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.

We have used a novel approach called expression selection to precisely define the hepatitis B virus (HBV) enhancer. Expression selection is based on a shuttle vector containing an enhancerless SV40 T antigen gene, the SV40 origin of replication and a plasmid replicon. This vector is linearized, ligated with the sonicated DNA to be analyzed and transfected into eukaryotic cells, where only plasmids which have incorporated an enhancer can express T antigen and therefore replicate. Vectors amplified by replication are selectively rescued in E. coli and their inserts analyzed. When we performed this protocol with HBV DNA we rescued two overlapping fragments of 166 and 214 bp which in HBV DNA map about 500 bp upstream of the core antigen mRNA initiation site and 1150 bp downstream of the surface antigen mRNA initiation site. These results were confirmed by conventional deletion mapping. When compared to the SV40 enhancer in nonhepatic cell lines, the HBV enhancer is only 5 to 10% as active; nevertheless, it also acts in an orientation-independent manner and in a position downstream of a gene. The HBV enhancer is situated in the coding region of the potential reverse transcriptase, and thus is the first enhancer identified to map in a protein-coding region.

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A large operon-type structure has been located between the g/tA and citB loci on the Bacillus subtilis chromosome. On the basis of the analysis of the 25 kb sequenced so far, it potentially encodes at least three large proteins which contain structural motifs associated with the subunits of all characterized peptide synthases. The amino acid recognition specificity of this new peptide synthase is discussed in the light of sequence homology with other synthases.Keywords : Bacillus subtilis, peptide synthase operon, racemase Micro-organisms produce a large number of peptides, generally as secondary metabolites, which have many important activities varying from antibacterial to antiviral or antifungal, and from anticarcinogenic to immunosuppressive. Regardless of their function, these peptides are synthesized either using the usual ribosomal system or through the action of complex multi-enzyme systems known as peptide synthases.It is now clear that although their products can vary considerably in length and amino acid composition, all peptide synthases studied so far share remarkable similarity in their structural organization and mechanism of action (Kleinkauf & von Dohren, 1990; Cosmina e t al., 1993). They are in fact organized into structural domains, each of which is responsible for the recognition and binding of a specific amino acid. Peptide synthesis takes place through subsequent reactions of thioester cleavage and amide bond formation.Bacillzrs szxbtilis produces a number of peptides and lipopeptides, both linear and cyclic (Zuber etal., 1993). Of these, only surfactin has been thoroughly characterized at the genetic level (Cosmina e t al., 1993; van Sinderen e t al., 1993). From gene sequence analysis and a variety of biochemical studies, it was found that surfactin synthase is a multi-subunit enzyme complex in which there are seven structural domains, corresponding to the number of amino acids present in surfactin.In this communication we report the identification in the B. mbtilis chromosome of a large operon encoding a new peptide synthase.The 15.4 kb fragment present in A clone AB21 (Fig. l) (Kunst & Devine, 1991), two additional A clones (A1A and A4B) were identified by D N A : DNA hybridization, the inserts of which overlap with the chromosomal fragment carried by AB21 (Fig. 1). Finally, an additional 4.3 kb overlapping fragment was isolated by chromosome walking (Glaser et a/., 1993) after cloning the AB21 HindIII-AcyI fragment into the suicide vector pDIA5304. Altogether, the three A clones and the pDIA5304 derivative contained an uninterrupted chromosomal region of approximately 25 kb.The identity of the 25 kb region carried by the A and plasmid vectors with the corresponding chromosomal area was confirmed by both restriction enzyme and Southern blot analyses. In particular, when AB21, A4B and A1A were digested with EcoRI and NotI, fragments of identical size were identified on an agarose gel. Furthermore, the large 3.0 kb EcoRI fragment of the three clones ( Fig. 1) hybridized with an intern...

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