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

Abstract

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 ...

Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E sigma F: identification of features of good E sigma F-dependent promoters

“…The Escherichia coli strain used for the construction of all plasmids was RR101, and all cloning work was carried out as previously described (2). Plasmid DNA from E. coli strains was isolated and purified by CsCI density gradient centrifugation as described previously (2). B. subtilis and E. coli strains were grown at 37°C in 2x YT medium (2); B. subtilis sporulation was carried out at 37°C in 2 x SG medium (7).…”

“…The B. subtilis strains and plasmids used in this work are listed in Table 1. The Escherichia coli strain used for the construction of all plasmids was RR101, and all cloning work was carried out as previously described (2). Plasmid DNA from E. coli strains was isolated and purified by CsCI density gradient centrifugation as described previously (2).…”

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

“…Due to the binding motif, the tlp promoter seemed to be controlled by the forespore-specific s F of C. acetobutylicum (Paredes et al, 2004;Jones, 2011). Downstream of each ssp gene a dyad-symmetrical DNA sequence is located, probably representing stem-loops of rho-independent transcription terminators (Connors et al, 1986;Vyas et al, 2011) (Fig. 3c).…”

N-terminal Amino Acid Residues Mediate Protein-Protein Interactions between DNA-bound α/β-Type Small, Acid-soluble Spore Proteins from Bacillus Species

“…The upstream primers were: SSPC (5Ј-CCATGGCTCAACAA AGTAGATC), SSPC⌬5 (5Ј-CCA-TGGCTAGATCAAACAACAATAATG), SSPC⌬10 (5Ј-CCATGGCTAAT-GATTTACTAATTCC), SSPC⌬11 (5Ј-CCATGGCTGATTTACT AATTC-CTCAAGC), SSPC⌬11-D13N (5Ј-CCATGGCTAATTTACTAATTCCT-CAA GC), SSPC⌬11-D13K (5Ј-CCATGGCTAAATTACTAATTCCTCA-AGCAGG), SSPC⌬14 (5Ј-CCATGGCTATTCCTCAAGCAGCTTCAGC); the downstream primer was SSPC2 (5Ј-AGCTGGATCCACCATTAGT-TCTGTATGG), complementary to nt 530 -547 in the sspC sequence (15). All upstream primers contained added NcoI restriction endonuclease sites (underlined residues) and the downstream primer contained a BamHI restriction site and 5Ј-flanking sequences (underlined residues), which were used for cloning purposes.…”

“…An NcoI restriction site was first introduced at the initiating methionine codon of the sspB gene by the PCR megaprimer method (17). The first round of PCR used the upstream primer SSPB1 (5Ј-ACGGCTAAGCTTTTTTTATT-TCTC), complementary to nt 173-196 of the sspB sequence (15), and the downstream primer SSPB-NCOI (5Ј-GAGTTTTGGTTAGCCATGGG-TAAAATCTCC), complementary to nt 357-386 of the sspB sequence (15) to generate a DNA fragment containing the sspB promoter with a site-directed mutation (underlined residue) creating the NcoI site. This PCR product was agarose gel-purified and used as the upstream primer in a second round of PCR with the downstream primer SSPB2 (5Ј-GGATCCCTTTTTTTCTAGGATATGTGGAGCAGG), complementary to nt 639 -665 of the sspB sequence (15) to generate a complete sspB gene with an added 3Ј BamHI site (underlined residues).…”