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

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 (greater than 65% sequence identity), the genes are not clustered but are located at 70 degrees (adjacent to glyB [sspB gene]), 115 degrees (between metC and pyr cluster [sspD gene]), 180 degrees (between metB and kauA [sspC gene]), and 260 degrees (between ilvC and aroG [sspA gene]) on the B. subtilis genetic map.     

Different small, acid-soluble proteins of the alpha/beta type have interchangeable roles in the heat and UV radiation resistance of Bacillus subtilis spores.

Spores of Bacillus subtilis strains which carry deletion mutations in one gene (sspA) or two genes (sspA and sspB) which code for major alpha/beta-type small, acid-soluble spore proteins (SASP) are known to be much more sensitive to heat and UV radiation than wild-type spores. This heat- and UV-sensitive phenotype was cured completely or in part by introduction into these mutant strains of one or more copies of the sspA or sspB genes themselves; multiple copies of the B. subtilis sspD gene, which codes for a minor alpha/beta-type SASP; or multiple copies of the SASP-C gene, which codes for a major alpha/beta-type SASP of Bacillus megaterium. These findings suggest that alpha/beta-type SASP play interchangeable roles in the heat and UV radiation resistance of bacterial spores.     

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

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 pJH101. 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 metC/D, with the order: sspF-sspD-metC/D-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.     

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

The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP-gamma 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-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 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-gamma 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-gamma type of protein.     

Condensation of the forespore nucleoid early in sporulation of Bacillus species.

Fluorescence microscopic examination coupled with digital videoimage analysis of 4',6-diamidino-2-phenylindole-stained sporulating cells of Bacillus megaterium or Bacillus subtilis revealed a striking condensation of the forespore nucleoid. While both mother cell and forespore compartments had equal amounts of DNA, the forespore nucleoid became greater than 2-fold more condensed than the mother cell nucleoid. The condensation of the forespore nucleoid began after only the first hour of sporulation, 2 to 3 h before expression of most forespore-specific genes including those for small, acid-soluble spore proteins, and was abolished in spo0 mutants but not in spoII or spoIII mutants. It is possible that this striking condensation of forespore DNA plays some role in modulating gene expression during sporulation.     

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.     

Effects of major spore-specific DNA binding proteins on Bacillus subtilis sporulation and spore properties

Sporulation of a Bacillus subtilis strain (termed alpha(-) beta(-)) lacking the majority of the alpha/beta-type small, acid-soluble spore proteins (SASP) that are synthesized in the developing forespore and saturate spore DNA exhibited a number of differences from that of the wild-type strain, including delayed forespore accumulation of dipicolinic acid, overexpression of forespore-specific genes, and delayed expression of at least one mother cell-specific gene turned on late in sporulation, although genes turned on earlier in the mother cell were expressed normally in alpha(-) beta(-) strains. The sporulation defects in alpha(-) beta(-) strains were corrected by synthesis of chromosome-saturating levels of either of two wild-type, alpha/beta-type SASP but not by a mutant SASP that binds DNA poorly. Spores from alpha(-) beta(-) strains also exhibited less glutaraldehyde resistance and slower outgrowth than did wild-type spores, but at least some of these defects in alpha(-) beta(-) spores were abolished by the synthesis of normal levels of alpha/beta-type SASP. These results indicate that alpha/beta-type SASP may well have global effects on gene expression during sporulation and spore outgrowth.     

Characterization of a polysaccharide deacetylase gene homologue (pdaB) on sporulation of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis ybaN (renamed pdaB) exhibits high similarity to those of several polysaccharide deacetylases. Northern hybridization analysis with sporulation sigma mutants indicated that the pdaB gene is transcribed by EsigmaE RNA polymerase and negatively regulated by SpoIIID. The pdaB mutant was deficient in spore formation. Phase- and electron microscopic observation showed morphological changes of spores in late sporulation periods. The pdaB spores that had lost their viability were empty. Moreover, GFP driven by the promoter of the sspE gene was localized in the forespore compartment for the wild type, but was localized in both the mother cell and forespore compartments for phase-gray/dark forespores of the pdaB mutant. This indicates that GFP expressed in the forespores of the mutant leaks into the mother cells. Therefore, PdaB is necessary to maintain spores after the late stage of sporulation.     

Salmonella typhimurium secreted invasion determinants are homologous to Shigella lpa proteins

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes ( sspB , sspC , sspD , and sspA ), located between spaT and prgH , which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins lpaB, lpaC, lpaD and lpaA. A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87,42 and 36 kDa. Complementation analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA . sspC and sspD , but not sspA are required for invasion. Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK , homologous to the Shigella lpa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63,59 and 22 kDa was isolated from culture supernatants of wild-type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri lpa proteins.     

Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis

The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the beta-galactosidase gene, lacZ, of Escherichia coli so that the expression of beta-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of beta-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID::lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spo0 lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make beta-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA----[spoIIG, spoIIE]----[spoIID, spoIIB, spoIIF].     

A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis

A series of plasmids has been constructed that can be used to fuse the beta-galactosidase gene (lacZ) of Escherichia coli to chromosomal genes of Bacillus subtilis. Insertion of the lacZ gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase (cat) gene. The latter is included, along with the lacZ gene, in a single DNA fragment or 'cartridge' that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned B. subtilis gene are described that enable the lac-cat cartridge to be inserted at specific sites, or at random, directly into the B. subtilis chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as phi 105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the lac-cat cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, lac fusions to genes in the sporulation operons spoIIA and spoVA.     

Dramatic increase in negative superhelicity of plasmid DNA in the forespore compartment of sporulating cells of Bacillus subtilis.

Plasmid pUB110, isolated from vegetative cells of Bacillus subtilis, has an average of 34 negative supertwists (tau av = -34). This value falls to -30 early in sporulation, and the plasmid in the mother cell compartment maintains a tau av of -30. However, the plasmid within the developing forespore becomes much more negatively supercoiled, reaching a tau av of -47 in the dormant spore. This increased negative supercoiling in the forespore plasmid takes place in parallel with the synthesis of small, acid-soluble spore proteins, alpha and beta; and the plasmid from spores lacking small, acid-soluble proteins alpha and beta has a tau av of -40. The large increase in negative supercoiling of spore plasmid was also observed with Bacillus megaterium and in B. subtilis containing a plasmid with an origin different from that of pUB110. During spore germination plasmid pUB110 rapidly relaxed back to the tau av value characteristic of vegetative cells. It is possible that the observed changes in forespore plasmid topology are involved in modulating gene expression, DNA photochemistry, or both of these parameters in this compartment.     

Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.