Detection of lateral heterogeneity in the cytoplasmic membrane of Bacillus subtilis.

Lateral heterogeneity in the cytoplasmic membrane of Bacillus subtilis was found by using density gradient centrifugation. Crude membranes (CM) present in the whole cell lysate were separated into three fractions of increasing density (F, CI, CII). Substantial difference exists in the amount of protein recovered from these fractions, the relative ratio being 15 : 35 : 50. The qualitative protein composition (by SDS-PAGE) of the fractions varies markedly as well. The lipid components extracted from the fractions are also distributed in different proportions, viz. 40 : 40 : 20. The spectrum of fatty acids (FA), detected in lipids of F fraction and analyzed by GC-MS exhibits the same profile as that found in CM; in contrast, fractions CI and CII undergo extensive FA reconstruction. Thermotropic behavior of fractions measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicates significant variations of microviscosity (r(s)) within the F, CI and CII fractions. The protein-to-lipid ratio plays evidently a key role in affecting the physical state of the cytoplasmic membrane. Microdomains of different density coexist in the membrane and exhibit heterogeneity in both chemical composition and "physical state"; the increased de novo synthesis of FA induced by the cold exclusively in fractions CI and CII indicates correlation with an altered physiological state of bacterial metabolism.

Different in vivo localization of the Escherichia coli proteins CspD and CspA.

Two Csp proteins (CspA and CspD) were fused to the green fluorescent protein GFP and expressed from their natural promoters or from an inducible promoter. Fluorescence microscopy and computerized image analysis indicate that in Escherichia coli growing at 37 degrees C CspD localizes in the nucleoid like the control H-NS while CspA occupies a polar position away from the nucleoid. Following cold shock CspA maintains its location, while CspD is not sufficiently expressed to permit its localization. The different localization of CspA and CspD indicates that these proteins play different roles in the cell in spite of their extensive structural similarity.

A possible role for L24 of Bacillus subtilis in nucleoid organization and segregation.

The condensation of DNA in bacterial nucleoids during cell cycle is a complex and dynamic process. Proteins displaying the physico-chemical properties of histones are known to contribute to this process. During a search for B. subtilis nucleoid associated proteins, HBsu and L24 were identified as the most abundant proteins in nucleoid containing fractions. Purified L24 binds and condenses DNA in vitro. In this paper we describe immunofluorescence studies that demonstrated that L24 is located at the poles of the nucleoids in exponentially growing cells. In contrast, the protein is dispersed in the cytoplasm during stationary phase. Moreover, overexpression of the rplX gene encoding L24 disrupts nucleoid segregation and positioning.

Characterization of LrpC DNA-binding properties and regulation of Bacillus subtilis lrpC gene expression.

The lrpC gene was identified during the Bacillus subtilis genome sequencing project. Previous experiments suggested that LrpC has a role in sporulation and in the regulation of amino acid metabolism and that it shares features with Escherichia coli Lrp, a transcription regulator (C. Beloin, S. Ayora, R. Exley, L. Hirschbein, N. Ogasawara, Y. Kasahara, J. C. Alonso, and F. Le Hégarat, Mol. Gen. Genet. 256:63-71, 1997). To characterize the interactions of LrpC with DNA, the protein was overproduced and purified. We show that LrpC binds to multiple sites in the upstream region of its own gene with a stronger affinity for a region encompassing P1, one of the putative promoters identified (P1 and P2). By analyzing lrpC-lacZ transcriptional fusions, we demonstrated that P1 is the major in vivo promoter and that, unlike many members of the lrp/asnC family, lrpC is not negatively autoregulated but rather slightly positively autoregulated. Production of LrpC in vivo is low in both rich and minimal media (50 to 300 LrpC molecules per cell). In rich medium, the cellular LrpC content is six- to sevenfold lower during the exponentional phase than during the stationary growth phase. Possible determinants and the biological significance of the regulation of lrpC expression are discussed.

The L17 ribosomal protein of Bacillus subtilis binds preferentially to curved DNA.

We searched in Bacillus subtilis for proteins that bind preferentially to curved DNA. Two proteins of 9 and 15 kDa displaying this property were purified from exponentially growing cells of B. subtilis strain 168. Sequencing of N-terminal amino acids identified them as the proteins HBsu and L17 respectively. The overproduction of L17 from B. subtilis in Escherichia coli was shown to have a strong effect on nucleoid morphology and segregation.

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.

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.

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.

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.

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.

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.

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.

Deoxyribonucleic acid-binding proteins in vegetative Bacillus subtilis: alterations caused by stage O sporulation mutations.

Deoxyribonucleic acid (DNA)-binding proteins have been compared between wild-type Bacillus subtilis and five sporulation mutants blocked at different stage O loci. Extracts from exponentially growing cells have been fractionated for proteins binding to single-stranded calf thymus DNA-cellulose and double-stranded B. subtilis DNA-cellulose. In nutrient broth, stage O mutations cause an accumulation of proteins with affinity for double-stranded DNA. Suppression of the mutation with extragenic suppressors relieves the accumulation. In minimal glucose medium, the stage O mutations also cause accumulation of proteins with affinity for double-stranded DNA, but the species accumulated are different from those of nutrient broth-grown cells. In neither case did stage O mutations affect proteins with affinity for single-stranded DNA. The results suggest that the products of stage O loci are functional and operative during vegetative growth.

Developmental modulation of deoxyribonucleic acid-binding proteins of Bacillus subtilis during sporulation stages.

The isolation of deoxyribonucleic acid (DNA)-binding proteins from various stages of growth and sporulation of Bacillus subtilis is described. After adsorption and elution from phosphocellulose, the proteins were fractionated according to their ability to adsorb to denatured calf thymus DNA-cellulose or native B. subtilis DNA-cellulose. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and purification was monitored by a nitrocellulose filter binding assay. Approximately 1% of the proteins in the crude extract adsorbed to denatured calf thymus DNA-cellulose and 0.1% adsorbed to native B. subtilis DNA-cellulose. Each class of proteins varied qualitatively and quantitatively as sporulation proceeded. Several proteins from the exponential phase of growth that bound to denatured DNA were lost by T(0), whereas at T(5) new polypeptides appeared. Fewer changes in the profile of proteins with affinity for native DNA were observed between exponential phase and T(0); however, the dominant species in these eluates were clearly different.