flow, and the epidemiology of aerially dispersed plant disease. IMPORTANCE: Enormous quantities and varieties of microorganisms are continually aerosolized and transported in the atmosphere, yet there is a limited understanding of the consequences to downwind ecosystems. While studying bacteria that survive extreme conditions at altitudes up to 29 km in the atmosphere, we discovered new species that have the capacity to cause disease in agriculturally relevant bean varieties. The hardiest isolate we characterized from the stratosphere is a member of the same species as an isolate from an agricultural source, which we have designated Curtobacterium aetherium . C. aetherium is a phytopathogen capable of enduring high-altitude and long-distance atmospheric transport while also possessing the potential to opportunistically infect crops in deposition locations.

When Carl Sagan and Ed Salpeter envisioned potential sinkers, floaters, and hunters living in Jupiter’s clouds in 1976, the nature of life in Earth’s atmosphere remained widely unknown. Decades later, research has revealed a remarkable variety of microorganisms in our atmosphere. However, the spectral features of airborne microbes as biomarkers for detecting atmospheric life remained a mystery. Here, we present the first reflectance spectra of biopigments of atmospheric microorganisms based on laboratory cultivars of seven microbial strains isolated from Earth’s atmosphere. We show their distinct UV-resistant biosignatures and their impacts on models of diverse planetary scenarios, using Habitable Worlds Observatory parameters. The reflectance of these biopigments from aerial bacteria creates the means to detect them on other Earth-like planets. It provides a paradigm shift that moves the search for life beyond the surface of a planet to ecosystems in atmospheres and clouds.

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; λ= 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 LD ₉₀ 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. Importance: Ultraviolet 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).