Switch protein alters specificity of RNA polymerase containing a compartment-specific sigma factor.

During sporulation in Bacillus subtilis, expression of developmental genes spoIVCB and cotD is induced in the mother cell compartment of the sporangium at morphological stages IV and V, respectively. A 27-kilodalton RNA polymerase sigma factor called sigma K (or sigma 27) has been found that causes weak transcription of spoIVCB and strong transcription of cotD. A 14-kD protein was also discovered that changes the specificity of sigma K-containing RNA polymerase, greatly stimulating spoIVCB transcription and markedly repressing cotD transcription. Both sigma K and the 14-kD protein are products of genes known to be required for expression of specific genes in the mother cell. Thus, sigma K directs gene expression in the mother cell and it is proposed that inactivation or sequestering of the 14-kD protein switches the temporal pattern of gene expression during the transition from stages IV to V of development.

Chromosomal rearrangement generating a composite gene for a developmental transcription factor.

Differential gene expression in the mother cell chamber of sporulating cells of Bacillus subtilis is determined in part by an RNA polymerase sigma factor called sigma K (or sigma 27). The sigma K factor was assigned as the product of the sporulation gene spoIVCB on the basis of the partial aminoterminal amino acid sequence of the purified protein. The spoIVCB gene is now shown to be a truncated gene capable of specifying only the amino terminal half of sigma K. The carboxyl terminal half is specified by another sporulation gene, spoIIIC, to which spoIVCB becomes joined inframe at an intermediate stage of sporulation by site-specific recombination within a 5-base pair repeated sequence. Juxtaposition of spoIVCB and spoIIIC need not be reversible in that the mother cell and its chromosome are discarded at the end of the developmental cycle. The rearrangement of chromosomal DNA could account for the presence of sigma K selectively in the mother cell and may be a precedent for the generation of cell type-specific regulatory proteins in other developmental systems where cells undergo terminal differentiation.

Regulation of sigma factor activity during Bacillus subtilis development.

Progression of Bacillus subtilis through a series of morphological changes is driven by a cascade of sigma (sigma) factors and results in formation of a spore. Recent work has provided new insights into the location and function of proteins that control sigma factor activity, and has suggested that multiple mechanisms allow one sigma factor to replace another in the cascade.

Fate of the SpoIIID switch protein during Bacillus subtilis sporulation depends on the mother-cell sigma factor, sigma K.

Sporulation of Bacillus subtilis involves the differentiation of two cell types, the mother cell and the forespore. Two key regulators of mother-cell gene expression are SpoIIID, a DNA-binding protein that activates or represses transcription of many different genes, and sigma K, a subunit of RNA polymerase that directs the enzyme to transcribe genes encoding proteins that form the spore coat. Previous studies showed that SpoIIID is needed to produce sigma K, but suggested that SpoIIID represses sigma K-directed transcription of genes encoding spore coat proteins. Here we show that a feedback loop connects the levels of sigma K and SpoIIID, such that production of sigma K leads to a decrease in the level of SpoIIID. The existence of the feedback loop was demonstrated by using antibodies prepared against SpoIIID to measure the level of SpoIIID during sporulation of wild-type cells, mutants defective in sigma K production, and a mutant engineered to produce sigma K earlier than normal. The feedback loop operates at the level of synthesis and/or stability of spoIIID mRNA, as demonstrated by measuring the level of spoIIID mRNA during sporulation of wild-type cells and mutants defective in sigma K production. Our results suggest that a rise in the level of sigma K during the stage (IV) of spore cortex formation causes a decrease in the level of SpoIIID, which, at least in part, establishes the switch to the stage V (spore coat formation) pattern of mother-cell gene expression.

Sporulation regulatory protein SpoIIID from Bacillus subtilis activates and represses transcription by both mother-cell-specific forms of RNA polymerase.

Mother-cell-specific gene expression during sporulation of Bacillus subtilis is controlled by sigma E and sigma K RNA polymerases. sigma E is required for the expression of genes during stage III (engulfment of the forespore), while sigma K is required for the expression of genes during stage IV (formation of the spore cortex) and stage V (formation of the spore coat). Previous studies indicated that SpoIIID could influence transcription by sigma K RNA polymerase in vitro. We demonstrate here that SpoIIID is a DNA-binding protein that recognizes specific sequences in the promoter regions and open reading frames of both sigma E- and sigma K-dependent genes. We also show that SpoIIID can activate or repress transcription by both forms of RNA polymerase. These results support the idea that the appearance and subsequent disappearance of SpoIIID plays a major role in controlling the mother-cell pattern fo gene expression during stages III to V of sporulation.

Regulation of the transcription of a cluster of Bacillus subtilis spore coat genes.

The pattern of transcription has been examined for a cluster of genes encoding polypeptides some or all of which are assembled into a cross-linked component of the Bacillus subtilis spore coat. Three promoters, designated PVWX, PX and PYZ, were indicated by reverse transcriptase mapping. On the basis of Northern hybridization, it appeared that the cotV, W and X genes were transcribed as a polycistronic mRNA from PVWX as well as a monocistronic cotX mRNA from Px. The cotY and cotZ genes are cotranscribed from the PYZ promoter with a smaller cotY mRNA resulting from premature termination or RNA processing. All four transcripts were synthesized late during sporulation and were not produced in mutants lacking sigma K, which directs RNA polymerase to transcribe genes in the mother-cell compartment of sporulating cells. The DNA-binding protein GerE, which affects transcription of many genes in the mother cell during the late stages of sporulation, was also shown to be involved. There was essentially no cotX mRNA in a gerE mutant and the amounts of cotVWX, cotYZ and cotY mRNAs were somewhat reduced. In vitro run-off transcription studies with sigma K RNA polymerase and GerE confirmed the presence of the three promoters, and directly showed that GerE was necessary for transcription from PX as well as enhanced transcription from the PVWX and PYZ promoters. The DNase I footprints of GerE for all three promoters were immediately upstream of the -35 regions. These GerE binding sites were compared to those in other GerE-responsive promoters and a larger consensus sequence for GerE binding was recognized. This complex transcriptional pattern of the cotVWXYZ cluster is probably necessary to ensure that an optimal amount of each protein is made for the assembly of the spore coat.

Identification of the promoter for a spore coat protein gene in Bacillus subtilis and studies on the regulation of its induction at a late stage of sporulation.

The cotA (pig) gene of Bacillus subtilis encodes a 65,000 Mr protein that is a component of the spore coat and is responsible for the brown pigment characteristic of colonies in which cells are undergoing sporulation. To study developmental regulation of the cotA gene, we identified its promoter and studied its transcription in a large number of mutants blocked at various stages of sporulation and germination. Deletion analysis showed that induction and efficient transcription of cotA required DNA sequences extending no more than 55 base pairs (bp) upstream (and no more than 130 bp downstream) from the 5' terminus of cotA mRNA. Transcription from the cotA promoter was found to be switched on at approximately the time (4 to 5 h after the onset of sporulation) of spore coat synthesis and deposition. Strikingly, this transcription was substantially inhibited in almost all asporogenous mutants blocked prior to the developmental stage (V) of spore coat formation. cotA transcription was also impaired in several stage V mutants but not in other stage V mutants or in mutants blocked in germination. The germination mutant gerE caused a several-fold overexpression of cotA. The dependence of cotA expression on so many genes required at early to intermediate stages of sporulation suggests that transcription of this spore coat gene is somehow coupled (directly or through several intervening steps) to a morphological or physiological feature(s) of the developing sporangium.

Sporulation regulatory protein GerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes.

The mother-cell line of gene expression during sporulation in Bacillus subtilis is a hierarchical cascade consisting of at least four temporally controlled gene sets, the first three of which each contain a regulatory gene for the next gene set in the pathway. gerE, a member of the penultimate gene set, is a regulatory gene whose products is required for the transcriptional activation of genes (coat protein genes cotB and cotC) in the last gene set. The gerE product also influences the expression of other members of the penultimate gene set (coat protein genes cotA and cotD appear to be repressed and activated, respectively). We now report that the purified product of gerE (GerE) is a DNA-binding protein that adheres to the promoters for cotB and cotC. We also show that GerE stimulates cotB and cotC transcription in vitro by RNA polymerase containing the mother-cell sigma factor sigma K. These findings support the view that GerE is a positively acting, regulatory protein whose appearance at a late stage of development directly activates the transcription of genes in the last known temporal class of mother-cell-expressed genes. In addition, GerE stimulates cotD transcription and inhibits cotA transcription in vitro by sigma K RNA polymerase, as expected from in vivo studies, and, unexpectedly, profoundly inhibits in vitro transcription of the gene (sigK) that encodes sigma K. The effects of GerE on cotD and sigK transcription are just the opposite of the effects exerted by the earlier-appearing, mother-cell regulatory protein spoIIID, suggesting that the ordered appearance of first SpoIIID, then GerE, ensures proper flow of the regulatory cascade controlling gene expression in the mother cell.

Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus.

A promoterless trp-lac fusion fragment was inserted near one end of the bacterial transposon Tn5 in the correct orientation to fuse lacZ gene expression to promoters outside Tn5. The resulting transposon, Tn5 lac, retains the kanamycin-resistance gene of Tn5 and transposes in Escherichia coli at 6% the frequency of Tn5 to many different sites in a bacteriophage lambda target. Expression of beta-galactosidase, the product of the lacZ gene, from Tn5 lac insertions in phage lambda depends both on insertion into a transcription unit in the correct orientation and on the regulation of the promoter of the transcription unit, verifying that by transposition Tn5 lac can fuse lacZ expression to outside promoters. An insertion of Tn5 lac in bacteriophage P1 was isolated and used to introduce Tn5 lac into Myxococcus xanthus, a bacterium that undergoes multicellular development. Stable kanamycin-resistant transductants are obtained that contain no P1 DNA sequences but have Tn5 lac inserted at different sites in the Myxococcus chromosome. Individual transductants express different levels of beta-galactosidase. A chromogenic substrate of beta-galactosidase, 5-bromo-4-chloro-3-indolyl beta-D-galactoside, is toxic in Myxococcus when cleaved in large amounts. In principle, Tn5 lac could be used to assay transcription in any bacterium in which Tn5 can transpose and beta-galactosidase can be measured.

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing sigmaA, the major sigma factor in growing cells.

Myxococcus xanthus is a Gram-negative bacterium that undergoes multicellular development upon starvation. We have developed a simple and rapid procedure for partial purification of RNA polymerase from growing M. xanthus cells, using heparin-agarose and DNA-cellulose chromatographies. In addition to core subunits, the enzyme contains one fairly abundant polypeptide of approximately 105 kDa. We have shown by Western blot analysis and protein sequencing that the 105-kDa polypeptide is sigmaA, the product of the M. xanthus sigA gene. Partially purified sigmaA RNA polymerase, or holoenzyme reconstituted from sigmaA and core RNA polymerase, transcribed in vitro the vegA and aphII genes that are known to be expressed in growing M. xanthus cells. Reconstituted sigmaA RNA polymerase produced vegA mRNA in vitro with the same 5' end as vegA mRNA produced in vivo, demonstrating that initiation of transcription was accurate in vitro. These results provide biochemical evidence that sigmaA is the major vegetative sigma factor of M. xanthus. To our knowledge, this is the first report of in vitro transcription of M. xanthus chromosomal genes, providing a foundation for further biochemical analysis of transcriptional regulatory mechanisms in a microbe that relies extensively on cell-cell interactions.

A feedback loop regulates the switch from one sigma factor to the next in the cascade controlling Bacillus subtilis mother cell gene expression.

Regulation of gene expression in the mother cell compartment of sporulating Bacillus subtilis involves sequential activation and inactivation of several transcription factors. Among them are two sigma factors, sigmaE and sigmaK, and a DNA-binding protein, SpoIIID. A decrease in the level of SpoIIID is thought to relieve its repressive effect on transcription by sigmaK RNA polymerase of certain spore coat genes. Previous studies showed that sigmaK negatively regulates the level of spoIIID mRNA. Here, it is shown that sigmaK does not affect the stability of spoIIID mRNA. Rather, sigmaK appears to negatively regulate the synthesis of spoIIID mRNA by accelerating the disappearance of sigmaE RNA polymerase, which transcribes spoIIID. As sigmaK begins to accumulate by 4 h into sporulation, the sigmaE level drops rapidly in wild-type cells but remains twofold to fivefold higher in sigK mutant cells during the subsequent 4 h. In a strain engineered to produce sigmaK 1 h earlier than normal, twofold less sigmaE than that in wild-type cells accumulates. SigmaK did not detectably alter the stability of sigmaE in pulse-chase experiments. However, beta-galactosidase expression from a sigE-lacZ transcriptional fusion showed a pattern similar to the level of sigmaE protein in sigK mutant cells and cells prematurely expressing sigmaK. These results suggest that the appearance of sigmaK initiates a negative feedback loop controlling not only transcription of spoIIID, but the entire sigmaE regulon, by directly or indirectly inhibiting the transcription of sigE.

Combined action of two transcription factors regulates genes encoding spore coat proteins of Bacillus subtilis.

During sporulation of Bacillus subtilis, spore coat proteins encoded by cot genes are expressed in the mother cell and deposited on the forespore. Transcription of the cotB, cotC, and cotX genes by final sigma(K) RNA polymerase is activated by a small, DNA-binding protein called GerE. The promoter region of each of these genes has two GerE binding sites. 5' deletions that eliminated the more upstream GerE site decreased expression of lacZ fused to cotB and cotX by approximately 80% and 60%, respectively but had no effect on cotC-lacZ expression. The cotC-lacZ fusion was expressed later during sporulation than the other two fusions. Primer extension analysis confirmed that cotB mRNA increases first during sporulation, followed by cotX and cotC mRNAs over a 2-h period. In vitro transcription experiments suggest that the differential pattern of cot gene expression results from the combined action of GerE and another transcription factor, SpoIIID. A low concentration of GerE activated cotB transcription by final sigma(K) RNA polymerase, whereas a higher concentration was needed to activate transcription of cotX or cotC. SpoIIID at low concentration repressed cotC transcription, whereas a higher concentration only partially repressed cotX transcription and had little effect on cotB transcription. DNase I footprinting showed that SpoIIID binds strongly to two sites in the cotC promoter region, binds weakly to one site in the cotX promoter, and does not bind specifically to cotB. We propose that late in sporulation the rising level of GerE and the falling level of SpoIIID, together with the position and affinity of binding sites for these transcription factors in cot gene promoters, dictates the timing and level of spore coat protein synthesis, ensuring optimal assembly of the protein shell on the forespore surface.

Overproducing the Bacillus subtilis mother cell sigma factor precursor, Pro-sigma K, uncouples sigma K-dependent gene expression from dependence on intercompartmental communication.

During sporulation of Bacillus subtilis, proteolytic activation of pro-sigma K and ensuing sigma K-dependent gene expression normally require the activity of many sporulation gene products. We report here that overproducing pro-sigma K at the onset of sporulation substantially uncouples sigma K-dependent gene expression from its normal dependency. Overproducing pro-sigma K in strains with a mutation in spoIIIG, spoIIIA, spoIIIE, or spoIVB partially restored sigma K-dependent gene expression in the mother cell and resulted in accumulation of a small amount of polypeptide that comigrated with sigma K, but these mutants still failed to form spores. In contrast, sporulation of spoIVF mutants was greatly enhanced by pro-sigma K overproduction. The products of the spoIVF operon are made in the mother cell and normally govern pro-sigma K processing, but overproduction of pro-sigma K appears to allow accumulation of a small amount of sigma K, which is sufficient to partially restore mother cell gene expression and spore formation. This spoIVF-independent mechanism for processing pro-sigma K depends on sigma E, an earlier-acting mother cell-specific sigma factor. The spoIIID gene, which encodes a mother cell-specific DNA-binding protein that is normally required for pro-sigma K production, was shown to be required for efficient pro-sigma K processing as well. bof (bypass of forespore) mutations bypassed this requirement for spoIIID, suggesting that SpoIIID is less directly involved in pro-sigma K processing than are spoIVF gene products. However, bof spoIIID double mutants overproducing pro-sigma K still failed to sporulate, indicating that SpoIIID serves another essential role(s) in sporulation in addition to its multiple roles in the production of sigma K.

Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions.

Certain developmental mutants of Myxococcus xanthus can be complemented extracellularly by wild-type cells. These mutants behave as if they are defective in cell-cell interactions that are required for development. There may be several different interactions because the mutants belong to four extracellular complementation groups (A, B, C, and D). We report here that B- and C- mutations change the pattern of gene expression during Myxococcus development as detected by transcriptional fusions to lacZ mediated by Tn5 lac. The mutant C locus reduced or abolished developmental beta-galactosidase expression from 15 lac fusions that normally begin to be expressed in wild-type cells after 6 hr of development. Expression of these C-dependent lac fusions was restored to C- mutants by adding wild-type cells. The C- mutation did not affect the expression of 10 lac fusions that normally begin to be expressed before 6 hr of development, indicating that the C-mediated cell-cell interaction is required beginning at about 6 hr of development. Cells require the B+ function very early in development because a B- mutation reduced or abolished developmental beta-galactosidase expression from all 26 lac fusions tested, including some that normally begin to be expressed at the onset of development. In a C- mutant and in a B- mutant, some lac fusions responded with reduced beta-galactosidase expression, whereas other fusions, which would normally begin beta-galactosidase expression at about the same time during development, expressed no beta-galactosidase, indicating that developmental genes within a given temporal class display different sensitivities to the absence of cell-cell interactions. Requirements for B+ and C+ function, as well as the previously described A+ function, appear to lie on the same developmental pathway.

A global analysis of developmentally regulated genes in Myxococcus xanthus.

Tn5 lac is a transposon that fuses the transcription of lacZ to exogenous promoters. We generated 2374 Tn5 lac insertion-containing strains of Myxococcus xanthus, a soil bacterium that undergoes multicellular development which culminates in the formation of spores. Thirty-six strains were identified that specifically increase beta-galactosidase expression at some particular time during development and these expression times range from minutes after starvation initiates development to 24 hr, when sporulation begins. Different maximum levels of beta-galactosidase expression were also observed and the maximum for many strains that begin beta-galactosidase expression late in development was observed only if spores were disrupted. Seven of the 36 strains display mild to severe defects in aggregation and/or sporulation, as did an additional five strains whose beta-galactosidase expression was not developmentally regulated. Restriction maps of the DNA adjacent to the Tn5 lac insertions that are developmentally regulated and/or cause developmental defects show that most of the 41 insertions are in different regions of the Myxococcus genome. The developmentally regulated Tn5 lac insertions described here provide a set of at least 29 new developmental markers for Myxococcus.

Intercellular signaling is required for developmental gene expression in Myxococcus xanthus.

Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta-galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant.

Defects in fruiting body development caused by Tn5 lac insertions in Myxococcus xanthus.

Mutations caused by insertions of Tn5 lac that block development are rare. At least six of the eight mutations examined appeared to be regulatory. Three of these were found to disrupt social motility, suggesting a particular importance for this function. One other occurred in a known cell-cell interaction gene, bsgA, and the remaining two were located in genes operative early in the developmental program.

Sporulation protein SpoIVFB from Bacillus subtilis enhances processing of the sigma factor precursor Pro-sigma K in the absence of other sporulation gene products.

Processing of inactive pro-sigma K to active sigma K in the mother cell compartment of sporulating Bacillus subtilis is governed by a signal transduction pathway emanating from the forespore and involving SpoIVFB in the mother cell. Coexpression of spoIVFB and sigK (encoding pro-sigma K) genes in growing B. subtilis or Escherichia coli enhanced pro-sigma K processing in the absence of other sporulation-specific gene products. The simplest explanation of these results is that SpoIVFB is a protease that processes pro-sigma K.

Effects of Bacillus subtilis sporulation regulatory protein SpoIIID on transcription by sigma K RNA polymerase in vivo and in vitro.

SpoIIID is a sequence-specific, DNA-binding protein that activates or represses transcription of different genes by sigma K RNA polymerase in vitro. A Bacillus subtilis strain engineered to produce both sigma K and SpoIIID during growth showed effects of SpoIIID on expression of sigma K-dependent genes that were consistent with the effects of a small amount of SpoIIID on transcription of these genes in vitro, indicating that the strain provides a simple, in vivo method to screen for effects of SpoIIID on transcription of sigma K-dependent genes.

Evidence that SpoIVFB is a novel type of membrane metalloprotease governing intercompartmental communication during Bacillus subtilis sporulation.

Processing of pro-sigma(K) in the mother cell compartment of sporulating Bacillus subtilis involves SpoIVFB and is governed by a signal from the forespore. SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments. Several conservative single amino acid changes in the HEXXH motif abolished function. However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function. Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence. Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases. A conservative change in this region of SpoIVFB abolished function. Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.