Molecular Microbiology (2005) 56 (4), 845���857 doi:10.1111/j.1365-2958.2005.04587.x �� 2005 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005 ? 2005 56 4845857 Review Article Bacillus subtilis antibioticsT. Stein Accepted 24 January, 2005. For correspondence. E-mail T.Stein@em.uni-frankfurt.de Tel. ( + 49) 69 7982 9522 Fax ( + 49) 69 7982 9527. MicroReview Bacillus subtilis antibiotics: structures, syntheses and specific functions Torsten Stein Institut f��r Mikrobiologie, Johann Wolfgang Goethe- Universit��t, Marie-Curie-Str. 9, 60439 Frankfurt/Main, Germany. Summary The endospore-forming rhizobacterium Bacillus sub- tilis ��� the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predomi- nantly peptides that are either ribosomally synthe- sized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally gen- erated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospho- lipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the ���pure��� anti-microbial action: Non-ribosomally pro- duced lipopeptides are involved in biofilm and swarm- ing development, lantibiotics function as pheromones in quorum-sensing, and a ���killing factor��� effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given. Introduction The rhizobacterium Bacillus subtilis (Sonenshein et al . 2001) has been used for genetic and biochemical studies for several decades, and is regarded as paradigm of Gram-positive endospore-forming bacteria (Moszer et al ., 2002). Several hundred wild-type B. subtilis strains have been collected, with the potential to produce more than two dozen antibiotics with an amazing variety of struc- tures. All of the genes specifying antibiotic biosyntheses combined amount to 350 kb however, as no strain pos- sesses them all, an average of about 4���5% of a B. subtilis genome is devoted to antibiotic production. One aim of this review is to give an updated summary of the struc- tures of all B. subtilis antibiotics, the biochemistry and genetic analysis of their biosynthetic pathways, as well as a survey on well-studied regulatory pathways. A further aim is to compile recent findings that demonstrate specific roles for B. subtilis antibiotics beyond the anti-microbial action ��� distinct antibiotics are involved in the morphology and physiology of B. subtilis and contribute to the survival of this organism in its natural habitat. The potential of B. subtilis to produce antibiotics has been recognized for 50 years. Peptide antibiotics repre- sent the predominant class. They exhibit highly rigid, hydrophobic and/or cyclic structures with unusual constit- uents like D -amino acids and are generally resistant to hydrolysis by peptidases and proteases (Katz and Demain, 1977 and references therein). Furthermore, cys- teine residues are either oxidized to disulphides and/or are modified to characteristic intramolecular C���S (thioet- her) linkages, and consequently the peptide antibiotics are insensitive to oxidation. Principally, two different bio- synthetic pathways for peptides allow the incorporation of such unusual (non-proteinaceous) constituents: (i) the non-ribosomal synthesis of peptides by large megaen- zymes, the non-ribosomal peptide synthetases (NRPSs) and (ii) the ribosomal synthesis of linear precursor pep- tides that are subjected to post-translational modification and proteolytic processing. Lantibiotics Peptide antibiotics with inter-residual thioether bonds as unique feature are outlined as lantibiotics (lanthionine- containing antibiotics) (Schnell et al ., 1988). Lanthionine formation occurs through post-translational modification (Fig. 1) of ribosomally synthesized precursor peptides including dehydration of serine and threonine residues, respectively, and subsequent addition of neighbouring cysteine thiol groups (for reviews, see Guder et al ., 2000

846 T. Stein �� 2005 Blackwell Publishing Ltd, Molecular Microbiology , 56 , 845���857 Jack and Jung, 2000 McAuliffe et al ., 2001). Based on structural properties two lantibiotic types are distinguish- able. Type A lantibiotics (21���38 amino acid residues) exhibit a more linear secondary structure and kill Gram- positive target cells by forming voltage-dependent pores into the cytoplasmic membrane. Remarkably, for the lan- tibiotic nisin produced by Lactococcus lactis it has been shown that the bactoprenol-bound ultimate peptidoglycan precursor lipid II represents both an important docking/ receptor molecule (Breukink et al ., 1999) and an intrinsic component of the lethal pore (Hasper et al ., 2004). Gram- positive lantibiotic producers exhibit efficient countermea- sures to obviate the action of their own products. Self- protection (immunity) against lantibiotics is based on ATP- binding cassette (ABC) transporter homologous proteins (LanFEG) that export the lantibiotic from the cytoplasmic membrane into the extracellular space (Stein et al ., 2003a 2005). Furthermore, several lantibiotic producers possess membrane-bound lipoproteins LanI, which exhibit a sequestering-like function that prevents high local concentrations of the lantibiotic close to the cytoplas- mic membrane and/or interferes with lantibiotic lipid II pore formation (Stein et al ., 2003a 2005 Koponen et al ., 2004). Subtilin, a 32-amino-acid pentacyclic lantibiotic (Fig. 2) is structurally related to the widely utilized biopreservative nisin (E 234) of L. lactis (Ross et al . 2002). The subtilin gene cluster specifies the subtilin prepeptide SpaS, SpaBC for post-translational lanthionine formation, and the translocator SpaT for export of the modified species. The extracellular B. subtilis serine proteases subtilisin (AprE), Wpra and Vpr are involved in subtilin processing (Corvey et al ., 2003). Subtilin immunity is mediated by the lipoprotein SpaI and the ABC translocator SpaFEG (Klein and Entian, 1994 Stein et al ., 2003a). The biosynthesis of subtilin is regulated by a positive feedback mechanism (Stein et al ., 2002a see also a general scheme of B. subtilis regulatory pathways of antibiotic biosynthesis in Fig. 4) in which extracellular subtilin activates the two component regulatory system SpaK (sensor histidine kinase) and SpaR (regulator protein) that binds to a DNA motif ( spa -box) promoting the expression of genes for subtilin biosynthesis ( spaS and spaBTC ) and immunity ( spaIFEG ) (Stein et al ., 2003b Kleerebezem, 2004). SpaRK expression is controlled by the sporulation tran- scription factor SigH, which itself is repressed during exponential growth by the transition-state regulator AbrB (Fawcett et al ., 2000). Thus, subtilin production appears to be dual controlled, to culture density in a quorum- sensing mechanism in which subtilin plays a pheromone- type role and in response to the growth phase (mediated by Abrb/SigH Stein et al. 2002b). The B. subtilis strain A1/3 produces ericin (Fig. 2 Stein et al ., 2002b). Surprisingly, the ericin gene cluster con- tains two structural genes, eriA and eriS , although the open reading frames (ORFs) are closely related to corre- sponding genes of the subtilin cluster. Ericin S and subtilin only differ in four amino acid residues, and expectedly the anti-microbial properties of both lantibiotics are compara- ble. However, ericin A has a different ring organization and 16 amino acid substitutions compared with ericin S. This compound becomes fully matured and is produced in equal quantities as ericin S. The need for only a single synthetase (EriBC) for two different products (ericin A/S) reflects the flexibility of lantibiotic pathways. The lantibiotic mersacidin (Fig. 2) belongs to the type B lantibiotics which exhibit a more globular structure. It inhibits cell wall biosynthesis by complexing lipid II (Br��tz et al ., 1997). The mersacidin gene cluster consists of the structural gene mrsA , as well as genes involved in post- translational modification ( mrsM and mrsD ), transport ( mrsT ), immunity ( mrsFEG ) and regulation ( mrsR1 mrsR2 , mrsK2 ). Whereas MrsR1 regulates mersacidin biosynthesis, the two-component regulatory system MrsR2/K2 appears to regulate the expression of the mersacidin immunity transporter specifying genes mrs- FGE (Guder et al ., 2002). Mersacidin production occurs from the beginning of the stationary phase however, the link between its mersacidin regulatory systems and the cellular regulation network of B. subtilis is yet unknown. Fig. 1. Proposed pathway for post-translational lanthionine forma- tion. The first step in lanthionine formation involves dehydration of L - serine and L -threonine residues in ribosomally generated prelantibi- otic peptides yielding 2,3-didehydroalanine and 2,3-didehydrobu- tyrine respectively. In the second step inter-residual thioether linkages are formed through stereospecific Michael-like additions of neigh- boured L -cysteine sulphydryl groups yielding meso -lanthionine and 3- methyllanthionine respectively. Note the a -carbon atom D-configura- tions of the formerly L -serine/ L -threonine residues grey boxes repre- sent formerly cysteines.