bacillus subtilis antibiotics: structures, syntheses and ... · bacillus subtilis antibiotics:...

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. [email protected]; Tel. (

+

49) 69 7982 9522; Fax(

+

49) 69 7982 9527.

MicroReview

Bacillus subtilis

antibiotics: structures, syntheses and specific functions

Torsten Stein

Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität, 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 antibioticswith an amazing variety of structures. The producedanti-microbial active compounds include predomi-nantly peptides that are either ribosomally synthe-sized and post-translationally modified (lantibioticsand lantibiotic-like peptides) or non-ribosomally gen-erated, as well as a couple of non-peptidic compoundssuch as polyketides, an aminosugar, and a phospho-lipid. Here I summarize the structures of all known

B.subtilis

antibiotics, their biochemistry and geneticanalysis of their biosyntheses. An updated summaryof well-studied antibiotic regulation pathways isgiven. Furthermore, current findings are resumed thatshow roles for distinct

B. subtilis

antibiotics beyondthe ‘pure’ anti-microbial action: Non-ribosomally pro-duced lipopeptides are involved in biofilm and swarm-ing development, lantibiotics function as pheromonesin quorum-sensing, and a ‘killing factor’ effectuatesprogrammed cell death in sister cells. A discussionof how these antibiotics may contribute to the survivalof

B. subtilis

in its natural environment is given.

Introduction

The rhizobacterium

Bacillus subtilis

(Sonenshein

et al

.2001) has been used for genetic and biochemical studiesfor several decades, and is regarded as paradigm ofGram-positive endospore-forming bacteria (Moszer

et al

.,2002). Several hundred wild-type

B. subtilis

strains havebeen collected, with the potential to produce more than

two dozen antibiotics with an amazing variety of struc-tures. All of the genes specifying antibiotic biosynthesescombined 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 ofthis review is to give an updated summary of the struc-tures of all

B. subtilis

antibiotics, the biochemistry andgenetic analysis of their biosynthetic pathways, as well asa survey on well-studied regulatory pathways. A furtheraim is to compile recent findings that demonstrate specificroles for

B. subtilis

antibiotics beyond the anti-microbialaction – distinct antibiotics are involved in the morphologyand physiology of

B. subtilis

and contribute to the survivalof this organism in its natural habitat.

The potential of

B. subtilis

to produce antibiotics hasbeen 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 tohydrolysis by peptidases and proteases (Katz andDemain, 1977; and references therein). Furthermore, cys-teine residues are either oxidized to disulphides and/orare modified to characteristic intramolecular C–S (thioet-her) linkages, and consequently the peptide antibioticsare insensitive to oxidation. Principally, two different bio-synthetic pathways for peptides allow the incorporation ofsuch unusual (non-proteinaceous) constituents: (i) thenon-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 modificationand proteolytic processing.

Lantibiotics

Peptide antibiotics with inter-residual thioether bonds asunique feature are outlined as lantibiotics (lanthionine-containing antibiotics) (Schnell

et al

., 1988). Lanthionineformation occurs through post-translational modification(Fig. 1) of ribosomally synthesized precursor peptidesincluding dehydration of serine and threonine residues,respectively, and subsequent addition of neighbouringcysteine 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 onstructural 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 poresinto the cytoplasmic membrane. Remarkably, for the lan-tibiotic nisin produced by

Lactococcus lactis

it has beenshown that the bactoprenol-bound ultimate peptidoglycanprecursor lipid II represents both an important docking/receptor molecule (Breukink

et al

., 1999) and an intrinsiccomponent 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 cytoplasmicmembrane into the extracellular space (Stein

et al

.,2003a; 2005). Furthermore, several lantibiotic producerspossess membrane-bound lipoproteins LanI, whichexhibit a sequestering-like function that prevents highlocal concentrations of the lantibiotic close to the cytoplas-mic membrane and/or interferes with lantibiotic lipid IIpore 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 subtilingene cluster specifies the subtilin prepeptide SpaS,SpaBC for post-translational lanthionine formation, andthe 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 thelipoprotein SpaI and the ABC translocator SpaFEG (Kleinand Entian, 1994; Stein

et al

., 2003a). The biosynthesisof 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 inFig. 4) in which extracellular subtilin activates the twocomponent regulatory system SpaK (sensor histidinekinase) and SpaR (regulator protein) that binds to a DNAmotif (

spa

-box) promoting the expression of genes forsubtilin 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 duringexponential growth by the transition-state regulator AbrB(Fawcett

et al

., 2000). Thus, subtilin production appearsto 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 (mediatedby 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 theopen reading frames (ORFs) are closely related to corre-sponding genes of the subtilin cluster. Ericin S and subtilinonly differ in four amino acid residues, and expectedly theanti-microbial properties of both lantibiotics are compara-ble. However, ericin A has a different ring organization and16 amino acid substitutions compared with ericin S. Thiscompound becomes fully matured and is produced inequal quantities as ericin S. The need for only a singlesynthetase (EriBC) for two different products (ericin A/S)reflects the flexibility of lantibiotic pathways.

The lantibiotic mersacidin (Fig. 2) belongs to the typeB lantibiotics which exhibit a more globular structure. Itinhibits cell wall biosynthesis by complexing lipid II (Brötz

et al

., 1997). The mersacidin gene cluster consists of thestructural gene

mrsA

, as well as genes involved in post-translational modification (

mrsM

and

mrsD

), transport(

mrsT

), immunity (

mrsFEG

) and regulation (

mrsR1mrsR2

,

mrsK2

). Whereas MrsR1 regulates mersacidinbiosynthesis, the two-component regulatory systemMrsR2/K2 appears to regulate the expression of themersacidin immunity transporter specifying genes

mrs-FGE

(Guder

et al

., 2002). Mersacidin production occursfrom the beginning of the stationary phase; however, thelink between its mersacidin regulatory systems and thecellular 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.

Bacillus subtilis

antibiotics

847



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, 845–857

MrsD, a member of the homo-oligomeric flavin-containingcysteine decarboxylases (HFCD) family, catalyses the oxi-dative decarboxylation of the C-terminal cysteine of themersacidin prepeptide. The dodecameric MrsD and itsclosely relative EpiD involved in epidermin biosynthesis of

Streptococcus epidermidis

represent the sole examplesof lantibiotic modifying enzymes with known three-dimen-sional structures (Blaesse

et al

., 2003).

Unusual lantibiotics

Sublancin 168 with a

b

-methyllanthionine bridge and –unusual for lantibiotics, two disulphide bridges (Fig. 2;Paik

et al

., 1998), acts preferentially against Gram-posi-tive bacteria. Its structural gene

sunA

(formerly

yolG

)belongs to the

B. subtilis

temperate bacteriophage SP

b

(Westers

et al

., 2003) and thus, sublancin and the ‘proph-age SP

b

-mediated bacteriocin’ (Hemphill

et al

., 1980) aremost probably the same compounds. An ABC transporter(SunT) and two thiol-disulphide oxidoreductases (BdbAB)

belong to the sublancin locus (Fig. 2). Only BdbB seemsto be dedicated for sublancin production, most probablyfor the formation of the disulphide bonds (Dorenbos

et al

.,2002). The BdbB paralogue BdbC protein is at least par-tially able to replace BdbB in sublancin production, butcontrariwise BdbB cannot complement the function ofBdbC (competence development), showing that these twoclosely related thiol-disulphide oxidoreductases havedifferent, but partly overlapping substrate specificities(Kunst

et al

., 1997; Dorenbos

et al

., 2002). The SP

b

locusincluding the sublancin gene cluster is not essential for

B. subtilis

survival (Westers

et al

., 2003). However, itcontains yet unidentified genes mediating resistanceagainst sublancin action. One attractive hypothesis is thatsublancin might contribute to the survival of bacterioph-age, e.g. that sublancin kills only non-lysogenized cellsand thus, enriching the per cent of a lysogenized

B. sub-tilis

population

.

Subtilosin A produced by several

B. subtilis

strains(Zheng

et al

., 1999; Stein

et al

., 2004) has a macrocyclic

Fig. 2.

Bacillus subtilis

lantibiotics, lantibiotic-like peptides and specifying gene clusters. The organization of gene clusters (boxed) specifying lantibiotic and lantibiotic-like peptides are given along with schematic structure representations of the matured peptides. Colour code: black, structural genes and genes specifying proteins involved in post-translational modification and transport; grey, regulatory genes; filled boxes, immunity genes. Numbers correspond to the size of the gene clusters (in kilobases, kb). A–

S

–A,

meso

-lanthionine; Abu–

S

–A, 3-methyllanthionine;

D

A, 2,3-didehydroalanine;

D

B, 2,3-didehydrobutyrine. For details, see the corresponding text.

848

T. Stein



,

56

, 845–857

structure (Fig. 2) with three inter-residual linkages (Marx

et al

., 2001) that have been elucidated as thioether bondsbetween cysteine sulphurs and amino acid alpha-carbons(Kawulka

et al

., 2004). It acts against a variety of Gram-positive bacteria, including

Listeria

(Zheng

et al

., 1999).The

sbo-alb

(anti-listerial bacteriocin) cluster encodesproteins AlbA (YwiA) most probably involved in post-trans-lational modification of presubtilosin, AlbF (YwhN) proba-bly acting in subtilosin processing and the subtilosinimmunity proteins AlbB–D (YwhQPO) (Zheng

et al

.,2000). Expression of the

sbo-alb

genes occurs understress conditions (Nakano

et al

., 2000) under AbrB control(Zheng

et al

., 1999; see also Fig. 4).

Non-ribosomal biosynthesized peptides

The non-ribosomal synthesis of peptide antibiotics iswidespread among bacteria and fungi (for recent reviews,see Sieber and Marahiel, 2003; Finking and Marahiel,2004; Walsh, 2004; and references therein). Large multi-enzymes, the NRPSs, that are composed of modularlyarranged catalytic domains (Fig. 3A), catalyse all neces-sary steps in peptide biosynthesis including the selectionand ordered condensation of amino acid residues. Eachelongation cycle in non-ribosomal peptide biosynthesisneeds the cooperation of three core domains. (i) Theadenylation domain (550 amino acid residues) selects itscognate amino acid and generates an enzymatically sta-bilized aminoacyl adenylate. This mechanism resemblesthe amino-acylation of tRNA synthetases during riboso-mal peptide biosynthesis. (ii) The thiolation or peptidylcarrier domain (80 aa) is equipped with a 4¢-phosphopan-

tetheine (PPan) prosthetic group to which the adenylatedamino acid substrate is transferred and thioesterifiedunder release of AMP. Thus, the PPan cofactor acts asthiotemplate and as a swinging arm to transport interme-diates between the various catalytic centres. The peptidylcarrier proteins are post-translationally converted frominactive apoforms to their active holoforms by dedicatedPPan transferases (Lambalot et al., 1996). (iii) The forma-tion of a new peptide bond is catalysed by condensationdomains (450 aa) located between each pair of adenyla-tion and peptidyl carrier domains. The linear assemblyline-like arrangement of multiple of such core units (i–iii)ensure the co-ordinated elongation of the peptide product.In most of the cases the non-ribosomal peptide biosyn-thesis is terminated by macrocyclization of the peptideproduct, whereby parts of the molecule distant in theconstructed linear peptide chain are covalently linked toone another (Kohli and Walsh 2003). Typically, such reac-tions are catalysed by thioesterase domains at the C-terminal end of the NRPS assembly line. The depictedmechanism of peptide biosynthesis has been outlined inthe concept of the ‘Multiple Carrier Model of NonribosomalPeptide Biosynthesis at Modular Multienzymatic Tem-plates’ (Stein et al., 1996). Mechanistically, NRPSs areclosely related to polyketide synthetases (PKSs), as bothmodular systems utilize multiple Ppan carriers for covalentbinding of monomers and growing chains. Both systemsare highly flexible in which naturally rearrangements canbe easily achieved within a relatively short period, permit-ting the random evolution of compounds that provideselective advantages. Striking examples for such flexibilityare the systems specifying the biosynthesis of the closely

Fig. 3. Summary of B. subtilis antibiotics.A. Non-ribosomally synthesized peptide antibiotics. In each line the producing B. subtilis strains, the genetic organization of the NRPSs (boxed), and schematic representations of produced peptide antibiotics and their possible isoforms are given. Amino acid residues, usually in L-configuration, are shown in the single-letter code, and residues in D-configuration are underlined; the fatty acid moieties are hatched and the number of their carbon atoms are indexed (Ci). For mycosubtilin synthetase the denotation of the NPRSs symbols is explicitly shown: mycA codes for an NRPS (449 kDa) encompassing domains for an acyl-ligase (AL), a ketosynthase (KS) and an acylmethyltransferase (AMT) followed by an elongation unit for asparagine (N). Each modularly arranged elongation unit contains a domain for adenylation of the amino acid substrate, a peptidyl carrier protein (PCP) and a condensation domain where the formation of a new peptide bond occurs. In the case of amino acids in D-configuration, the NRPSs contain an additional epimerase domain. Numbers correspond either to the size of the gene clusters (in kb) or to the derived molecular mass of the NRPSs (in kDa).1Surfactin consists of a heptapeptide moiety bonded to the carboxyl and hydroxyl groups of a b-hydroxy fatty acid. Its production is widely distributed among B. subtilis, pumilus, licheniformis and amyloliquefaciens strains and thus, a disconcerting variety of surfactin isoforms have been described under different synonyms such as bacircine, halo- and isohalobactin, lichenysin A/G, daitocin and pumilacidin (summarized in Peypoux et al., 1999; Kalinovskaya et al., 2002).2The iturine lipopeptide family share a b-amino fatty acid as integral constituent, positions 1–3 of the peptide moiety (L-Asx-D-Tyr-D-Asx) and an additional D-amino acid at position 6.3Fengycin (plipastatin) consists of a b-hydroxy fatty acid connected to the N-terminus of a decapeptide including four D-amino acid residues and the rare amino acid L-ornithine. The C-terminal residue of the peptide moiety is linked to the tyrosine residue at position 3, forming the branching point of the acylpeptide and the eight-membered cyclic lactone.4NPRSs can be involved in producing compounds other than antibiotics: Corynebactin (DHB-Gly-Thr)3 produced by Corynebacterium glutamicum (Budzikiewicz et al., 1997) is a 12-membered trilactone macrocyclic ring composed of three threonine residues, each connected to dihydroxybu-tyrate (DHB) via glycine spacers; the B. subtilis product has been renamed to bacillibactin (May et al., 2001). Corynebactin/bacillibactin acts as a siderophore; complexing of ferric iron occurs by the six hydroxyl groups of the DHB moieties.B. Structure representations of further non-ribosomally synthesized B. subtilis peptide antibiotics and miscellaneous antibiotics (Wilson et al., 1987; Hilton et al., 1988; Kitajima et al., 1990; Kugler et al., 1990; Majumder et al., 1988; Pinchuk et al., 2002; Tamehiro et al., 2002; Inaoka et al., 2004).

Bacillus subtilis antibiotics 849

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857

A

B

1988

850 T. Stein


related compounds of the iturin family (see Fig. 3A). Thus,NRPSs and PKSs are per se extremely amenable togenetic manipulations, providing powerful tools forfuture development and production of novel peptides,polyketides and hybrid compounds with new properties.The huge potential of NRPSs and PKSs in the generationof novel drugs has been excellently reviewed elsewhere(Sieber and Marahiel, 2003; Finking and Marahiel, 2004;Walsh, 2004).

Non-ribosomally generated amphipathic lipopeptideantibiotics with condensed b-hydroxyl or b-amino fattyacids are widespread in B. subtilis. Variations in lengthand branching of the fatty acid chains and amino acidsubstitutions lead to remarkable product microheteroge-neity (Kowall et al., 1998). The lipoheptapeptide surfactin(Fig. 3A) is the most powerful biosurfactant known – a20 mM solution lowers the surface tension of water from72 to 27 mN m-1; it exerts a detergent-like action on bio-logical membranes (Carrillo et al., 2003), and is distin-guished by its exceptional emulsifying, foaming, anti-viraland anti-mycoplasma activities (reviewed by Peypouxet al., 1999). Surfactin is biosynthesized by the threeNRPSs SrfA–C (Peypoux et al., 1999); the thioesterase/acyltransferase enzyme SrfD stimulates the initiation ofthis process (Steller et al., 2004). The mechanism of sur-factin excretion is fully unknown, as an active transporterhas not been found, implying passive diffusion across thecytoplasmic membrane. Surfactin resistance is providedby YerP, the first example of a RND (resistance, nodulationand cell division) family multidrug efflux pump in Gram-positive bacteria (Tsuge et al., 2001a). The regulation ofsurfactin biosynthesis is closely connected to the compe-tence development pathway (Marahiel et al., 1993;reviewed in Hamoen et al., 2003; see also Fig. 4). Naturalcompetence defines the ability for exogenous DNAuptake. Remarkably, the comS gene involved in B. subtiliscompetence development is located within and out offrame of the srfA gene that specifies surfactin synthetase(Fig. 3A). The expression of both srfA and comS is regu-lated via a complex network that governs cellular differen-tiation, including quorum sensing via extracellular ComXand the two-component regulatory system ComPA(reviewed in Hamoen et al., 2003). Thus, B. subtilis ele-gantly uses a single quorum-sensing pathway for theDNA-uptake system and surfactin production. It is con-ceivable that competence development in order to assim-ilate external DNA is a microbial attempt to ensure themaintenance of genetic information beyond the individualcell. Additionally, uptake of external DNA can be used toincrease the genetic diversity of the bacterial population.

The iturin family encompasses the closely related cycliclipoheptapeptides mycosubtilin, the iturines and the bacil-lomycins (Fig. 3A) with strong anti-fungal and haemolyticbut only limited anti-bacterial activities (Thimon et al.,

1995). They are synthesized by the closely related NRPSsmycosubtilin (Duitman et al., 1999), iturin (Tsuge et al.,2001b) and bacillomycin (Moyne et al., 2004) synthetase.

Fengycin (synonymous to plipastatin) combines severalexceptional structural properties: cyclization, branchingand unusual constituents (Fig. 3A). Fengycin specificallyacting against filamentous fungi (Vanittanakom et al.,1986) is biosynthesized by fengycin synthetase encom-passing the five NRPSs Fen1–Fen5 encoded by ppsA–E(Steller et al., 1999).

Remarkably, although genes specifying surfactin andfengycin synthetase are conserved within the B. subtilis168 genome (Kunst et al., 1997), the corresponding anti-biotics are not produced. Surfactin production depends onthe PPan transferase Sfp (Nakano et al., 1992) whichconverts the inactive apoforms of surfactin and fengycinsynthetase to their active holoforms (Lambalot et al.,1996). However, the sfp allel of the 168 strain specifiesan inactive protein due to a frameshift mutation (Mootzet al., 2001). Accordingly, the introduction of a native sfpallel into B. subtilis 168 provoked surfactin (Nakano et al.,1992) and fengycin (plipastatin) (Tsuge et al., 1999)production.

The biosynthesis of the dipeptide bacilysin (Fig. 3B; L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) depends onthe ywfBCDEFGH cluster (Inaoka et al., 2003). Theunusual epoxy-modified amino acid anti-capsin is proba-bly generated through the action of a prephenate dehy-dratase and an aminotransferase encoded by ywfBG,respectively, as a branching off from prephenate of thearomatic amino acid pathway (Hilton et al., 1988). Genes

Fig. 4. Regulatory pathways of antibiotic biosynthesis in B. subtilis. Survey of the regulatory pathways for the biosynthesis of the B. subtilis antibiotics subtilin, subtilosin, bacilysin, surfactin, the killing factor Skf and the spore-associated anti-microbial polypeptide TasA. The scheme is simplified in terms of the regulation of competence development, which has been elaborately summarized by Hamoen et al. (2003); for details, see the corresponding text. A B. subtilis cell is symbolized by a lipid bilayer; compounds acting as pheromone are boxed; membrane-localized sensor histidine kinases are symbolized as circles. Positive and negative regulation of gene expression is indicated by arrows and T-bars respectively. For clarity, the repression of AbrB on sbo-alb and tasA was omitted.



bacDE (ywfEF) have been shown to encode the functionsof amino acid ligation and bacilysin immunity respectively(Steinborn et al., 2004). Bacilysin production is regulatedon different levels (see also Fig. 4), negatively by GTP viathe transcriptional regulator CodY (Inaoka et al., 2003)and AbrB (Yazgan et al., 2003). Positive regulation occursby guanonsine 5¢-diphosphate 3¢-diphosphate (ppGpp)(Inaoka et al., 2003) and a quorum-sensing mechanismthrough the peptide pheromone PhrC (Yazgan et al.,2003).

Miscellaneous antibiotic compounds

The genome of B. subtilis 168 contains the pksA–S –locus with a remarkable size of 76 kb, that specifies aPKS–homologous system (Kunst et al., 1997). Specula-tive products might be the polyketides difficidin (Fig. 3B;Wilson et al., 1987) or bacillaene (empirical formulaC35H48O7; Patel et al., 1995). However, B. subtilis 168 doesnot produce polyketides, presumably due to the mutatedsfp gene (see above). It has been very recently shownthat the biosynthesis of difficidin and bacillaene in B. sub-tilis A1/3 is dependent on a Sfp-homologous PPan trans-ferase (Hofemeister et al., 2004). Thus, Sfp in B. subtilis168 might also be involved in the phosphopantetheinyla-tion of polyketide synthase acyl carrier domains.

A series of new antibiotics have been recently isolatedfrom well-known B. subtilis strains. These include bac-ilysocin (Fig. 3B), an anti-microbial phospholipid, that canbe isolated from B. subtilis 168 cells by extraction withbutanol (Tamehiro et al., 2002). Most probably bacilysocinis derived from the major B. subtilis phospholipid phos-phatidylglycerol through YtpA-catalysed acyl ester hydrol-ysis (Tamehiro et al., 2002). Amicoumacins (Fig. 3B) areproduced by several B. subtilis strains excluding the 168strain (Pinchuk et al., 2002). Their anti-bacterial and anti-inflammatory activities, as well as their action on Helio-bacter pylori make the amicoumacins attractive for thetreatment of chronic gastritis and peptic ulcer in humans(Pinchuk et al., 2001). Very recently, Inaoka et al. (2004)showed the production of the aminosugar antibiotic 3,3¢-neotrehalosadiamine (NTD), dormant in the wild-typestrain, that can be induced by a rifampicin-resistant phe-notype of the RNA polymerase. The operon specifyingNTD biosynthesis encompasses the genes ntdABC(yhjLKJ). NTD acts as an autoinducer for its own biosyn-thesis genes via the regulator protein NtdR encoded byntdR (yhjM) (Inaoka et al., 2004). The transition-phase,spore-associated 31 kDa TasA protein exhibits a broadspectrum of anti-microbial activity. TasA together withyqxM and sipW constitutes a transition-phase operon(under positive control of Spo0A/SigH, and under repres-sion of AbrB; see Fig. 4) that could play a protective role

during B. subtilis sporulation (Stover and Driks, 1999).Further B. subtilis antibiotics are summarized in Fig. 3B.

Specific biological functions of distinct B. subtilis antibiotics

Microbes produce an amazing variety of antibiotics and,moreover, possess multidrug-type resistance genes, bothsuggesting dynamic ‘intermicrobial warfares’. Conse-quently, the classification of anti-microbials as competitiveweapons against other microorganisms has influencedour view for several decades. However, antibiotics areoften produced by specific strains and, thus, are not oblig-atory for the general survival of the genera per se. Twoimportant questions that arise are: (i) why antibiotics arebiosynthesized and (ii) are there any biological roles forantibiotics beyond the ‘pure’ anti-microbial action? Theefforts for antibiotic production are enormous, in particularif one reminds that most of antibiotic biosyntheses areregulated by mechanisms shared with other starvation-induced activities (see also Fig. 4) such as sporulation,genetic competence development and production of extra-cellular degradative enzymes (Katz and Demain, 1977;Losick et al., 1986; Marahiel et al., 1993). Therefore, it isinconceivable that the intricate reaction sequences of anti-biotic biosyntheses would have been retained in naturewithout benefit to the organism.

Rhizobacteria are present in the soil in an average ofabout 108 cells per gram, and from the soil, they aretransferred to various associated environments includingplants, foods, animals, marine and freshwater habitats(Priest, 1993). One of the main representative, the ‘hay-bacterium’ B. subtilis produces more than two dozen anti-biotics. If all pathways are considered, their productionrequires more than 350 kb (NRPSs, 200 kb; PKSs, 76 kb;lantibiotics, 50 kb; others >20 kb), corresponding to aremarkable 10% of the annotated ORFs. It should beemphasized that all investigated B. subtilis strains pro-duce individual antibiotic cocktails encompassing only aportion of the compounds depicted above; the average ofa B. subtilis genome that is devoted to antibiotic produc-tion is about 4–5%. The potential of a given B. subtilisstrain for antibiotic syntheses is comparable with Bacillusamyloliquefaciens (six operons of 306 kb, 7.5% of thegenome; Koumoutsi et al., 2004) but stays behind thepotential of Streptomycetes such as Streptomycetes aver-mitilis (25 operons of 560 kb corresponding to 6.4% of thegenome; Omura et al., 2001). The marked differences ofB. subtilis strains with regards to their produced antibioticspectra suggest that the antibiotic specifying loci musthave been recent acquisitions. Horizontal exchange ofgenetic material enabled via uptake of phage, plasmid ornaked DNA by genetically competent cells is a feasiblepossibility for this divergence. Presumably, accommoda-

852 T. Stein


tion of genes specifying antibiotic biosyntheses and/orresistance determinants would be beneficial for the B.subtilis cells and thus, enriching the fraction of a popula-tion that is comprised of antibiotic producing and/or toler-ant cells. One example of B. subtilis for the acquisition ofphage DNA is the sublancin specifying gene cluster withinthe prophage SPb locus (Dorenbos et al., 2002; Westerset al., 2003). Remarkably, the closely related gene clusterfor subtilin and ericin biosynthesis inhabit identical geneloci in B. subtilis strains ATCC 6633 and A1/3 (Fig. 2),suggesting that they have evolved from a common ances-tor (Stein et al., 2002b) and/or that they might be inter-changeable genetic elements. Presumably also NRPSsspecifying genes might be interchangeable among differ-ent B. subtilis strains, as for example mycosubtilin andfengycin synthetase genes in B. subtilis ATCC 6633(Duitman et al., 1999) and A1/3 (Hofemeister et al., 2004)have been found in identical loci respectively. Further-more, the srf loci of B. subtilis 168 (Kunst et al., 1997) andB. amyloliquefaciens (Koumoutsi et al., 2004) are identi-cal, supporting the idea that NRPSs are also interchange-able among different Bacilli.

A couple of antibiotics have been found to be producedby a great variety of B. subtilis strains (subtilosin, surfactin,bacilysin); others are produced strain-specifically(lantibiotics subtilin, ericin and mersacidin). However,systematic studies that survey the complete spectrum ofantibiotic activities by different B. subtilis strains (e.g. inthe A1/3 strain; Hofemeister et al., 2004) are rare. Pinchuket al. (2002) investigated 51 Bacillus strains isolated fromdifferent habitats, from which 47 have been identified asB. subtilis, among them 11 amicoumacin producer. Sur-factin production is widely spread among B. subtilis (Leen-ders et al., 1999; Peypoux et al., 1999; Vater et al., 2002;Hofemeister et al., 2004), a property that is shared withclosely related Bacilli such as amyloliquefaciens (Kou-moutsi et al., 2004), circulans (Hsieh et al., 2004) andpumilus (Kalinovskaya et al., 2002) strains.

Altogether, it seems to be that B. subtilis is outstandingin the genus Bacillus with regards to its potential to pro-duce so many different antibiotics. However, B. subtilis isby far the most commonly investigated Bacillus genus,and the large number of known B. subtilis antibiotics mightreflect the numerousness of natural isolates and studies.Also other Bacilli such as Bacillus brevis (brevistin,edeines, gramicidines, tyrocidin) or B. amyloliquefaciens(Koumoutsi et al., 2004) produce a couple of antibiotics,although their number seems to minor as compared withB. subtilis. Otherwise, it is tempting to speculate that thefrequent occurrence of B. subtilis among other Bacillusstrains in natural isolates might be also a consequence ofthe benefits of the produced compounds. Unfortunately,the originally B. subtilis 168 Marburg strain systematicallyinvestigated and used as a model system for Gram-

positive organisms has been cultivated in the laboratoryfor several decades, and more alarmingly, was exposedto X-rays in the mid-1940s (Burkholder and Giles, 1947).This strain does not produce lipopeptides or polyketides,and consequently, important contributions of these com-pounds to the morphology of B. subtilis might have beenoverlooked or underestimated in previous studies.

Lipopeptide antibiotics are among the most frequentlyproduced B. subtilis antibiotics. They as well as otheramphiphilic compounds such as the phospholipid bac-ilysocin are low-molecular-mass surfactants that are ableto alter the physical and/or chemical properties at inter-faces. Three possible roles for such bioemulsifiers havebeen proposed: (i) an increase of the surface area ofhydrophobic water-insoluble growth substrates, (ii) anincrease in the bioavailability of hydrophobic substratesby increasing their apparent solubility and (iii) an influenceon the attachment and detachment of microorganisms toand from surfaces (Rosenberg and Ron, 1999). It is easyto imagine that these roles would have strong influenceon the survival of B. subtilis in its natural habitat, the soiland the rhizosphere. In this respect, the non-ribosomallygenerated anionic lipoheptapeptide surfactin is by far themost prominent and best-investigated representative.

Many bacteria exhibit two distinct lifestyles, a free-float-ing planktonic mode for rapid proliferation and spread intonew territories and a sessile biofilm mode. Biofilms arehighly structured microbial communities that adhere tosurfaces and constitute the majority of bacteria in mostnatural and pathogenic ecosystems (for recent reviews,see Harshey, 2003; Hall-Stoodley et al., 2004; Stanleyand Lazazzera, 2004). Cell motility in colonies, swarming,involves differentiation of vegetative cells into hyperflagel-lated ‘swarmer cells’ that undergo rapid and co-ordinatedpopulation migration across solid surfaces ( Shapiro,1998; Fraser and Hughes, 1999). The swarming motilityof B. subtilis is strictly dependent on the production ofsurfactin (Kinsinger et al., 2003), an observation madewith undomesticated strains (Kearns and Losick, 2003;Kearns et al., 2004). However, surfactin production is nec-essary but not sufficient for swarming, in which at leastthe factors swrAB, swrC (synonymous to the surfactinresistance gene yerP) and efp are additionally involved(Kearns et al., 2004). B. subtilis biofilm formation (Brandaet al., 2004) is dependent on the transcription factorsSpoOA (Hamon and Lazazzera, 2001), sigma-H and AbrB(Hamon et al., 2004). As these transcription factors arealso involved in the regulation of several antibiotic biosyn-theses (Fig. 4), antibiotic production in a biofilm is con-ceivable. It has been recently documented that thecolonization of plant roots by B. subtilis is associated withsurfactin production and biofilm formation, and strikingly,surfactin protected the plant against the infection by thepathogen Pseudomonas syringae (Bais et al., 2004).



Results from a very recent study imply that the surfactins,and not other lipopeptides like the bacillomycins, enablethe natural isolate B. subtilis A1/3 to form biofilms (Hofe-meister et al., 2004). A close correlation between antibi-otic production and biofilm formation in other bacilli (Yanet al., 2003) or the observation that surface-active rham-nolipid surfactants affect the architecture of biofilms inPseudomonas aerginosa (Davey et al., 2003) suggeststhat biofilm-associated antibiotic/surfactant production ismore widely distributed than previously thought. Interest-ingly, surfactin is also able to inhibit biofilm formation ofother bacteria (Bais et al., 2004), and even the humanpathogen Salmonella enterica (Mireles et al., 2001). Theanti-microbial and fungicidal action of lipopeptides in addi-tion to surfactin (fengycin, iturin, bacillomycin) might beadvantageous for B. subtilis cells to eliminate competitorsin the same habitat. It seems to be that the production ofthese lipopeptides (e.g. bacillomycin in B. subtilis A1/3;Hofemeister et al., 2004) is articulately delayed (late sta-tionary phase) as compared with surfactin (transitionbetween exponential and stationary growth). Altogether, itis worth to further consider the use of B. subtilis, anubiquitously occurring ‘safe’ microorganism, in agricultureas natural fungicide and plant growth-promoting microor-ganism (reviewed in Nicholson, 2002) and/or decontami-nation of solid surfaces (Rosenberg and Ron, 1999).

Nutrient-limited B. subtilis cells are able to sporulate,an elaborate process that results in the release of anendospore from the terminally differentiated, apoptoticmother cell (Errington, 2003). Strikingly, Branda et al.(2001) documented that sporulation is tightly intertwinedwith the development of highly ordered and surface-asso-ciated cell clots, ‘fruiting-bodies’, that are characterized byspore-specific gene expression. The formation of similaraerial hyphae in multicellular organism like fungi need thegeneration of surface-active molecules (Wösten et al.,1999; Kodani et al., 2004). Three genes are involved in B.subtilis ‘fruiting body’ formation (Branda et al., 2001):yveQ and yveR seem to encode exopolysaccharide bio-synthetic enzymes, and sfp specifies a PPan transferase.As Sfp can modify the surfactin and fengycin NRPSs andthe PKS synthase (see above), its influence on fruiting-body formation is most probably exerted by one or moresurface-active products of these NRPS and/or PKSsystems. Importantly, fruiting bodies are only formed byundomesticated, natural B. subtilis isolates, which againemphasizes the importance of carrying out investigationswith other than laboratory or laboratory-acclimatizedstrains (general aspects are reviewed in Palkova, 2004).

Bacillus subtilis sporulation is governed by the regula-tory protein Spo0A. Gonzalez-Pastor et al. (2003) discov-ered that Spo0A is also involved in the regulation of twohighly interesting operons, namely skf (sporulation killingfactor) and sdp (sporulation delay protein) (Fawcett et al.,

2000; Molle et al., 2003). Early sporulating B. subtilis cells(Spo0A-active) produce and export the antibiotic-like kill-ing factor Skf, to which they are immune, and that causeslysis of non-sporulating (Spo0A-inactive) sister cells – amechanism designated as ‘cannibalism of siblings’(Gonzalez-Pastor et al., 2003). Remarkably, Skf (YbcO)exhibits also anti-microbial activity, in particular againstthe rice pathogen Xanthomonads (Lin et al., 2001). Thesporulation delay protein Sdp acts cooperatively with Skfand effectuates programmed cell death in Spo0A-inactivecells, and furthermore, Sdp holds up sporulation withinSpo0A producer cells (Gonzalez-Pastor et al., 2003). Thenutrient scavenge of lysed sister cells is beneficial forSpo0A-active Skf/Sdp-producing cells, a mechanism thatallows them to keep growing rather than to complete theenergy-consuming last resort sporulation pathway.

We become increasingly aware that single-cell microor-ganisms display sophisticated social behaviours: prokary-otic B. subtilis cells live in complex communities wherethey co-ordinate gene expression and group behaviourthrough different quorum-sensing pathways (Shapiro,1998). The collective cell death of a subpopulation can beseen as ‘altruistic suicide’, as a consequence of develop-mental processes which would ensure the survival of theremaining unharmed and/or better-adapted cells. Such amechanism might be one of the clues to understand theclassical question: why are antibiotic production andsporulation so often related to one another (Katz andDemain, 1977; Marahiel et al., 1993). Although antibioticsare not obligatory for sporulation, the biosyntheses of acouple of them are regulated by factors shared with thesporulation process (Fig. 4). It is conceivable that AbrB-regulated antibiotics that are consequently induced inSpo0A-active cells (e.g. subtilin, subtilosin, bacilysin, sur-factin) are also involved in the action against non-sporu-lating (Spo0A-inactive) sister cells. However, the directregulation of the skf cluster by Spo0A (Fawcett et al.,2000; Gonzalez-Pastor et al., 2003) clearly distinguishesSkf from other B. subtilis antibiotics. It is remarkable thatthe B. subtilis lantibiotics subtilin (Stein et al., 2002a) andericin (J. Hofemeister, pers. comm.), both autoregulatedvia two-component regulatory systems, function as pher-omones for quorum sensing (Stein et al., 2002a; Kleer-ebezem, 2004). It has to be elucidated whether quorumsensing via lantibiotics is restricted to only a handful B.subtilis strains or whether it is wider distributed than actu-ally known. Notably, we have begun to understand thatdistinct B. subtilis antibiotics and antibiotic-like com-pounds play crucial roles in communal development andcontribute to the survival of B. subtilis in its natural habitat.It is to be expected that future studies will give us adetailed and more integrated understanding of the chal-lenging biological functions of anti-microbial compoundsof Bacillus and other organisms.

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