Cloning of the gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium.

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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 ...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cloning and nucleotide sequencing of genes for three small, acid-soluble proteins from Bacillus subtilis spores

Connors¹,

Mason²,

Setlow³

1986

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“…DNA fragments were separated by agarose gel electrophoresis, and individual fragments were isolated as previously described (5). The 0.65-kilobase (kb) MboI fragment containing the coding sequence of the B. megaterium SASP-B gene was also isolated as previously described (9).…”

Section: Methodsmentioning

confidence: 99%

“…B. subtilis DNA (50 ,ug) was digested with EcoRI and PvuII and run on a 1% agarose gel, and the DNA in regions of the gel containing fragments ranging in size from 1.0 to 1.8 and 3.5 to 5 kb was isolated as previously described (5). EcoRI linkers were added to the purified DNA, the samples were digested with EcoRI, and linker fragments were removed by spermine precipitation (11).…”

Section: Methodsmentioning

confidence: 99%

Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis

Hackett¹,

Setlow²

1987

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The BaciUus subtilis gene (sspE) which codes for small acid-soluble spore protein -y (SASP-,y) was cloned, and its chromosomal location (650, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP--y is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs. The SASP--y gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is -340 nucleotides long. The results of hybridization of an sspE gene probe to Southern blots of B. subtilis DNA suggested that there is only a single gene coding for the SASP-,y type of protein in B. subtilis. This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B. subtilis chromosome, with concomitant loss of the wild-type gene. This sspE deletion strain sporulated well, but lacked the SASP-y type of protein.

“…We decided to use this approach for two reasons. (i) Previous work had strongly suggested that SASP genes would not be transcribed in Escherichia coli from their own promoters (4,14), and (ii) several SASP genes, even if transcribed in E. coli, are either translated very poorly or the final protein is unstable (3,7,9,14). DNA (17).…”

mentioning

confidence: 99%

Cloning and nucleotide sequence of the Bacillus megaterium gene coding for small, acid-soluble spore protein B

Hackett¹,

Setlow²,

Setlow³

1986

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The Bacillus megaterium gene coding for small, acid-soluble spore protein (SASP) B was cloned and its nucleotide sequence was determined. The amino acid sequence predicted from the DNA sequence was identical to that determined previously for SASP B, with the exception of the amino-terminal methionine predicted from the gene sequence which is presumably removed posttranslationally and an asparagine residue predicted at position 21 which was originally identified as an aspartate residue. The mRNA encoded by the SASP B gene is synthesized for only a discrete period midway in sporulation, in parallel with mRNAs coding for other SASPs. The small size of the SASP B mRNA (365 nucleotides) indicated that the mRNA is monocistronic. The SASP B gene itself hybridized strongly to only one band in Southern blots of restriction enzyme digests of B. megaterium DNA, suggesting that the SASP B gene is not a member of a highly conserved multigene family, as is the case for other SASP genes.