The ability to construct defined genetic mutations in many bacteria is difficult and limited. Transposon mutagenesis is often highly efficient, but is not site specific, thus selections are often needed to identify mutants of interest. The construction of arrayed mutant libraries would help to fill this need, though these libraries are costly and time consuming. To enable easier construction of arrayed libraries we developed a workflow and methodology using a hierarchical barcoding scheme to identify mutants within a multiwell plate. We applied this method to the marine Alphaproteobacterium Ruegeria pomeroyi DSS-3 and created a library with over 2,800 disrupted genes.

Here, we report the genome sequences of three bacterial isolates that were cultured from aerosol samples collected at altitudes of 18 to 29 km above sea level. The isolates tolerate desiccation and shortwave UV radiation and are members of the actinobacterial genera Curtobacterium and Modestobacter and the betaproteobacterial genus Noviherbaspirillum .

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.

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 ssDNA can overcome the limitations of traditional recombineering methods which rely on the exogenous delivery of editing template. By modifying a previously reported plasmid-based system for fully in vivo ssDNA 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 expression of the system’s functional components, combined with targeted counter-selection 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 re-targeting, our system provides an easy-to-use method for rapid, efficient construction of desired mutants.

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 LD90in 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.