Determination of the chromosomal locations of four Bacillus subtilis genes which code for a family of small, acid-soluble spore proteins

Abstract

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...

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

“…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). This was also found previously, when pJH101 derivatives were integrated into the B. subtilis chromosome (2,5,12).…”

“…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).…”

“…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).…”

Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains

“…Site-specific integration of foreign DNA linked to chromosomal sequences by homologous recombination has been described for many microorganisms and used to generate insertion mutants (32,33), to map and clone genes (6,18,30), to introduce defined deletions (17, 34), and to stabilize and amplify important traits (25,38).…”

“…Induction of this phospho-13-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-13-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-13-glucosidase that also hydrolyzes lactose-6-phosphate.Site-specific integration of foreign DNA linked to chromosomal sequences by homologous recombination has been described for many microorganisms and used to generate insertion mutants (32,33), to map and clone genes (6,18,30), to introduce defined deletions (17, 34), and to stabilize and amplify important traits (25,38).Recently, integration systems have been developed for Lactococcus lactis, a gram-positive bacterium widely used in industrial food fermentations, whereby randomly cloned chromosomal DNA (26), prophage sequences (5), natural plasmid DNA (13, 36), transposon DNA (4, 19) and insertion elements (37, 40) were used to provide the required homology. In some cases, multicopy integration of sequences to a level of about 15 copies per chromosome has been obtained, but their stability was low in the absence of selective pressure (5,26).…”