Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

PBSX and skin are two unusual genetic elements resident on the Bacillus subfilis chromosome. PBSX is a phage-li ke element located a t approximately 100" which is induced by the SOS response and results in cell lysis with the release of phage-like particles. The phage particles contain bacterial chromosomal DNA and kill sensitive bacteria without injecting DNA. The skin element is located at approximately 230" on the chromosome and is positioned within the sigK open reading frame (ORF). It is excised at a particular stage of sporulation, leading to reconstitution of the complete sigK gene. In this paper, w e show that there are phage-like operons present in the skin element which are highly homologous to the region of PBSX comprising part of the control region and the late operon. These operons are similar in terms of their gene organization, the percentage identity of the products of homologous ORFs and the positioning and strengths of ribosome-binding sites for each ORF. Although this high degree of conservation suggests that the phage-like operons in skin can be expressed, expression of the late operon was not detected during exponential growth, during sporulation or after induction of the SOS response. However two non-phage-like operons in the skin element are expressed and have distinct expression profiles that are dependent on the growth and developmental status of the cell.

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they arexepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show thatxepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression ofxhlB (encoding the putative holin) together withxlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

The influence of DNA structure on topoisomerase I-DNA interaction has been investigated using a high affinity binding site and mutant derivatives thereof. Parallel determinations of complex formation and helix structure in the absence of superhelical stress suggest that the interaction is intensified by stable helix curvature. Previous work showed that a topoisomerase I binding site consists of two functionally distinct subdomains. A region located 5' to the topoisomerase I cleavage site is essential for binding. The region 3' to the cleavage site is covered by the enzyme, but not essential. We report here that the helix conformation of the latter region is an important modulator of complex formation. Thus, complex formation is markedly stimulated, when an intrinsically bent DNA segment is installed in this region. A unique pattern of phosphate ethylation interferences in the 3'-part of the binding site indicates that sensing of curvature involves backbone contacts. Since dynamic curvature in supercoiled DNA may substitute for stable curvature, our findings suggest that topoisomerase I is able to probe DNA topology by assessment of writhe, rather than twist.