Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu

Abstract

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 204 degrees 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-beta-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.