A homologous class of histonelike proteins which are believed to wrap the DNA and to condense the chromosome into highly folded nucleoid structures has been identified in different bacterial species. Bacillus subtilis encodes a homodimeric DNA-binding protein called HBsu. We have cloned the corresponding gene (hbs) on a 3.8-kb fragment. The gene was subcloned to a 1-kb fragment, sequenced, and characterized. It encodes a 92-amino-acid protein with a predicted molecular mass of 9,884 Da. Fortunately, analysis of the DNA sequence downstream of the 3' end of hbs revealed the location of the first 19 amino acid residues of MtrA. This finding located the hbs gene unequivocally to the 5' end of the mtr operon at about 2040 on the standard genetic map of B. subtilis. Northern (RNA) blot analysis and primer extension studies indicated the presence of two distinct hbs transcripts, which were found to be initiated at two different sites located about 160 bases apart. Several attempts to replace the hbs gene in the B. subtilis chromosome with a cat-interrupted copy (hbs::cat) through marker replacement recombination were unsuccessful. In order to study whether hbs is an essential gene, we have constructed a strain containing a truncated copy of the gene behind its own promoter and another intact copy under control of the isopropyl-,-D-thiogalactopyranoside (IPTG)-inducible spac-1 promoter. In this strain (BM19), normal growth was found to depend on IPTG, whereas in the absence of IPTG, growth was severely affected. These results suggest an essential role for the hbs gene product for normal growth in B. subtilis.

The solution structure of the histone-like DNA-binding protein, HBsu, from Bacillus subtilis in 2 mM sodium cacodylate, pH 7.5, is sensitive to the ionic strength of the buffer. This was shown by circular dichroism measurements at different concentrations of sodium chloride and potassium fluoride. The stability of HBsu is also influenced; at HBsu concentrations of about 0.1 mg -ml-', melting temperatures of 32°C and 55°C were found in the absence of potassium fluoride and in the presence of 0.5 M potassium fluoride, respectively, exhibiting drastic ionic-strength-dependent differences in the temperature-induced unfolding of HBsu.Furthermore, at low ionic strength, circular dichroism spectra vary markedly depending on the HBsu concentration in the approximate range 0.2-3 mg * ml-'. Such protein-concentrationdependent differences in the spectra were not observed in the presence of 0.5 M potassium fluoride. Very similar circular dichroism spectra of HBsu and the histone-like DNA-binding protein of Bacillus stearothermophilus (HBst) at high ionic strength, indicate comparable structures of both proteins under these conditions. Estimation of the secondary structure content from the circular dichroism spectra yields data which are in satisfactory agreement with the values obtained from the crystal structure of HBst.Transition temperatures of 45 "C and 61 "C were found in differential scanning calorimetric measurements performed with HBsu in potassium-fluoride-free buffer and in the presence of 0.5 M potassium fluoride, respectively. The thermodynamic data point to the melting of native HBsu dimers into two denatured monomers.The histone-like DNA-binding protein (HU) is ubiquitous in the eubacterial kingdom [l] and is also found in archaebacterial species [2,3]. It is composed of about 90 amino acids with a molecular mass of 9500 Da and occurs in most organisms as a dimer with identical subunits. In both Escherichia coli [4] and Salmonella typhimurium [5], however, the HU protein is encoded by two genes, and the protein exists predominantly in the heterodimeric form in vitro. The sequences of HU proteins from different bacterial species [ by the ability to produce bead-like structures [13, 141, but recent data show localization of the HU protein on the outer, less-organized regions of the bacterial genome [15]. According to the limited abundance of HU proteins in bacterial cells, only about one-sixth of the genome can be packed into nucleosome-like particles [l]. Due to non-specific DNA binding, and probably due to protein-induced DNA bending, the E. coli HU protein influences DNA replication and transcription initiation in vitro [16, 171. Although less well studied than their counterparts in E. coli, the Bacillus HU proteins have proven to be valuable tools for investigating bacterial DNA organisation. They are homodimeric and their host organism is capable of undergoing a simple differentiation process. The three-dimensional structure of the histone-like protein from the thermophilic Bacillus stearothermophilus, HBs...

A synthetic gene encoding the histone‐like DNA‐binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild‐type protein. N‐terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli.. Site‐directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA‐binding parameters by fluorescence spectroscopy. According to gel‐retardation experiments, the mutant protein [Phe47 → Trp]HBsu shows identical DNA binding to wild‐type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47 → Trp]HBsu binds double‐stranded DNA with a dissociation constant in the micromolar range. Computer‐assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47 → Trp]HBsu appears to contact three or four base pairs of DNA. These results are in pritial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.

The binding of Bacillus globigii HB protein to synthetic deoxyoligonucleotides of different length and sequence has been studied by polyacrylamide gel electrophoresis. Without detectable sequence specificity the protein binds to single-stranded and double-stranded DNA. Under the conditions employed, binding of HB protein to deoxyoligonucleotides with six or less nucleotides per strand cannot be detected whle eight or more nucleotide units per strand of single-stranded DNA or base pairs of double-stranded DNA are sufficient for binding.The complete amino acid sequence of HB protein has been determined by manual Edman degradation of tryptic peptides. Like most DNA-binding proteins of its class, HB protein does not contain cysteine, tyrosine or tryptophan residues. The primary structure of HB protein shows 84% homology with the sequence of the related DNA-binding protein I1 from Bacillus stearothermophilus.As in eukaryotic cells, the DNA in both bacterial and archaebacterial organisms is associated with proteins. These often basic polypeptides are thought to be involved in the aggregation of the chromosomal DNA. While in eukaryotes at least the lowest organizational unit of the chromatin, the nucleosome, is quite well characterized [l], neither all the components nor the structure of the prokaryotic nucleoprotein complex [2 -61 are known with certainty.The non-specifically DNA-binding HB protein from Bacillus globigii is a representative of the most extensively characterized group of prokaryotic proteins engaged in the organization of DNA. The proteins of this group have been classified as DNA-binding proteins I1 (DNABP 11) [7]. Proteins belonging to this family have been isolated from more than ten different organisms, including thermophilic bacteria and archaebacteria. There is appreciable sequence homology between them [8, 91, but their primary structures are not related to those of the eukaryotic hstones or high-mobility group proteins. Some of the DNABP I1 have also been shown to be immunologically related to each other [lo, 111. Whereas at least two genes code for DNABP I1 in Escherichia coli [12, 131, in most bacteria only one species of the protein has been found. Binding of the proteins to DNA in vitro causes a condensation of the nucleic acid and the formation of beaded structures [lo, 11, 14, 151. The copy number of about 100000 per bacterial cell, however, does not allow the entire chromosome to be covered by DNABP 11. The three-dimensional structure of the DNABP I1 from Bacillus stearothermophilus has recently been elucidated [16].Like other members of this family, HB protein is a basic, thermostable polypeptide [ll]. HB protein is present as a Abbreviations. ssDNA, single-stranded DNA; dsDNA, doubledimer in solution and has been shown to bind to bacteriophage lambda DNA in vitro. Its binding to polymeric DNA and RNA has been studied by electron microscopy, fluorescence spectroscopy and sedimentation analysis [ l l , 171. Here we have used polyacrylamide gel electrophoresis in a system similar to that...

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