Isolation of Thermophilic Mutants of Bacillus subtilis and Bacillus pumilus and Transformation of the Thermophilic Trait to Mesophilic Strains

Abstract

~ ~ ~~Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 "C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 "C and 70 "C at a frequency of less than 10-'O. The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of to lo-' for single markers and about lo-' for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about lo-', suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes. I N T R O D U C T I O NEvery organism has its own optimal temperature for growth, being either a mesophile, a thermophile or a psychrophile. The factors that dictate the optimum growth temperature have not been established. Identification of genes controlling growth temperatures of these different classes of organisms by genetic means, e.g. transformation, should provide information that will improve our understanding of the mechanisms that determine the range of temperatures over which different organisms can grow. McDonald & Matney (1 963) claimed the transformation of a characteristic which permitted a strain of Bacillus subtilis to grow at temperatures as high as 55 "C to other B. subtilis strains whose upper temperature limit for growth was 50 "C. Furthermore, Friedman & Mojica (1978) and Lindsay & Creaser (1975) both claimed transformation of the thermophilic trait from thermophilic 'Bacillus caldolyticus' or Bacillus stearoth~r~ic~philus to mesophilic B. subtilis. These observations have not been substantiated. The major difficulty in characterizing the thermophilic trait has been a lack of genetic evidence, because the organisms used for these studies were not genetically characterized.In this communication we report the isolation of thermophilic mutants from two genetically marked mesophilic species (B. suhtilis and Bacillus purnilus) and transformation of the thermophilic trait to mesophilic strains. Differences in their gene expression and behaviour over a range of temperatures are discussed. M E T H O D S5nc.torItr. The B. .stihtili,s strains used were 168 DB-1 from Dr Roy Doi, University of Texas, Arlington, Tx., USA : 1 A348 irpC,? rpoBSOO, resislant to 50 1.18 rifampicin ml-' , and I E4(pLS I3), resistant to 50 pg lincomycin ml-'. from the Bacillus Genetic Stock Center, Ohio State University, USA ; and 168 BR I5 I 1j.s-3 trpC2, requiring Iysine and tryptophan. The B. p r t t i i / t / s strains used were Bp203 Arg-, requiring arginine. and wild-type BpB

Temperature spans for growth: Hypothesis and discussion

“…Several genetic approaches which appear to be successful ?,ave been reported [44,451. Droffner and Yamamoto (45] concluded from their Bacillus mutants that t-~v~ genes are responsible for the conversion into thermophiles. A chemical oxidation to the disulfide increased the thermostability drastically, whereas the reduction ~tto SH-groups made the enzyme as labile as the wild-type enzyrc.~ [46].…”

Toxin-producing Escherichia coli isolates from composting pig waste

“…The process of composting has been divided into mesophilic, thermophilic and curing stages (Droffner and Yamamoto, 1991). Coliform organisms such as E. coli are expected to be killed during the thermophilic stage of composting as temperature ≥ 55 °C are recorded at this stage (Ryckeboer et al, 2003;Tiquia, 2005).…”

Transcriptional Activity at Supraoptimal Temperature of Growth in the Antarctic Psychrotrophic Bacterium Pseudomonas syringae

“…Mutations in a single critical gene (e.g., in temperature-sensitive mutants) can lower the maximum temperature of growth, but the factor(s) determining the whole limit of growth is not known. A few studies have been carried out in this direction [7,12,14,20], but none of them have been followed up with definitive conclusions.…”