Branched pattern of regulatory interactions between late sporulation genes in Bacillus subtilis.

We measured the synthesis of dipicolinic acid (DPA) during sporulation in spo mutants of Bacillus subtilis by a sensitive biological assay based on cross-feeding of a spoVF mutant strain and also chemically. Many spo mutations, including several that block sporulation at stage III, did not prevent synthesis of DPA but instead prevented its incorporation into the spore. In general, strains with mutations in loci that are expressed in the spore compartment synthesized DPA, whereas strains with mutations in most of the loci that are expressed in the mother-cell compartment did not. Transcription of the gerE gene, as measured by DNA-RNA hybridization, followed a dependence pattern very similar to that of DPA synthesis. However, the dependence patterns of the two operons show that at about stage IV of sporulation there is a branch in the sequence of operon expression in the mother cell. One branch leads through spoVC to synthesis of DPA synthetase, and the other leads through spoVD to expression of gerE.

Cloning and dependence pattern of the sporulation operon spoVH.

The spoVH locus, involved in the sporulation of Bacillus subtilis, was cloned in derivatives of the temperate bacteriophage luminal diameter 105. Two recombinant phages were obtained which contained 4.2 kilobases of chromosomal DNA. Both phages only partially complemented a mutation in the spoVH operon, spoVH516. Nevertheless, analysis of the cloned locus with integrational plasmids showed that the complete operon had been cloned. A spoVH'-lacZ transcriptional fusion was constructed, and this indicated that the spoVH operon was expressed 2.25 h after the start of sporulation. The distribution of beta-galactosidase in sporulating cells containing a spoVH'-lacZ fusion showed that spoVH was expressed in the spore compartment; lac fusion experiments were also used to study spoVH expression in the presence of other sporulation mutations. Expression of spoVH was prevented by mutations in any of the stage 0 or stage II loci and also by mutations in spoIIIA, spoIIIB, and spoIIIE. A similar pattern of dependence was found previously for the expression of spoVA, which is also expressed in the spore compartment.

Nucleotide sequence of the sporulation operon, spoIIIE, of Bacillus subtilis.

A fragment of Bacillus subtilis DNA 3490 bp long, capable of complementing spoIIIE mutations, was sequenced. The region of the fragment that encodes functions required for sporulation was delimited using integrational plasmids. Sequencing showed that this region contained an operon with two open reading frames together with associated ribosome-binding sites. The deduced translation products would be polypeptides of 518 and 252 amino acid residues. Several sequences resembling promoters recognized by RNA polymerase containing sigma 29 occur in the region preceding the larger open reading frame. Although no transcription-termination signal was identified downstream of the smaller coding region, analysis with integrational plasmids and determination of the size of spoIIIE messenger RNA suggest that the locus does not contain a third gene.

Regulation of stage II of sporulation in Bacillus subtilis.

A mutation, spo-87, in the spo0J locus of Bacillus subtilis allows appreciable transcription of spoIIA, spoIID and spoIIG and later operons, even though most of the cells are morphologically blocked at stage 0 and the incidence of heat-resistant spores is about 1 per 10(4) cells. This mutation therefore appears to disengage the genetic control of sporulation from the morphological changes to which it should be connected. spoIIA and spoIIG are transcribed independently of one another. However, the products of both operons are needed for the activation of spoIID which occurs later. This indicates a convergence of parallel pathways of operon expression. We have also shown that nonsense mutations in spoIIAC (which codes for a sigma factor of RNA polymerase) prevent transcription of spoIID; by contrast, missense mutations in the same gene allow transcription of spoIID.

The possible DNA-binding nature of the regulatory proteins, encoded by spoIID and gerE, involved in the sporulation of Bacillus subtilis.

The predicted polypeptide products of two genes, spoIID and gerE, which appear to be concerned in the regulation of spore formation in Bacillus subtilis have been compared by modelling methods with known DNA-binding proteins. The results indicate that both polypeptides may have DNA-binding properties and the conclusion is drawn that this may account for their regulatory action.

Dependent sequences of gene expression controlling spore formation in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple form of differentiation that involves the formation of a two-cell organism; mother cell and prospective spore. It is regulated by at least 50 operons, some mono- and some polycistronic. These are expressed in a dependent sequence which branches at an early stage so that there are independent lines of expression in both cells; further ramifications then occur in both cell compartments. Three DNA-binding proteins and two sigma factors have so far been identified as probable regulators of the dependent sequence. Most of the operons of the sequence are expressed in the first four hours and the last two stages occur mainly by self-assembly of proteins.

Longevity of microorganisms in natural environments.

Bacterial endospores are exceptional among living systems in respect to their resistance to adverse environmental conditions, notably to heat and dessication, and consequently might be expected to show crytobiosis of very long duration. This report considers recent observations indicating that the maximal longevity of bacterial spores is probably much greater than previously believed.

Synthesis of spoIIA and spoVA mRNA in Bacillus subtilis.

The expression of the spoIIA and spoVA sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoVA loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.

Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis.

A spoIIA::lacZ gene fusion has been used to investigate the dependence pattern of expression of the spoIIA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the hybrid gene, begins to appear about 30 to 60 min after the induction of sporulation. spoIIA expression is dependent upon the products of all of the known spoO loci but on none of the 'later' loci tested. The beta-galactosidase activity falls after 1.5 h in Spo+ cells and in late-blocked mutants, but continued accumulation of the enzyme occurs in certain stage II mutants. Kinetic experiments suggest that the fall in activity may be, in part, the result of regulation at the level of translation. Mutations in several loci, spo0J, spoIIIF and spoVIC, delay expression of the operon by 1-3 h. The significance of these results in terms of models for the control of gene expression during sporulation is discussed.

Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis.

A spoVAA::lacZ gene fusion has been used to study expression of the spoVA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoVA mutations are manifested. spoVA expression is dependent on all of the known spo0 and spoII loci and on some of the 'early' spoIII loci, but not on 'later' loci. Several lines of evidence suggest that spoVA expression occurs only in the spore compartment. The implications of this observation for models of the overall regulation of gene expression during sporulation are discussed.

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].

Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIIC in spo mutants of Bacillus subtilis: a branched pathway of expression of sporulation operons.

The sporulation gene spoIIIC from Bacillus subtilis was fused to the lacZ gene from Escherichia coli, so that the transcription of the lacZ gene was under the control of the spoIIIC promoter. Production of beta-galactosidase, under conditions of sporulation, was then used as an indicator to study the expression of spoIIIC in relation to other sporulation loci. Expression of spoIIIC, which occurred only in the mother cell compartment, was prevented by mutations in all of the stage 0 and stage II loci, and also by spoIIID, spoIVA and spoIVB mutations. By contrast, the last three operons are not needed for expression of spoVA, which has previously been shown to be spore-specific. In consequence, a branched pathway of gene expression is proposed. One branch leads to expression of spoVA within the spore compartment, the other to expression of spoIIIC in the mother cell.

The nucleotide sequence and the transcription during sporulation of the gerE gene of Bacillus subtilis.

We have determined the nucleotide sequence of a 1496 bp stretch of Bacillus subtilis chromosome that complements the gerE36 mutation. The sequence contains three open reading frames. One of these is part of the sdhC gene, the other two, whose functions are unknown, are capable of encoding proteins with Mr values of approximately 17,000 and 8500. The use of integrational plasmids to delimit the gerE transcriptional unit shows that the gerE locus consists of one gene, which encodes a polypeptide of 74 amino acid residues and which is switched on after t3 during sporulation of wild-type B. subtilis 168.

Cloning and sequencing of a gene from Bacillus amyloliquefaciens that complements mutations of the sporulation gene spoIID in Bacillus subtilis.

A segment of DNA from Bacillus amyloliquefaciens, which complemented a mutant sporulation gene, spoIID68, in Bacillus subtilis, was cloned into a derivative of the temperate bacteriophage phi 105. The segment of DNA included an entire structural gene and complemented the mutation spoIID298, in addition to spoIID68, in B. subtilis. The nucleotide sequence of the gene from B. amyloliquefaciens was determined and compared with that of the B. subtilis gene; 74% homology was found in the coding region. Amino acid primary sequences derived from the nucleotide sequences of the two genes were also compared. The gene from B. amyloliquefaciens coded for a protein of 344 amino acid residues, one more than the protein coded by the corresponding gene from B. subtilis. Comparison of the primary amino acid sequences of the two genes showed that 78% of the residues were completely conserved and 8% were semi-conserved. Variation, however, was not random, i.e. some segments were much more highly conserved than others. Both proteins had a hydrophobic region at the N-terminus.

spoIID operon of Bacillus subtilis: cloning and sequence.

The locus spoIID, involved in the sporulation of Bacillus subtilis, was cloned into derivatives of the temperate phage phi 105. Two recombinant phages were obtained which contain chromosomal DNA covering 1.6 kbp. They are both able to complement mutations spo-68 and spo-298. These mutations, which were believed to be in different loci, spoIID and spoIIC respectively, were shown to be closely linked, and both map at the position assigned to spoIID on the genetic map of B. subtilis. The sequence of 1656 bp carrying the spoIID locus was determined. Only one open reading frame was found; this codes for a protein of 343 amino acids. It is preceded by a ribosome binding site and possible recognition sequences for sigma 32- and sigma 29-RNA polymerases. Studies of the locus by means of integrational plasmid vectors defined the outer limits of the transcriptional unit. These results are completely compatible with the sequence data. The combination of sequence and mapping and the information obtained by the use of integrational plasmids confirm that the spoIID locus functions as a monocistronic operon.

spoVIC, a new sporulation locus in Bacillus subtilis affecting spore coats, germination and the rate of sporulation.

A mutation near cysB on the Bacillus subtilis chromosome marks a new sporulation locus, spoVIC. It causes spores to germinate more slowly than those of the wild-type under all conditions and, from indirect evidence, it does not appear to alter the affinity for the germinant L-alanine. The mutant spores have some deficiency of coat proteins (particularly the alkalisoluble coat protein, Mr = 12 000) and the spore coat layers are disorganized. The mutant strain grows normally and sporulates normally until stage II, after which its sporulation is delayed by about 2 h compared to that of the wild-type. This delay results in the prolonged synthesis of some coat proteins and the late synthesis of others. The abnormal coat may be the cause of the germination deficiency. A double mutant strain carrying the spoVIC610 mutation together with gerE36 sporulates slowly. Its spores have very little coat protein, are sensitive to heat, lysozyme and organic solvents, but germinate as well as the strain carrying the spoVIC mutation alone. The role of the spore coat in germination is discussed in the light of these findings.

Protease production during sporulation of germination mutants of Bacillus subtilis and the cloning of a functional gerE gene.

Early in sporulation, cells of wild-type Bacillus subtilis produce three proteases (b, c and d) with monomeric Mr values of about 65 000, 53 000 and 43 500, and a further protease, e (Mr about 30 000) at the time of coat assembly. An additional protease, f (Mr about 15 000) appears transiently in sporangia at about the time of spore release. Three strains with defective spore coats were examined for alterations in sporulation proteases. A strain carrying the gerE36 mutation produces b, c and d normally, fails to produce e and accumulates f on or in its spores. A strain carrying the spoVIC610 mutation produces normal quantities of proteases b, c and d, but has a reduced amount of proteases e and f. A strain carrying both the gerE36 and the spoVIC610 mutations accumulates neither protease e nor f. The wild-type allele of the gerE gene was cloned in the vector, phage phi 105J9. Complementation tests with the cloned gene showed that the gerE36 mutation is recessive to the wild-type allele.

Cloning of the Bacillus subtilis spoIIA and spoV A loci in phage phi 105DI:1t.

A 6.95 kb HindIII-generated DNA fragment from Bacillus subtilis 168 was inserted into the DNA of phage phi 105DI:1t. The recombinant phage (phi 105DS1) contained DNA of 33.8 kb as compared with 35.2 kb for phi 105DI:1t and 39.2 kb for the wild-type phage. In the presence of helper phage, phi 105DS1 complemented both spoIIA and spoV A mutations in B. subtilis.

Genetic and phenotypic characterization of a cluster of mutations in the spoVA locus of Bacillus subtilis.

Twenty-nine mutants blocked during stage V of sporulation have been isolated following directed mutagenesis of the lys-1 region of the Bacillus subtilis 168 chromosome. All of a sample of eight mutants tested are unaffected in sporulation marker events up to stage IV but did not produce dipicolinic acid. They produced stable 'phase white' spores that were released from the mother cell, and were partially resistant to toluene and lysozyme but sensitive to chloroform and heat. Mutation spoV A89, known to be in the lys-1 region, showed similar phenotypic characteristics. Three-factor transformation crosses and recombination indices showed that the new mutations and spoV A89 lie in a single linkage group, which maps between lys-1 and another sporulation locus, spoIIA. The size of the spoV A locus is such that it probably contains several genes, and these may be contiguous with the cluster of genes included within the spoIIA locus.

Identification of a new sporulation locus, spoIIIF, in Bacillus subtilis.

We have isolated a mutant of Bacillus subtilis, strain 590, which is blocked at stage III of sporulation. The spo mutation which is carried by this strain is linked to pheA by transformation and defines a previously unidentified locus, spoIIIF. The spoIIIF locus is contiguous with the spoVB locus, in which a mutation causes a block at stage V of sporulation. We also give a detailed genetic map of the pheA region of the chromosome.