Chromosomal inactivation of Bacillus subtilis exfusants: a prokaryotic model of epigenetic regulation.

Epigenetic mechanisms are not exclusively reserved to eukaryotic organisms. They are also observed in prokaryotes. As described first by Hotchkiss and Gabor, protoplast fusion between strains of Bacillus subtilis produces heterodiploid cells. Heterodiploidy is associated with the inactivation of one of the chromosomes. To study the physical structure of the fusion product and the molecular mechanisms of inactivation, we constructed heterodiploid clones containing two chromosomes labeled by a NotI restriction fragment length polymorphism. In the progeny, we identified haploid recombinant clones that contain a chromosome carrying large regions of inactivated DNA. Studies of both recombinants of the latter kind and heterodiploid cells indicated that chromosomal inactivation was not determined by alteration of the inactivated nucleotide sequence, but was probably due to a modification in the structure of the bacterial chromatin.

Characterization of an lrp-like (lrpC) gene from Bacillus subtilis.

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilis lrp-like-gene is a bona fide Lrp protein-the first one to be detected in gram-positive bacteria. When expressed in E. coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B subtilis Lrp-like protein plays a role in the growth phase transition.

Overproduction, purification and characterization of the HPB12-L24 ribosomal protein of Bacillus subtilis.

HPB12-L24 was previously described as a bifunctional histone-like and ribosomal protein in Bacillus subtilis. In order to confirm the identity of HPB12 and L24, and to study the properties of this protein, the rplX gene of B. subtilis encoding L24 has been overexpressed in Escherichia coli by an efficient protein overproduction system. A simple and rapid purification scheme using ammonium sulfate precipitation and cation-exchange chromatography is presented. 10 mg of pure L24 per g of Escherichia coli cells were obtained. The purified recombinant protein L24 is heat-stable, acid-soluble and binds preferentially supercoiled DNA like protein HPB12. These results confirm the identity of HPB12 and L24. Overexpression of rplX led to gross alterations of cell morphology and to an abnormal shape of nucleoids.

Noncomplementing diploids from Bacillus subtilis protoplast fusion: relationship between maintenance of chromosomal inactivation and segregation capacity.

Fusions of Bacillus subtilis protoplasts from two genetically marked strains produce noncomplementing heterodiploid bacteria. These noncomplementing diploids (Ncds) carry both parental chromosomes, but only one is expressed. Fusion products of strains polymorphic for NotI restriction sites provide new physical evidence to support the conclusion that Ncds are not an artifact of cross feeding or cell adhesion. We show that reversible chromosomal inactivation can only account for the biparental trait of unstable Ncds. Two types of cells were recovered from the late progeny of unstable Ncds: Ncds with irreversible chromosome silencing (stable Ncds) and secondary recombinants that displayed a genomic mosaic NotI profile. Segregants from an unstable Ncd population gave rise to two viable haploid cell types. By contrast, stable Ncds segregated into a population of viable and inviable haploid cells. We propose that the latter are derived from irreversible chromosome silencing. Our results indicate that clonal populations of stable Ncds are heterogenous and suggest that segregation and inactivation are independent parameters.

Suppression of the Bgl+ phenotype of a delta hns strain of Escherichia coli by a Bacillus subtilis antiterminator binding site.

Bacillus subtilis, like Escherichia coli, possesses several sets of genes involved in the utilization of beta-glucosides. In E. coli, all these genes are cryptic, including the genes forming the bgl operon, thus leading to a Bgl- phenotype. We screened for B. subtilis chromosomal DNA fragments capable of reverting the Bgl+ phenotype associated with an E. coli hns mutant to the Bgl- wild-type phenotype. One B. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from the bgl gene. Deletion studies as well as subcloning experiments allowed us to prove that the putative B. subtilis of the E. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein of E. coli bgl operon by our putative B. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.

The Bacillus subtilis nucleoid-associated protein HPB12 strongly compacts DNA.

The HPB12 protein from the nucleoid of Bacillus subtilis was previously described, and its DNA binding properties have been reported previously (V. Salti, F. Le Hégarat, and L. Hirschbein, Biochim. Biophys. Acta 1009:161-167, 1989). The DNA-HPB12 complexes were examined by electron microscopy. They appeared as short, slightly curved rods whereas naked DNA showed no compaction. Since only a small number of complexes with an intermediate degree of folding were observed, it appears that the nucleoid-associated protein HPB12 binds cooperatively to DNA, confirming Salti et al. (V. Salti, F. Le Hégarat, and L. Hirschbein, Biochim. Biophys. Acta 1009:161-167, 1989), and gives rise to a tightly compacted DNA-protein complex. N-terminal sequencing of purified HPB12 showed that all but one of the first 26 amino acids were identical to those of the L24 ribosomal protein.

Establishment of a new replicon generated from an integrational plasmid and a cryptic pUB110 origin-like region in Bacillus subtilis.

A Bacillus subtilis integrational plasmid pVG4 (7.3 kb) was constructed. It was composed of a part of pBR322 (Ap, OriC), a part of pUB110 (Nm), and a part of the spoOA gene. The origin region of pUB110 had been deleted over 1.4 kb. Surprisingly, the replicative plasmids (8 kb) were generated by transformation of B. subtilis BG83 with pVG4 on selection with Nm. Analysis of the total DNA indicated the presence of repeated DNA and that pVG4 did not appear to integrate at the spoOA locus. The new replicon termed pYV exhibited a quite different restriction pattern from pVG4 due to dramatic DNA rearrangements. Although some components initially present in pVG4 were either present in pYV (Ap, oriC, and Nm) or lost (spoOA), other ones were newly gained such as a chromosomal fragment and a pUB110-type origin-like region. The latter was identified by restriction sites mapping and limited sequence analyses. By PCR amplification, the origin region of pYV was shown to be present as a cryptic sequence in the chromosome of strain BG83. The chromosomal fragment integrated into pYV was at least 0.25 kb long and located in a 19-kb SfiI fragment mapping at 106 degrees. We propose that the establishment of the new replicon pYV is the result of a genetic recombination between the pUB110 part present in the integrational plasmid pVG4 and the cryptic origin region of pUB110 harbored in the chromosome of the recipient strain BG83 in relation with a particular role of neomycin selection.

Purification and characterization of the HU-like protein HPB9 from the Bacillus subtilis nucleoid.

The Bacillus subtilis HPB9 is the major heat-stable and acid-soluble protein associated with the nucleoid isolated at low ionic strength. The abundance of the protein in the cell is estimated to about 20,000 monomers per cell (Salti et al. (1985) J. Gen. Microbiol. 131, 581-590). The protein cross reacts specifically with the antiserum against the Bacillus globigii HBg. Moreover, HPB9 is able to introduce negative supercoiling in a relaxed covalently closed circular DNA, in the presence of topoisomerase I as demonstrated by one and two-dimensional electrophoresis. These results indicate that the nucleoid associated protein HPB9 is an HU-like protein and could be involved in the DNA compaction.

Application of the mini-mu phage for the isolation of lac transcriptional fusions in Bacillus subtilis genes.

A cassette containing a selectable cat gene and the lacZ gene without its own promoter has been incorporated into the mini-Mu bacteriophage genome. This mini-Mu derivative, referred to as mMu-Bs, can be used in Escherichia coli for the generation of lacZ transcriptional fusions to Bacillus subtilis genes cloned into plasmids. The resultant fusions can be analyzed in B. subtilis either as multicopy plasmids or as a single copy integrated via a Campbell-like recombination into the wild-type locus of the cloned fragment.

The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis.

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non-complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows. (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously. (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.

Purification and properties of the DNA-binding protein HPB12 from Bacillus subtilis nucleoid.

We report the purification to homogeneity of a 12 KDa protein (HPB12) present in the nucleoids of Bacillus subtilis. From the purification data the abundance of the protein was estimated to about 20,000 monomers per cell. The HPB12 protein is heat-stable and acid-soluble and binds preferentially to supercoiled and linearized double-stranded DNAs. The binding of the protein to the supercoiled DNA occurs very rapidly and appears to be cooperative. Moreover, the complexes are extremely stable and do not dissociate after 90 min. These properties are consistent with a role of the HPB12 protein in the structure of the B. subtilis chromosome.

RecE-dependent lysogenic induction in the absence of repressor in Bacillus subtilis non-complementing diploids.

The RecE protein of Bacillus subtilis, known to be required for induction of the SOS response and of phi 105 prophage, was shown to be involved in mitomycin C induction of B. subtilis diploid lysogens carrying a silent phi 105 prophage in their unexpressed chromosome. These stable non-complementing diploid lysogens, formed by protoplast fusion and regeneration, did not synthesize repressor, so that the induction observed must have resulted from RecE-dependent activation of the prophage rather than from RecE-dependent inactivation of repressor. Mitomycin C treatment does not induce permanent expression of the silent chromosome, so the activation seems to be temporary, perhaps reflecting the action of an SOS function under RecE control.

Stabilized non-complementing diploids (Ncd) from fused protoplast products of B. subtilis.

Non-complementing diploids (Ncd) displaying the parental phenotype can be selected from polyethylene glycol (PEG)-treated fused polyauxotrophic protoplasts of Bacillus subtilis. These bacteria carry the two parental genomes, but only one of them is phenotypically expressed, the other being replicated but not expressed. Cellular cloning and DNA-DNA in situ hybridization led to the discovery of non-complementing diploid cells which at first sight could have been considered as parental haploids. The new class of stabilized Ncd (10(-7) segregants) can be obtained either directly after the primary fusion event or from segregating Ncd after further growth. The totally inactive chromosome of a stable Ncd can be activated after PEG-induced self fusion. DNA-mediated transformation studies using crude stable Ncd lysates as DNA donors show low frequencies for the genetic markers from the 'silent' chromosome. Contrary to the unstable Ncd situation, however, these frequencies remain low even with purified donor DNA. The differences in the transformation properties of the non-expressed markers are correlated to Ncd clone stability. These facts suggest that chromosome inactivation in PEG-induced fusion involves at least a two-stage process. The first would be reversible and the second irreversible, thus preserving the inactive chromosome state.

Isolation and characterization of small heat-stable acid-soluble DNA-binding proteins from Bacillus subtilis nucleoids.

Small heat-stable, acid-soluble proteins (HASP) have been isolated from Bacillus subtilis nucleoids obtained from cell lysates of low ionic strength and lysozyme concentration. They were identified by their ability to bind homologous and heterologous native and denatured DNA. Four major species, of 8.5, 12, 23 and 26 kDal, were found. Their affinity for DNA was moderate as measured by the sensitivity to ionic strength of the DNA-protein complex (0.1-0.4 M-NaCl). Partial digestion by micrococcal nuclease of the 'low ionic strength nucleoids' released a DNA fragment of 80-120 bp. The data reported here indicate that small basic proteins, together with other components such as RNA, cations and polyamines, may be involved in the compaction of the prokaryotic genome.

In vitro transcription from Bacillus subtilis nucleoids by homologous RNA polymerase.

The effect of DNA structural features on RNA synthesis was investigated. Purified Bacillus subtilis nucleoids templated from vegetative cells were transcribed by the homologous RNA polymerase using Hg-UTP as one of the nucleotide substrates. Low RNA polymerase/DNA ratios were used during transcription in order to avoid nonspecific initiations. The rate of synthesis of total RNA was 40% greater on nucleoid templates than on naked DNA. The proportion of asymmetric transcript synthesized on nucleoid templates (HvsL strand transcripts) was close to that observed in vivo, whereas with naked DNA this value was at least 3-times lower. The percentage of rRNA relative to the total RNA, synthesized in the in vitro system with the nucleoid template, approaches the rate of the in vivo transcription. The size of the RNA synthesized on nucleoids was large and heterogeneous while with naked DNA it was homogeneous and of about 6 S. Our results suggest that the supercoiled, folded nucleoids may retain some of the structural features responsible for the regulation of RNA synthesis in vivo.

Absence of functional RNA encoded by a silent chromosome in non-complementing diploids obtained from protoplast fusion in Bacillus subtilis.

The molecular basis for lack of phenotypic expression of one chromosome in Bacillus subtilis non-complementing diploid clones (Ncd cells) was investigated. Correlations between chromosomal inactivation and absence of functional transcripts were determined with wild-type prophage phi 105 or a thermoinducible mutant phi 105 cts23, on either the expressed or the silent chromosome. It appears that no significant amount of phage mRNA is detectable in Ncd cells carrying the prophage in the inactive chromosome. However, phi 105 mRNA represents 0.23% of total cellular mRNA in an Ncd strain with the prophage in the expressed chromosome and 0.28% in the parental lysogenic strain. The lack of an mRNA repressor of phi 105 prophage from the silent chromosome was confirmed by the absence of repressor activity in Ncd clones with a temperature sensitive mutant phi 105 located in the silent chromosome. After heat induction, no phage production was observed. As expected these clones do not exhibit phi 105 immunity when superinfected with the same phage. The combined data of the present and previous work suggest that control of phenotypic suppression of Ncd strains should, at the transcription level, involve a different DNA tertiary organisation in one of the two chromosomes.

Isolation and characterization of the nucleoid of non-complementing diploids from protoplast fusion in Bacillus subtilis.

Nucleoids of non-complementing diploids (Ncd) from protoplast fusion of B. subtilis were isolated. Their purified DNA banded in neutral CsCl gradient as a single unimodal peak of buoyant density 1.711 g/cm3, a value which is similar to that of the DNA purified from the original parental strains, suggesting that methylation of bases is not a significant factor in chromosome inactivation. Nucleoids released from a Ncd clone give two peaks in a sucrose gradient with a characteristic S value for each nucleoid. That is in contrast to nucleoids from the haploid parents whose sedimental patterns show only one peak. Both nucleoid preparations from Ncd strains assayed for transformation activity show the fast sedimenting nucleoid devoid of transformation activity while the slow nucleoid was active in transformation for the alleles carried by the genome which is expressed in vivo. Both nucleoids of the Ncd strains are transcribed in vivo. The RNA associated with the inactive chromosome is synthesized by the RNA polymerase of the active one. This study provides evidence that inactivation of one parental genome in the Ncd strain may be related with the tertiary organization of its DNA.

Identification of flagellin associated with the Bacillus subtilis folded chromosome.

We have analyzed the nature and contents of a major protein, P36, in the nucleoid of the Bacillus subtilis wild type and an isogenic mutant devoid of flagella. It appears that deoxyribonucleic acid-P36 complex is flagellin present as membrane-associated flagella.

Folded chromosomes of vegetative Bacillus subtilis: composition and properties.

The isolation of folded DNA from Bacillus subtilis, a Gram positive bacterium is described. When the lysis was achieved with 1 M NaCl a slow sedimenting nucleoid was obtained (1600-2000 S). Conversely, when the lysis was achieved with 0.2 M NaCl a fast sedimenting nucleoid was obtained (3500-4000 S). The yield of folded DNA was between 80 to 90 % of the total lysate DNA. Both nucleoids contained the same amount of RNA, but the relative proportions of lipids and proteins were different. Folded chromosomes were prepared in the presence of spermidine: artifactual protein binding is shown to be unlikely. Electrophoresis of nucleoid proteins showed a dominant polypeptide (MW = 36,000), which remained associated with DNA after sarcosyl treatment and could be partially removed by heat mediated DNA unfolding. In vitro transcription by endogenous RNA polymerase bound to the fast sedimenting-nucleoid was rifamycin resistant; the template capacity of the fast sedimenting-nucleoid was compared with that of the completely unfolded chromosomes.