Four genes (ssp genes) coding for small, acid-soluble spore proteins of Bacillus megaterium and the gene for the protease that cleaves them during germination were cloned in the integratable plasmid pJHlOl. Each plasmid was integrated into the B. megaterium chromosome by a Campbell-type mechanism, allowing mapping of all five genes. The gene for the small, acid-soluble spore protein-specific protease (gpr) mapped near rib, and the sspA gene mapped between argA and hisA. The three other genes of the spp gene family (sspB, -D, and -F) all mapped near metCID, with the order: sspF-sspD-metCID-hemA-argO-sspB. While neither gpr nor sspF has been mapped in B. subtilis, the positions of the sspA, -B, and -D loci are similar in B. megaterium and B. subtilis, suggesting that the members of this multigene family have not recently undergone significant movement on the chromosome. It appears that more gene rearrangement has occurred in the flanking genes than has occurred in the ssp family of genes producing the small, acid-soluble spore proteins.Approximately 20% of the protein of Bacillus megaterium spores is made up of a group of small, acid-soluble spore proteins (SASP) which are synthesized only during sporulation (6). The SASP are degraded early in spore germination, providing amino acids for protein synthesis at this time, and their degradation is initiated by a protease that recognizes a specific amino acid sequence present in all SASP (6). 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).The multigene nature of the SASP-A/C gene family as well as a single SASP-B gene have also been found in B. subtilis, and to date five SASP genes have been cloned and mapped in this organism (2,3,12). Strikingly, all five genes, including four SASP-A/C-type genes, are scattered about the chromosome (12). Major questions concerning these SASP genes, in particular, the multigene family of type A/C, include how and when they arose in evolution and how they became scattered on the chromosome. Data that might lead to answers to these questions include the chromosomal locations of SASP genes in an organism somewhat diverged from B. subtilis. Since genes coding for eight SASP (ssp genes), as well as the gene for the sequence-specific protease which acts on SASP during spore germination (gpr gene), have been cloned from B. megaterium (6-9, 11; M. D. Sussman and P. Setlow, unpublished results), it seemed worthwhile to devise methods to map these genes.Considerable progress has been made in the mapping of the B. megaterium chromosome, using phage MP13; to date, approximately 40% of the chromosome has been joined by linkage groups, and a group of strains has been constructed to fa...