A putative new peptide synthase operon in Bacillus subtilis: partial characterization

Abstract

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...

Cloning, Sequencing, and Characterization of the Iturin A Operon

“…At present, we are able to compare very similar operons, such as surfactin operons of B. subtilis (3) and two lichenysin operons of Bacillus licheniformis (13,45), plipastatin (37,38) and fengycin (16) operons, and iturin A and mycosubtilin (4) operons. These comparisons may provide good techniques for elucidating the boundary of the functional domain that directs engineered peptide synthesis (22,31,33,34).…”

“…The presence of the ␤-amino fatty acid is the most striking characteristic of the iturin A group and distinguishes this group from the other two groups. The operons that encode surfactin (3), plipastatin-fengycin (16,37,38,40), and mycosubtilin (4), which is a member of the iturin A group, have been sequenced and characterized. In particular, a study of the mycosubtilin operon of B. subtilis ATCC 6633 (4) showed that MycA, a novel template enzyme which has functional domain homology to ␤-ketoacyl synthetase and amino transferase and amino adenylation, was present, which implied that MycA is responsible for incorporation of the ␤-amino fatty acid.…”

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains

“…It may prove to be a useful tool in understanding the primary structures of peptides produced by gene clusters of as-yet-unknown function, such as results from the genome sequencing project of Bacillus subtilis (52). It is also of great interest for crystallographic studies, since such proteins should be as small as possible.…”

Purification and Characterization of PBP4a, a New Low-Molecular-Weight Penicillin-Binding Protein from Bacillus subtilis

“…Z34883 in the EMBL database). Therefore, the amplified fragment contains the sequence encoding the mature PBP4a with an additional N-terminal methionine as well as the endogenous stop codons and putative terminator sequence (25).…”

“…Successively referred to as pbp (25) and dacC (19), this gene was overexpressed in Escherichia coli by Pedersen et al (19), who showed that dacC does indeed encode a membrane-bound PBP migrating between PBP4 and PBP5 of B. subtilis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein is now referred to as PBP4a.…”