Three Bacillus subtilis genes (termed sspA, sspB, and sspD) which code for small, acid-soluble spore proteins (SASPs) have been cloned, and their complete nucleotide sequence has been determined. The amino acid sequences of the SASPs coded for by these genes are similar to each other and to those of the SASP-1 of B. subtilis (coded for by the sspC gene) and the SASP-A/C family of B. megaterium. The sspA and sspB genes are expressed only in sporulation, in parallel with each other and with the sspC gene. Two regions upstream of the postulated transcription start sites for the sspA and B genes have significant homology with the analogous regions of the sspC gene and the SASP-A/C gene family. Purification of two of the three major B. subtilis SASPs (a and j) and determination of their amino-terminal sequences indicated that the sspA gene codes for SASP-a and that the sspB gene codes for SASP-j. This was confirmed by the introduction of deletion mutations into the cloned sspA and sspB genes and transfer of these deletions into the B. subtilis chromosome with concomitant loss of the wild-type gene.A total of 10 to 20% of the protein of dormant spores of various Bacillus species is made up of a group of small, acid-soluble spore proteins (SASPs) (8). The SASPs are synthesized only during sporulation under transcriptional control and are the products of an extensive, divergent multigene family (8). During spore germination the SASPs are rapidly degraded to free amino acids, and these amino acids support much of the protein synthesis during the early minutes of this developmental period. While the amino acid storage function of the SASPs seems clear and has parallels in other dormant systems, it is possible that the SASPs serve other functions as well. This idea is attractive because the SASPs make up 25 to 50% of the protein of the spore core or protoplast and are present at a total concentration above 25 mg/ml (19). Indeed, there is circumstantial evidence that the SASPs are involved in the resistance of the dormant spores to . To definitively answer questions on the role of SASPs the dormant spores, the isolation of mutants with mutations in genes which code for major SASPs would be of obvious utility. However, the isolation of such mutants by classical procedures does not seem feasible. Consequently, we decided to generate SASP gene mutants in vitro by using cloned SASP genes and then to reintroduce the mutated genes into the bacterial chromosome with concomitant loss of the wild-type gene. We previously reported the cloning and nucleotide sequence of one SASP gene (originally termed the SASP-1 gene, now called the sspC gene [2]) from Bacillus subtilis (3), and in this communication we report the cloning and sequence of genes coding for three additional B. subtilis SASPs (sspA, sspB, and sspD genes) and show that the sspA and sspB genes code for two of the three major B. subtilis SASPs, ot and ,B, respectively. As described in the accompanying communication, the mnemonic ssp (spore-specific protein) will be used to ...

The chromosomal locations of four genes which code for small, acid-soluble spore proteins (SASP) in Bacillus subtilis have been determined. Although these four genes code for extremely homologous small, acid-soluble spore proteins (>65% sequence identity), the genes are not clustered but are megaterium there are at least seven genes coding for SASPs with greater than 65% amino acid sequience identity and that all of these genes are expressed (4). This identification of an extended, divergent multigene family in a procaryote is a novel finding and raises some obvious questions, one of which is the chromosomal location of these genes: i.e., whether they are clustered or scattered. A definitive answer to this question has been difficult to obtain with B. megaterium. Consequently, we have turned to the genetically well characterized organism B. subtilis, from which we have recently cloned four closely related genes, each of which codes for a SASP (1, 2). In this communication we report the determination of the chromosomal position of these four B. subtilis genes, Which we term ssp genes. The mnemonic ssp (spore-specific protein) is used for SASP genes, even though this abbreviation has been used before to describe a mutation resulting in the defective excision of spore-specific photoproducts (7). We thought that since the latter mutation has never been mapped, a more descriptive abbreviation could be found for such mutations (e.g., spe Construction of plasmids for integration into the chromosome. (i) sspA. Plasmid pMC3 containing a 0.7-kb EcoRIPvuII fragment from the sspA gene region was isolated as previously described (1). The sspA gene-containing fragment was cut out of this plasmid with EcoRI and HindIIl, giving a 0.7-kb fragment, of which a few nucleotides (including the HindIII site and a PstI site) were derived from pUC9 (1). This fragment (0.2 jig) was ligated with pJH101 (1 jig) that had been cut with HiindIII and EcoRI, and the mixture was used to transform E. coli RR101 to ampicillin and chloramphenicol resistance. The transformants were then screened for tetracycline sensitivity. The plasmids carried by several colonies were screened by restriction enzyme digestion, and one was chosen for further study. This plasmid contains ca. 125 nucleotides of the coding sequence of sspA plus ca. 575 nucleotides of downstream flanking sequence.(ii) sspB. Plasmid pHC201 was constructed as described in the accompanying communication (1). This plasmid contains the 0.7-kb EcoRI fragment from the sspB gene region, including ca. 75 base pairs of coding sequence, integrated in plasmid pJHiO1.(iii) sspC (previously termed the SASP-1 gene [2]). The cloned 3.2-kb EcoRI fragment containing the complete sspC gene was isolated as previously described and cleaved with HindIlI into 2.5-kb (containing only flanking sequence) and 0.7-kb (containing coding and flanking sequence) fragments (2). The 2.5-kb fragment was isolated, 0.3 jig was ligated with 1 ,ug of pJH101 which had been cut with EcoRI plus HindlIl, the mixture was used t...

The first Bacilus subtilis small, acid-soluble spore protein (SASP) gene has been cloned by using previously cloned B. megaterium SASP genes as DNA-DNA hybridization probes. Determination of the DNA sequence of the B. subtilis SASP gene showed that it codes for a 72-residue protein (termed SASP-1) containing a single spore protease cleavage site as well as other sequences conserved in BaciUus megaterium SASPs A, C, C-1, C-2, and C-3. The B. subtlis SASP-1 genes's coding sequence is preceded by a potential Bacilus ribosome-binding site, and is followed by a sequence that could form a stem-and-loop structure characteristic of transcription termination sites. Upstream from the coding sequence there are no obvious homologies with other B. subtlis sporulation genes, but similarities with B. megatenium SASP genes are evident. SASP-1 mRNA (290 bases long) is absent from vegetative cells, but appears midway in sporulation and then disappears. The cloned SASP-1 gene hybridizes to three bands other than the SASP-1 gene itself in EcoRI or Hindm digests of B. subtilis DNA. Presumably these other bands represent SASP genes related to the SASP-1 gene, and we have been able to detect at least three such proteins in B. subtilis spores.

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