Genetic analysis of Bacillus stearothermophilus by protoplast fusion

Abstract

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...

Application of a recN sequence similarity analysis to the identification of species within the bacterial genus Geobacillus

“…However, more recent measurements (Nazina et al, 2001) show that the G+C content for G. Group 6B includes NUB3621 (=BGSC 9A5), doubtless the most well-characterized Geobacillus strain from a genetic standpoint. Systems for plasmid transformation (Wu & Welker, 1989), generalized transduction (Welker, 1988) and protoplast fusion (Chen et al, 1986) have been described for this strain, and data generated from those studies have revealed a circular genetic map (Vallier & Welker, 1990). Although the research literature describes NUB3621 as G. stearothermophilus, the recN and 16S rRNA gene sequence analysis presented in Fig.…”

Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation

“…Aeromonas hydrophila RS61-6, Pseudomonas aeruginosa PA-3-11, Escherichia coli DH5aF', and B. subtilis BR151 were grown in Luria broth (LB) medium (Gibco BRL) at 37°C. Bacillus stearothermophilus NUB 3621 was grown at 65°C in LB medium supplemented with Castenholtz salts and trace elements as described by Chen et al (4). Haloferax volcanii DS2 was grown at 37°C in a yeast-tryptone-salts medium described by Nieuwlandt et al (23).…”

Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription

“…The bacterial strains and plasmids used in this study are described in Table 1. B. stearothermophilus cultures were grown in modified Luria-Bertani (LB) medium (4), minimal glucose (MG) medium (4), and MG medium supplemented with 0.1% Bacto-Casamino Acids (Difco Laboratories, Detroit, Mich.) (MGCA medium). MG medium that did not contain NH4NO3 and nitrilotriacetate (nitrogen-free MG medium) was used in some experiments.…”