Genes for Bacillus megaterium small, acid-soluble spore proteins: cloning and nucleotide sequence of three additional genes from this multigene family

Abstract

Three genes coding for small, acid-soluble spore proteins (SASP) were cloned from Bacillus megaterium, using previously cloned B. megaterium SASP genes (SASP-C and -C-3) as DNA-DNA hybridization probes. One gene (SASP-A) codes for the A protein, a previously identified major SASP. The other two (termed genes for SASP-C-4 and -C-5) are extremely similar in much of their nucleotide sequence to the previously cloned B. megaterium SASP-C-2 gene. The proteins coded for by all these SASP genes had extensive sequence homology with each other and with those coded for by the B. megaterium SASP-C, -C-1, -C-2, and -C-3 genes. Their coding sequences are preceded by strong ribosome-binding sites and are followed by regions of dyad symmetry which presumably are transcription stop sites. The SASP-A, -C-4, and -C-5 genes are expressed in parallel during sporulation, and their transcription start points were localized by the size of the mRNAs produced. The sequences localized 10 and 35 base pairs upstream from the transcription start points show significant homology with the analogous regions of the SASP-C, -C-1, -C-2, and -C-3 genes. The identification of seven closely related SASP genes in B. megaterium indicates that the SASP are the products of a very extensive multigene family.

Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease

“…1, lane b). This is exactly the result predicted if this pJH101 derivative had integrated into the chromosome by a Campbell-type mechanism at the site of the SASP-A gene (7). Similar analyses of drug-resistant clones derived from transformation with pJH101 derivatives carrying the SASP-C, SASP-C-3, SASP-C-4, or gpr gene indicated that these plasmids had also integrated into the chromosome by a Campbell-type mechanism at the site of the cloned gene carried on the transforming plasmid (data not shown).…”

“…There are two major types of SASP in B. megaterium, type A/C and type B, which differ significantly from each other in amino acid sequence (6,11). While there is only a single type B SASP gene, there are at least seven genes which code for type A/C SASP of extremely similar amino acid sequence, with two of these genes coding for the two major SASP of this type, termed SASP-A and SASP-C in B. megaterium (6)(7)(8)11). …”

“…Consequently, we now term the B. megaterium SASP-C and -C-3 genes the sspB and sspD genes. The B. megaterium SASP-C-4 gene is not the homolog of the other cloned B. subtilis A/C-type ssp gene (termed sspC [3,7]); consequently, we have termed the subtilis, the sspB and sspD genes map at 70°C and 115°C on the chromosome, respectively (2), with the argO locus at 100°C (15). Strikingly, in B. megaterium, the sspB and sspD loci are also weakly linked to the argO locus and on opposite sides (Table 1; Fig.…”

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

“…Whereas several other weakly hybridizing bands were seen in some digests (lanes d to f), the relative intensity of these bands was very much lower than that of the major band. In contrast, the various members of the SASP A and C gene family cross-hybridized much more strongly even under more restrictive hybridization conditions (55°C) (2,3,8). It is, of course, possible that there are multiple SASP B-like genes in B. megaterium but that they are not highly conserved and thus hybridize only very weakly to the SASP B probe.…”

“…The amino acid sequences of these proteins are known, and those of SASP A and C are extremely similar, whereas that of SASP B is much less closely related (14)(15)(16)(17). This system appears even more complex at the gene level because, in addition to the genes coding for SASP A and C, there are five other genes that code for proteins with amino acid sequences extremely similar (70 to 90% sequence identity) to those of SASP A and C (7)(8)(9). All of these closely related SASP Aand C-like genes are expressed in parallel midway in sporulation, although some (SASP A and C genes) are expressed at much higher levels than others (7)(8)(9).…”