Thermostability and superhelicity of plasmid DNA in Bacillus stearothermophilus.

The thermostability of the staphylococcal plasmids pC194 and pUB110 and their antibiotic-resistance determinants was examined upon transfer to Bacillus stearothermophilus CU21. Plasmid pGS13, a pUB110 derivative carrying the chloramphenicol acetyltransferase (CAT) gene of pC194, could be maintained up to the maximum growth temperature (68 degrees C) by selection for chloramphenicol resistance. In the absence of selective pressure, pGS13 was lost at temperatures above 60 degrees C. Segregational instability of pGS13 was accompanied by a progressive loss of negative superhelicity at elevated temperatures. Thermostable mutants of pGS13 were isolated by screening for expression of the antibiotic-resistance determinants after growth under non-selective conditions. These mutants were found to contain an insertion of a 1.7 kb DNA sequence derived from the cryptic B. stearothermophilus plasmid pBS02. Increased thermostability correlated with preservation of plasmid superhelicity at elevated temperatures.

Synthesis and secretion of a heat-stable carboxymethylcellulose from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus.

The cellulase gene celA of Clostridium thermocellum coding for the thermostable endoglucanase A was transferred from Escherichia coli to Bacillus subtilis 168 and B. stearothermophilus CU21 using plasmids derived from the Bacillus vector pUB110. When the structural part of the gene was joined to a pUB110 promoter the recombinant plasmids (pSE102, pSE105) were stably maintained and expressed carboxymethylcellulose (CMCase) activity. In B. stearothermophilus CU21 (pSE105) the clostridial CMCase was produced over a wide temperature range up to the maximal growth temperature (68 degrees C). In contrast to E. coli, all of the CMCase synthesized in bacilli was released into the culture medium. About 50% of the extracellular protein secreted by B. subtilis 168 (pSE102) carrying the celA gene consisted of endoglucanase A. These findings demonstrate the feasibility of producing cellulolytic enzymes from thermophilic anaerobes in bacilli.