Efficient and reliable protoplasting, regeneration, and fusion techniques were established for the prototrophic strain Bacillus stearothermophilus NUB36. Auxotrophic mutants were isolated, and protoplast fusion was used to construct isogenic mutant strains and for chromosomal mapping. Markers were mapped using two-, threeP., and four-factor crosses. The order of the markers was hom-1-thr-1-his-1(gly-1 or gly-2)-pur-1-pur-2. These markers may be analogous to hom, thrA, hisA, glyC, and purA markers on the Bacilus subtilis chromosome. No analogous pur-l marker has been reported in B. subtlis. The relative order of three of the markers (hom-1-thr-1-gly-1) was independently confirmed by transduction.Insights into the molecular mechanisms of thermophily have been hampered because virtually nothing is known about the genetic basis of thermophily. This gap in our knowledge is related to the lack of an efficient genetic exchange system in a thermophilic bacterial species.Research on thermophile genetics has been confined mostly to Bacillus stearothermophilus and related thermophilic bacilli. Of the various traditional modes of gene transfer, DNA-mediated transformation was used for screening experiments because conjugation has not been demonstrated in the genus Bacillus and transduction occurs at too low a frequency to be useful for preliminary studies.Over the past 20 years, a majority of the standard strains of B. stearothermophilus have been screened for genetic transformation by using procedures that were developed for B. subtilis. To our knowledge however, there are only two published reports of genetic transformation in this organism (17,30). The low frequency of transformation and the lack of reproducibility precluded the use of these systems for genetic studies.The lack of success encountered in these studies underlines the relevance of certain practical considerations in the development of a genetic exchange system in a strain of interest. The first consideration is that the standard strains of B. stearothermophilus are most likely noncompetent. Only a few bacterial strains have evolved a mechanism for the uptake and expression of DNA, and even in the bestcharacterized bacterial transformation systems, many wildtype strains are noncompetent. In others, only certain auxotrophic mutants are competent. A second consideration is the need for a battery of auxotrophic markers to facilitate the genetic analysis of B. stearothermophilus. A few mutants of B. stearothermophilus have been isolated, but a majority of these are either antibiotic-resistant mutants or mutants lacking a particular enzyme activity (19). There is only one report on the isolation of auxotrophic mutants (25). This is not surprising since most standard strains of B. stearothermophilus have relatively complex nutritional requirements (3, 5). In the absence of prototrophic strains that grow well in a minimal medium, it is not possible to isolate auxotrophic * Corresponding author. (12) for Bacillus licheniformis were used to screen the strains for tr...

Introduction. Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world. Aim. We aim to introduce a CRISPR–Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species. Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR–Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa. Results. We have constructed plasmids carrying a CRISPR–Cas9 system based on that of S. pyogenes , which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR–Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site. Conclusion. We have constructed shuttle plasmids carrying a CRISPR–Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.