Analysis of genes involved in biosynthesis of the lantibiotic subtilin

Abstract

333:276-278, 1988). The most important lantibiotics are subtilin and the food preservative nisin, which both have a very similar structure. By using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA region adjacent to spaS was isolated from Bacillus subtilis. Sequence analysis of a 4.9-kb fragment revealed several open reading frames with the same orientation as spaS. Downstream of spaS, no reading frames were present on the isolated XbaI fragment. Upstream of spaS, three reading frames, spaB, spaC, and spaT, were identified which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin. The SpaT protein derived from the spaT sequence was homologous to hemolysin B of Escherichia coli, which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC revealed subtilin-negative mutants, whereas spaT gene disruption mutants still produced subtilin. Remarkably, the spaT mutant colonies revealed a clumpy surface morphology on solid media. After growth on liquid media, spaT mutant cells agglutinated in the mid-logarithmic growth phase, forming longitudinal 3to 10-fold-enlarged cells which aggregated. Aggregate formation preceded subtilin production and cells lost their viability, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtilin transport.

The biology of lantibiotics from the lacticin 481 group is coming of age

“…Such biosynthetic machineries differ significantly from those of the nisin, epidermin and Pep5 groups (former class AI), which include two modification enzymes, LanB and LanC (Table 2). LanB is the dehydratase that converts serine and threonine into Dha and Dhb residues, and LanC is the cyclase responsible for the subsequent (Me)Lan formation Engelke et al, 1992;Klein et al, 1992;Schnell et al, 1992;Kuipers et al, 1993Kuipers et al, , 2004de Vos et al, 1995;Meyer et al, 1995;Bierbaum et al, 1996a;Entian & de Vos, 1996;Kupke & Götz, 1996;Siezen et al, 1996;Karakas Sen et al, 1999;Guder et al, 2000;McAuliffe et al, 2001a;Koponen et al, 2002;Twomey et al, 2002;Okeley et al, 2003;Chatterjee et al, 2005b;Patton & van der Donk, 2005;Li et al, 2006). Whereas LanC proteins are homologous to the C-terminal parts of LanM proteins, LanB and LanM proteins do not display significant sequence similarities (Kupke & Götz, 1996;Siezen et al, 1996;Uguen et al, 2000).…”

“…Nisin and subtilin LanB and LanC form a multimeric complex with the membrane transporter LanT (Siegers et al, 1996;Kiesau et al, 1997). Furthermore, nisin and subtilin LanT proteins are devoid of protease domains (Klein et al, 1992;Engelke et al, 1992;Siezen et al, 1996). Nisin and subtilin leader peptides are cleaved extracellularly by a LanP protein and by unspecific extracellular proteases, respectively (van der Meer et al, 1993;Entian & de Vos, 1996;Corvey et al, 2003;Kuipers et al, 2004; Table 2).…”

“…AMS proteins are composed of three domains: an N-terminal cysteine protease domain, a central membrane-spanning domain with four or six transmembrane segments, and a C-terminal ATP-binding domain. Both the protease and the ATP-binding domains are et al (1992, 1994); Klein et al (1992Klein et al ( , 1993; Kuipers et al (1993Kuipers et al ( , 1995; van der Meer et al…”

“…As mentioned above, additional modification enzymes are necessary for the biosynthesis of lacticin 3147, epidermin, mersacidin, and probably lactocin S and cinnamycin. All lantibiotics, except for epidermin and cinnamycin, for which the situation is unclear Schnell et al, 1992;Peschel et al, 1997;Widdick et al, 2003), are secreted via a LanT transporter (CylB for cytolysin), but in most of the cases the LanT protein is devoid of a protease domain, and a separate LanP protein (CylA for cytolysin) is used to cleave off the leader peptide Engelke et al, 1992;Klein et al, 1992;Kuipers et al, 1993Kuipers et al, , 2004Meyer et al, 1995;Entian & de Vos, 1996;Siegers et al, 1996;Siezen et al, 1996;Kiesau et al, 1997;Skaugen et al, 1997;Dougherty et al, 1998;Altena et al, 2000). The subtilin precursor differs by being cleaved by unspecific surface proteases of the Bacillus subtilis producer strain rather than by a specific LanP enzyme Corvey et al, 2003), whereas both cytolysin precursor peptides are first cleaved by the protease domain of CylB, secreted, and then cleaved again by CylA Segarra et al, 1991;Booth et al, 1996;Coburn & Gilmore, 2003).…”

Characterization of lactosporin, a novel antimicrobial protein produced byBacillus coagulansATCC 7050

“…PCR was used to investigate the possible relatedness of the antimicrobial protein produced by B. coagulans ATCC 7050 with the known bacteriocins produced by Bacillus spp. including coagulin produced by B. coagulans I 4 (Hyronimus et al 1998), and subtilin and subtilosin, which are produced by B. subtilis (Klein et al 1992;Stein et al 2004). Genomic DNA was extracted from overnight cultures of B. coagulans and B. subtilis ATCC 6633.…”