THE DNA-BINDING PROTEIN HBSU IS ESSENTIAL FOR NORMAL GROWTH AND DEVELOPMENT IN BACILLUS-SUBTILIS

The hbs operon of Bacillus subtilis comprises a single gene which is localized between the spoIVA and mtrA open reading frames, and is situated at 204-degrees of the standard map of B subtilis. Expression of hbs is initiated from two distinct promoters called P1 and P2. The transcription initiation sites have been mapped by primer extention analysis. Sequences upstream from P1 show a -35 and -10 region which may be recognized by the vegetative form of RNA polymerase E-sigma(A), whereas sequences upstream from P2 may be recognized by either E-sigma(C) or E-sigma(H) minor forms of RNA polymerase. In vegetative cells, hbs is highly and equally transcribed from both promoters, P1 and P2. In contrast, in sporulating cells, hbs is expressed predominantly from P2. In order to study the physiological role of HBsu, we must overcome our failure to interrupt the hbs gene within the B subtilis chromosome by using a previously constructed strain, BM19, bearing hbs under the control of the IPTG-inducible spac-1 promoter. In this strain, growth was found to depend highly on hbs expression. In the absence of IPTG, growth was strongly affected culminating in a filamentous cell morphology. Although sporulation in IPTG-uninduced BM19 cells was poor, due to the limited cell growth, the outgrowth of those spores was delayed by 1 h. In contrast, in the presence of IPTG, a condition that induces hbs expression, normal outgrowth of spores was observed. The proposed essentiality of the hbs gene product for growth and development in B subtilis may be attributed to its interaction with replication and transcription as a consequence of its facility to wrap DNA and to condense the chromosome into nucleosomelike structures. A comparative sequence analysis of HBsu with 18 homologous histonelike proteins of diverse origin demonstrated their high conservation throughout evolution.