The Bacillus subtilis nucleoid-associated protein HPB12 strongly compacts DNA

Abstract

The HPB12 protein from the nucleoid of BaciUlus subtilis was previously described, and its DNA binding properties have been reported previously (V. Salti, F. Le Hegarat, and L. Hirschbein, Biochim. Biophys. Acta 1009: [161][162][163][164][165][166][167] 1989). The DNA-HPB12 complexes were examined by electron microscopy. [161][162][163][164][165][166][167] 1989), and gives rise to a tightly compacted DNA-protein complex. N-terminal sequencjng of purified HPB12 showed that all but one of the first 26 amino acids were identical to those of the L24 ri66somal protein.The bacterial nucleoid consists mostly of DNA, with some RNA and a variety of proteins (3,29). Many of the nucleoidassociated proteins described for Escherichia coli and Bacillus spp. have physicochemical properties similar to those of histones (24, 28; for review, see references 6 and 30). The ability of these proteins, however, to organize the DNA into a prokaryotic type of chromatin (17) within the cell has not been ascertained because it is difficult to isolate completely intact nucleoids from cells.We previously described HPB12 as a 12-kDa, basic, heatstable, DNA-binding protein from the Bacillus subtilis nucleoid (22). In order to determine the functions of this protein, it was purified and its DNA binding properties were characterized. We observed that HPB12 binds preferentially to doublestranded supercoiled DNA in a cooperative mode. The complexes are stable for more than 1.5 h (33).Because of the difficulty in working with natural, undistorted DNA contained in entirely intact B. subtilis nucleoids (3), we studied in vitro the binding of purified HPB12 to purified pBR322 plasmid. In this way, we could observe changes in DNA conformation due to the associated protein.In this paper, we further describe the DNA binding properties of HPB12 by visualizing the HPB12-DNA complexes by electron microscopy. The electron microscopy data confirm the cooperative mode of binding of HPB12 to supercoiled doublestranded DNA, as deduced from prior biochemical experiments. Moreover, these results clearly demonstrate that HPB12 strongly compacts the DNA.Sequencing of purified HPB12 led us to the hypothesis that HPB12 is also a constituent of the ribosome and is the first bifunctional ribosomal protein described for gram-positive bacteria. * MATERIALS AND METHODSPurification of HPB12. HPB12 was purified from exponentially growing cells of B. subtilis 168M trpC2 as described previously (33).Evaluation of the amount of HPB12 per nucleoid. The amount of HPB12 in the nucleoids was estimated from densitometric measurements made on the heat-stable acid-soluble protein fraction of the nucleoid (32). The estimation was made as follows. HPB12 corresponds to 7.1% of the nucleoid heat-stable acid-soluble proteins, which are themselves 1.5% of the total cell protein. Assuming that the total protein content of one cell is about 1.55 x 10-13 g (12), the amount of HPB12 in the nucleoid is 0.9 x 10-16 g. This corresponds to 10,000 monomers per nucleoid. The minimal amount of HP...

Association of the histone-like protein HBsu with the nucleoid of Bacillus subtilis

“…The major heat-stable, acid-soluble protein, called HPB9, was found to be identical to HBsu (21). A second protein of this class, HPB12, with an estimated abundance of 20,000 monomers per cell, was found to be identical to the ribosomal protein L24 (2). It was proposed to participate in nucleoid formation by binding DNA as well as nucleoidassociated RNA.…”

Purification, Cloning, and Preliminary Characterization of aSpiroplasma citri Ribosomal Protein with DNA Binding Capacity

“…The bifunctional nature and the presence of DNA binding motifs in some ribosomal proteins has been reported and has led to speculations on the origin of the ribosomal proteins (58 -64). It is interesting to note that in B. subtilis the HPB12-L24 protein has been described as a bifunctional ribosomal protein (L24) with histone-like properties and DNA binding activity (63,65,66).…”