Structural and germination defects of Bacillus subtilis spores with altered contents of a spore coat protein.

The start sites for transcription and translation of a Bacillus subtilis spore coat protein gene, cotT, were determined. The CotT protein was synthesized as a 10.1-kDa precursor which was processed to a coat polypeptide of 7.8 kDa. Insertional inactivation of the cotT gene resulted in spores with an altered appearance of the inner coat layers and slow germination in response to a germination solution containing fructose, glucose, and asparagine. Rates of germination in L-alanine and in Penassay broth were the same as that of the wild type. A strain containing the cotT gene on a low-copy-number plasmid produced spores with an excess of CotT precursor and a thickening of the striated inner coat. These spores responded poorly to all of the germinants mentioned above. A site-directed mutation of the codon at the processing site of CotT resulted in the accumulation of the precursor in sporulating cells and on the spores, but there was no effect on the germination rates or solvent resistance of these spores. Both the lack and the overproduction of CotT led to subtle alterations in the structure of the inner spore coat and in the capacity of spores to respond to particular germinants.

Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease.

A colony screening procedure was devised to detect Bacillus subtilis mutants containing temperature-sensitive trypsin-like intracellular protease activity. The enzyme was characterized as a non-sulfhydryl serine protease on the basis of inhibitor studies. It was also inhibited by D- or L-histidine but not by any other amino acid tested. The long-term survival at 45 degrees C of these mutants in a minimal salts medium was decreased, with rapid lysis occurring within 24 h. A D-histidine function in long-term survival and inhibition accounted for the presence of additional protease mutants among survivors of histidine auxotrophs selected for their ability to utilize D-histidine. In addition to being lysed when incubated at 45 degrees C under nongrowth conditions, all of the protease mutants had a decreased rate of protein turnover and produced spores deficient in a major low-molecular-weight spore coat polypeptide. The morphology of the undercoat layers was altered, but there was no effect on spore heat resistance or on germination. The missing spore coat polypeptide appeared to be processed from a larger precursor by cleavage to produce N-terminal histidine. A defect in this protease could account for the lack of processing and thus the absence of this polypeptide in spore coats.