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Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005? 2005564845857Review ArticleBacillus subtilis antibioticsT. Stein
Molecular Microbiology (2005) 56(4), 845–857
doi:10.1111/j.1365-2958.2005.04587.x
MicroReview
Bacillus subtilis antibiotics: structures, syntheses and
specific functions
Torsten Stein
Institut für Mikrobiologie, Johann Wolfgang GoetheUniversität, Marie-Curie-Str. 9, 60439 Frankfurt/Main,
Germany.
Summary
The endospore-forming rhizobacterium Bacillus subtilis – 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 predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics
and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds
such as polyketides, an aminosugar, and a phospholipid. 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 produced lipopeptides are involved in biofilm and swarming 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
Accepted 24 January, 2005. For correspondence. E-mail
Fax
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Tel.
(+49) 69 7982 9522;
(+49) 69 7982 9527.
© 2005 Blackwell Publishing Ltd
two dozen antibiotics with an amazing variety of structures. All of the genes specifying antibiotic biosyntheses
combined amount to 350 kb; however, as no strain possesses 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 structures 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 represent the predominant class. They exhibit highly rigid,
hydrophobic and/or cyclic structures with unusual constituents like D-amino acids and are generally resistant to
hydrolysis by peptidases and proteases (Katz and
Demain, 1977; and references therein). Furthermore, cysteine residues are either oxidized to disulphides and/or
are modified to characteristic intramolecular C–S (thioether) linkages, and consequently the peptide antibiotics
are insensitive to oxidation. Principally, two different biosynthetic pathways for peptides allow the incorporation of
such unusual (non-proteinaceous) constituents: (i) the
non-ribosomal synthesis of peptides by large megaenzymes, the non-ribosomal peptide synthetases (NRPSs)
and (ii) the ribosomal synthesis of linear precursor peptides 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 (lanthioninecontaining 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
Fig. 1. Proposed pathway for post-translational lanthionine formation. The first step in lanthionine formation involves dehydration of Lserine and L-threonine residues in ribosomally generated prelantibiotic peptides yielding 2,3-didehydroalanine and 2,3-didehydrobutyrine respectively. In the second step inter-residual thioether linkages
are formed through stereospecific Michael-like additions of neighboured L-cysteine sulphydryl groups yielding meso-lanthionine and 3methyllanthionine respectively. Note the a-carbon atom D-configurations of the formerly L-serine/L-threonine residues; grey boxes represent formerly cysteines.
Jack and Jung, 2000; McAuliffe et al., 2001). Based on
structural properties two lantibiotic types are distinguishable. Type A lantibiotics (21–38 amino acid residues)
exhibit a more linear secondary structure and kill Grampositive target cells by forming voltage-dependent pores
into the cytoplasmic membrane. Remarkably, for the lantibiotic 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). Grampositive lantibiotic producers exhibit efficient countermeasures to obviate the action of their own products. Selfprotection (immunity) against lantibiotics is based on ATPbinding 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 cytoplasmic 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 transcription 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 quorumsensing mechanism in which subtilin plays a pheromonetype 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 contains two structural genes, eriA and eriS, although the
open reading frames (ORFs) are closely related to corresponding 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 comparable. 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 (Brötz
et al., 1997). The mersacidin gene cluster consists of the
structural gene mrsA, as well as genes involved in posttranslational 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 mrsFGE (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.
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
Bacillus subtilis antibiotics 847
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;
DA, 2,3-didehydroalanine; DB, 2,3-didehydrobutyrine. For details, see the corresponding text.
MrsD, a member of the homo-oligomeric flavin-containing
cysteine decarboxylases (HFCD) family, catalyses the oxidative decarboxylation of the C-terminal cysteine of the
mersacidin prepeptide. The dodecameric MrsD and its
closely relative EpiD involved in epidermin biosynthesis of
Streptococcus epidermidis represent the sole examples
of lantibiotic modifying enzymes with known three-dimensional 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-positive bacteria. Its structural gene sunA (formerly yolG)
belongs to the B. subtilis temperate bacteriophage SPb
(Westers et al., 2003) and thus, sublancin and the ‘prophage SPb-mediated bacteriocin’ (Hemphill et al., 1980) are
most probably the same compounds. An ABC transporter
(SunT) and two thiol-disulphide oxidoreductases (BdbAB)
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
belong to the sublancin locus (Fig. 2). Only BdbB seems
to be dedicated for sublancin production, most probably
for the formation of the disulphide bonds (Dorenbos et al.,
2002). The BdbB paralogue BdbC protein is at least partially able to replace BdbB in sublancin production, but
contrariwise BdbB cannot complement the function of
BdbC (competence development), showing that these two
closely related thiol-disulphide oxidoreductases have
different, but partly overlapping substrate specificities
(Kunst et al., 1997; Dorenbos et al., 2002). The SPb locus
including the sublancin gene cluster is not essential for
B. subtilis survival (Westers et al., 2003). However, it
contains yet unidentified genes mediating resistance
against sublancin action. One attractive hypothesis is that
sublancin might contribute to the survival of bacteriophage, e.g. that sublancin kills only non-lysogenized cells
and thus, enriching the per cent of a lysogenized B. subtilis population.
Subtilosin A produced by several B. subtilis strains
(Zheng et al., 1999; Stein et al., 2004) has a macrocyclic
848 T. Stein
structure (Fig. 2) with three inter-residual linkages (Marx
et al., 2001) that have been elucidated as thioether bonds
between cysteine sulphurs and amino acid alpha-carbons
(Kawulka et al., 2004). It acts against a variety of Grampositive bacteria, including Listeria (Zheng et al., 1999).
The sbo-alb (anti-listerial bacteriocin) cluster encodes
proteins AlbA (YwiA) most probably involved in post-translational modification of presubtilosin, AlbF (YwhN) probably acting in subtilosin processing and the subtilosin
immunity proteins AlbB–D (YwhQPO) (Zheng et al.,
2000). Expression of the sbo-alb genes occurs under
stress 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 is
widespread among bacteria and fungi (for recent reviews,
see Sieber and Marahiel, 2003; Finking and Marahiel,
2004; Walsh, 2004; and references therein). Large multienzymes, the NRPSs, that are composed of modularly
arranged catalytic domains (Fig. 3A), catalyse all necessary steps in peptide biosynthesis including the selection
and ordered condensation of amino acid residues. Each
elongation cycle in non-ribosomal peptide biosynthesis
needs the cooperation of three core domains. (i) The
adenylation domain (550 amino acid residues) selects its
cognate amino acid and generates an enzymatically stabilized aminoacyl adenylate. This mechanism resembles
the amino-acylation of tRNA synthetases during ribosomal peptide biosynthesis. (ii) The thiolation or peptidyl
carrier domain (80 aa) is equipped with a 4¢-phosphopan-
tetheine (PPan) prosthetic group to which the adenylated
amino acid substrate is transferred and thioesterified
under release of AMP. Thus, the PPan cofactor acts as
thiotemplate and as a swinging arm to transport intermediates between the various catalytic centres. The peptidyl
carrier proteins are post-translationally converted from
inactive apoforms to their active holoforms by dedicated
PPan transferases (Lambalot et al., 1996). (iii) The formation of a new peptide bond is catalysed by condensation
domains (450 aa) located between each pair of adenylation and peptidyl carrier domains. The linear assembly
line-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 biosynthesis is terminated by macrocyclization of the peptide
product, whereby parts of the molecule distant in the
constructed linear peptide chain are covalently linked to
one another (Kohli and Walsh 2003). Typically, such reactions are catalysed by thioesterase domains at the Cterminal end of the NRPS assembly line. The depicted
mechanism of peptide biosynthesis has been outlined in
the concept of the ‘Multiple Carrier Model of Nonribosomal
Peptide Biosynthesis at Modular Multienzymatic Templates’ (Stein et al., 1996). Mechanistically, NRPSs are
closely related to polyketide synthetases (PKSs), as both
modular systems utilize multiple Ppan carriers for covalent
binding of monomers and growing chains. Both systems
are highly flexible in which naturally rearrangements can
be easily achieved within a relatively short period, permitting the random evolution of compounds that provide
selective advantages. Striking examples for such flexibility
are 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 Lconfiguration, 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 Dconfiguration, 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).
1
Surfactin 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).
2
The 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.
3
Fengycin (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.
4
NPRSs 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 dihydroxybutyrate (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).
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
Bacillus subtilis antibiotics 849
A
B
1988
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
850 T. Stein
related compounds of the iturin family (see Fig. 3A). Thus,
NRPSs and PKSs are per se extremely amenable to
genetic manipulations, providing powerful tools for
future development and production of novel peptides,
polyketides and hybrid compounds with new properties.
The huge potential of NRPSs and PKSs in the generation
of novel drugs has been excellently reviewed elsewhere
(Sieber and Marahiel, 2003; Finking and Marahiel, 2004;
Walsh, 2004).
Non-ribosomally generated amphipathic lipopeptide
antibiotics with condensed b-hydroxyl or b-amino fatty
acids are widespread in B. subtilis. Variations in length
and branching of the fatty acid chains and amino acid
substitutions lead to remarkable product microheterogeneity (Kowall et al., 1998). The lipoheptapeptide surfactin
(Fig. 3A) is the most powerful biosurfactant known – a
20 mM solution lowers the surface tension of water from
72 to 27 mN m-1; it exerts a detergent-like action on biological membranes (Carrillo et al., 2003), and is distinguished by its exceptional emulsifying, foaming, anti-viral
and anti-mycoplasma activities (reviewed by Peypoux
et al., 1999). Surfactin is biosynthesized by the three
NRPSs SrfA–C (Peypoux et al., 1999); the thioesterase/
acyltransferase enzyme SrfD stimulates the initiation of
this process (Steller et al., 2004). The mechanism of surfactin excretion is fully unknown, as an active transporter
has not been found, implying passive diffusion across the
cytoplasmic membrane. Surfactin resistance is provided
by YerP, the first example of a RND (resistance, nodulation
and cell division) family multidrug efflux pump in Grampositive bacteria (Tsuge et al., 2001a). The regulation of
surfactin biosynthesis is closely connected to the competence development pathway (Marahiel et al., 1993;
reviewed in Hamoen et al., 2003; see also Fig. 4). Natural
competence defines the ability for exogenous DNA
uptake. Remarkably, the comS gene involved in B. subtilis
competence development is located within and out of
frame of the srfA gene that specifies surfactin synthetase
(Fig. 3A). The expression of both srfA and comS is regulated via a complex network that governs cellular differentiation, including quorum sensing via extracellular ComX
and the two-component regulatory system ComPA
(reviewed in Hamoen et al., 2003). Thus, B. subtilis elegantly uses a single quorum-sensing pathway for the
DNA-uptake system and surfactin production. It is conceivable that competence development in order to assimilate external DNA is a microbial attempt to ensure the
maintenance of genetic information beyond the individual
cell. Additionally, uptake of external DNA can be used to
increase the genetic diversity of the bacterial population.
The iturin family encompasses the closely related cyclic
lipoheptapeptides mycosubtilin, the iturines and the bacillomycins (Fig. 3A) with strong anti-fungal and haemolytic
but only limited anti-bacterial activities (Thimon et al.,
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.
1995). They are synthesized by the closely related NRPSs
mycosubtilin (Duitman et al., 1999), iturin (Tsuge et al.,
2001b) and bacillomycin (Moyne et al., 2004) synthetase.
Fengycin (synonymous to plipastatin) combines several
exceptional structural properties: cyclization, branching
and unusual constituents (Fig. 3A). Fengycin specifically
acting against filamentous fungi (Vanittanakom et al.,
1986) is biosynthesized by fengycin synthetase encompassing the five NRPSs Fen1–Fen5 encoded by ppsA–E
(Steller et al., 1999).
Remarkably, although genes specifying surfactin and
fengycin synthetase are conserved within the B. subtilis
168 genome (Kunst et al., 1997), the corresponding antibiotics are not produced. Surfactin production depends on
the PPan transferase Sfp (Nakano et al., 1992) which
converts the inactive apoforms of surfactin and fengycin
synthetase to their active holoforms (Lambalot et al.,
1996). However, the sfp allel of the 168 strain specifies
an inactive protein due to a frameshift mutation (Mootz
et al., 2001). Accordingly, the introduction of a native sfp
allel 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; Lalanine-[2,3-epoxycyclohexano-4]-L-alanine) depends on
the ywfBCDEFGH cluster (Inaoka et al., 2003). The
unusual epoxy-modified amino acid anti-capsin is probably generated through the action of a prephenate dehydratase and an aminotransferase encoded by ywfBG,
respectively, as a branching off from prephenate of the
aromatic amino acid pathway (Hilton et al., 1988). Genes
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
Bacillus subtilis antibiotics 851
bacDE (ywfEF) have been shown to encode the functions
of amino acid ligation and bacilysin immunity respectively
(Steinborn et al., 2004). Bacilysin production is regulated
on different levels (see also Fig. 4), negatively by GTP via
the transcriptional regulator CodY (Inaoka et al., 2003)
and AbrB (Yazgan et al., 2003). Positive regulation occurs
by guanonsine 5¢-diphosphate 3¢-diphosphate (ppGpp)
(Inaoka et al., 2003) and a quorum-sensing mechanism
through 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 a
PKS–homologous system (Kunst et al., 1997). Speculative products might be the polyketides difficidin (Fig. 3B;
Wilson et al., 1987) or bacillaene (empirical formula
C35H48O7; Patel et al., 1995). However, B. subtilis 168 does
not produce polyketides, presumably due to the mutated
sfp gene (see above). It has been very recently shown
that the biosynthesis of difficidin and bacillaene in B. subtilis A1/3 is dependent on a Sfp-homologous PPan transferase (Hofemeister et al., 2004). Thus, Sfp in B. subtilis
168 might also be involved in the phosphopantetheinylation of polyketide synthase acyl carrier domains.
A series of new antibiotics have been recently isolated
from well-known B. subtilis strains. These include bacilysocin (Fig. 3B), an anti-microbial phospholipid, that can
be isolated from B. subtilis 168 cells by extraction with
butanol (Tamehiro et al., 2002). Most probably bacilysocin
is derived from the major B. subtilis phospholipid phosphatidylglycerol through YtpA-catalysed acyl ester hydrolysis (Tamehiro et al., 2002). Amicoumacins (Fig. 3B) are
produced by several B. subtilis strains excluding the 168
strain (Pinchuk et al., 2002). Their anti-bacterial and antiinflammatory activities, as well as their action on Heliobacter pylori make the amicoumacins attractive for the
treatment 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-type
strain, that can be induced by a rifampicin-resistant phenotype of the RNA polymerase. The operon specifying
NTD biosynthesis encompasses the genes ntdABC
(yhjLKJ). NTD acts as an autoinducer for its own biosynthesis genes via the regulator protein NtdR encoded by
ntdR (yhjM) (Inaoka et al., 2004). The transition-phase,
spore-associated 31 kDa TasA protein exhibits a broad
spectrum of anti-microbial activity. TasA together with
yqxM and sipW constitutes a transition-phase operon
(under positive control of Spo0A/SigH, and under repression of AbrB; see Fig. 4) that could play a protective role
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
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, both
suggesting dynamic ‘intermicrobial warfares’. Consequently, the classification of anti-microbials as competitive
weapons against other microorganisms has influenced
our view for several decades. However, antibiotics are
often produced by specific strains and, thus, are not obligatory for the general survival of the genera per se. Two
important questions that arise are: (i) why antibiotics are
biosynthesized and (ii) are there any biological roles for
antibiotics beyond the ‘pure’ anti-microbial action? The
efforts for antibiotic production are enormous, in particular
if one reminds that most of antibiotic biosyntheses are
regulated by mechanisms shared with other starvationinduced activities (see also Fig. 4) such as sporulation,
genetic competence development and production of extracellular degradative enzymes (Katz and Demain, 1977;
Losick et al., 1986; Marahiel et al., 1993). Therefore, it is
inconceivable that the intricate reaction sequences of antibiotic biosyntheses would have been retained in nature
without benefit to the organism.
Rhizobacteria are present in the soil in an average of
about 108 cells per gram, and from the soil, they are
transferred to various associated environments including
plants, foods, animals, marine and freshwater habitats
(Priest, 1993). One of the main representative, the ‘haybacterium’ B. subtilis produces more than two dozen antibiotics. If all pathways are considered, their production
requires more than 350 kb (NRPSs, 200 kb; PKSs, 76 kb;
lantibiotics, 50 kb; others >20 kb), corresponding to a
remarkable 10% of the annotated ORFs. It should be
emphasized that all investigated B. subtilis strains produce individual antibiotic cocktails encompassing only a
portion of the compounds depicted above; the average of
a B. subtilis genome that is devoted to antibiotic production is about 4–5%. The potential of a given B. subtilis
strain for antibiotic syntheses is comparable with Bacillus
amyloliquefaciens (six operons of 306 kb, 7.5% of the
genome; Koumoutsi et al., 2004) but stays behind the
potential of Streptomycetes such as Streptomycetes avermitilis (25 operons of 560 kb corresponding to 6.4% of the
genome; Omura et al., 2001). The marked differences of
B. subtilis strains with regards to their produced antibiotic
spectra suggest that the antibiotic specifying loci must
have been recent acquisitions. Horizontal exchange of
genetic material enabled via uptake of phage, plasmid or
naked DNA by genetically competent cells is a feasible
possibility for this divergence. Presumably, accommoda-
852 T. Stein
tion of genes specifying antibiotic biosyntheses and/or
resistance determinants would be beneficial for the B.
subtilis cells and thus, enriching the fraction of a population that is comprised of antibiotic producing and/or tolerant cells. One example of B. subtilis for the acquisition of
phage DNA is the sublancin specifying gene cluster within
the prophage SPb locus (Dorenbos et al., 2002; Westers
et al., 2003). Remarkably, the closely related gene cluster
for subtilin and ericin biosynthesis inhabit identical gene
loci in B. subtilis strains ATCC 6633 and A1/3 (Fig. 2),
suggesting that they have evolved from a common ancestor (Stein et al., 2002b) and/or that they might be interchangeable genetic elements. Presumably also NRPSs
specifying genes might be interchangeable among different B. subtilis strains, as for example mycosubtilin and
fengycin 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. Furthermore, the srf loci of B. subtilis 168 (Kunst et al., 1997) and
B. amyloliquefaciens (Koumoutsi et al., 2004) are identical, supporting the idea that NRPSs are also interchangeable among different Bacilli.
A couple of antibiotics have been found to be produced
by 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 of
antibiotic activities by different B. subtilis strains (e.g. in
the A1/3 strain; Hofemeister et al., 2004) are rare. Pinchuk
et al. (2002) investigated 51 Bacillus strains isolated from
different habitats, from which 47 have been identified as
B. subtilis, among them 11 amicoumacin producer. Surfactin production is widely spread among B. subtilis (Leenders et al., 1999; Peypoux et al., 1999; Vater et al., 2002;
Hofemeister et al., 2004), a property that is shared with
closely related Bacilli such as amyloliquefaciens (Koumoutsi et al., 2004), circulans (Hsieh et al., 2004) and
pumilus (Kalinovskaya et al., 2002) strains.
Altogether, it seems to be that B. subtilis is outstanding
in the genus Bacillus with regards to its potential to produce so many different antibiotics. However, B. subtilis is
by far the most commonly investigated Bacillus genus,
and the large number of known B. subtilis antibiotics might
reflect 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 with
B. subtilis. Otherwise, it is tempting to speculate that the
frequent occurrence of B. subtilis among other Bacillus
strains in natural isolates might be also a consequence of
the benefits of the produced compounds. Unfortunately,
the originally B. subtilis 168 Marburg strain systematically
investigated and used as a model system for Gram-
positive organisms has been cultivated in the laboratory
for several decades, and more alarmingly, was exposed
to X-rays in the mid-1940s (Burkholder and Giles, 1947).
This strain does not produce lipopeptides or polyketides,
and consequently, important contributions of these compounds to the morphology of B. subtilis might have been
overlooked or underestimated in previous studies.
Lipopeptide antibiotics are among the most frequently
produced B. subtilis antibiotics. They as well as other
amphiphilic compounds such as the phospholipid bacilysocin are low-molecular-mass surfactants that are able
to alter the physical and/or chemical properties at interfaces. Three possible roles for such bioemulsifiers have
been proposed: (i) an increase of the surface area of
hydrophobic water-insoluble growth substrates, (ii) an
increase in the bioavailability of hydrophobic substrates
by increasing their apparent solubility and (iii) an influence
on the attachment and detachment of microorganisms to
and from surfaces (Rosenberg and Ron, 1999). It is easy
to imagine that these roles would have strong influence
on the survival of B. subtilis in its natural habitat, the soil
and the rhizosphere. In this respect, the non-ribosomally
generated anionic lipoheptapeptide surfactin is by far the
most prominent and best-investigated representative.
Many bacteria exhibit two distinct lifestyles, a free-floating planktonic mode for rapid proliferation and spread into
new territories and a sessile biofilm mode. Biofilms are
highly structured microbial communities that adhere to
surfaces and constitute the majority of bacteria in most
natural and pathogenic ecosystems (for recent reviews,
see Harshey, 2003; Hall-Stoodley et al., 2004; Stanley
and Lazazzera, 2004). Cell motility in colonies, swarming,
involves differentiation of vegetative cells into hyperflagellated ‘swarmer cells’ that undergo rapid and co-ordinated
population migration across solid surfaces ( Shapiro,
1998; Fraser and Hughes, 1999). The swarming motility
of B. subtilis is strictly dependent on the production of
surfactin (Kinsinger et al., 2003), an observation made
with undomesticated strains (Kearns and Losick, 2003;
Kearns et al., 2004). However, surfactin production is necessary but not sufficient for swarming, in which at least
the factors swrAB, swrC (synonymous to the surfactin
resistance gene yerP) and efp are additionally involved
(Kearns et al., 2004). B. subtilis biofilm formation (Branda
et al., 2004) is dependent on the transcription factors
SpoOA (Hamon and Lazazzera, 2001), sigma-H and AbrB
(Hamon et al., 2004). As these transcription factors are
also involved in the regulation of several antibiotic biosyntheses (Fig. 4), antibiotic production in a biofilm is conceivable. It has been recently documented that the
colonization of plant roots by B. subtilis is associated with
surfactin production and biofilm formation, and strikingly,
surfactin protected the plant against the infection by the
pathogen Pseudomonas syringae (Bais et al., 2004).
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
Bacillus subtilis antibiotics 853
Results from a very recent study imply that the surfactins,
and not other lipopeptides like the bacillomycins, enable
the natural isolate B. subtilis A1/3 to form biofilms (Hofemeister et al., 2004). A close correlation between antibiotic production and biofilm formation in other bacilli (Yan
et al., 2003) or the observation that surface-active rhamnolipid surfactants affect the architecture of biofilms in
Pseudomonas aerginosa (Davey et al., 2003) suggests
that biofilm-associated antibiotic/surfactant production is
more widely distributed than previously thought. Interestingly, surfactin is also able to inhibit biofilm formation of
other bacteria (Bais et al., 2004), and even the human
pathogen Salmonella enterica (Mireles et al., 2001). The
anti-microbial and fungicidal action of lipopeptides in addition to surfactin (fengycin, iturin, bacillomycin) might be
advantageous for B. subtilis cells to eliminate competitors
in the same habitat. It seems to be that the production of
these lipopeptides (e.g. bacillomycin in B. subtilis A1/3;
Hofemeister et al., 2004) is articulately delayed (late stationary phase) as compared with surfactin (transition
between exponential and stationary growth). Altogether, it
is worth to further consider the use of B. subtilis, an
ubiquitously occurring ‘safe’ microorganism, in agriculture
as natural fungicide and plant growth-promoting microorganism (reviewed in Nicholson, 2002) and/or decontamination 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 an
endospore from the terminally differentiated, apoptotic
mother cell (Errington, 2003). Strikingly, Branda et al.
(2001) documented that sporulation is tightly intertwined
with the development of highly ordered and surface-associated cell clots, ‘fruiting-bodies’, that are characterized by
spore-specific gene expression. The formation of similar
aerial hyphae in multicellular organism like fungi need the
generation 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 biosynthetic enzymes, and sfp specifies a PPan transferase.
As Sfp can modify the surfactin and fengycin NRPSs and
the PKS synthase (see above), its influence on fruitingbody formation is most probably exerted by one or more
surface-active products of these NRPS and/or PKS
systems. Importantly, fruiting bodies are only formed by
undomesticated, natural B. subtilis isolates, which again
emphasizes the importance of carrying out investigations
with other than laboratory or laboratory-acclimatized
strains (general aspects are reviewed in Palkova, 2004).
Bacillus subtilis sporulation is governed by the regulatory protein Spo0A. Gonzalez-Pastor et al. (2003) discovered that Spo0A is also involved in the regulation of two
highly interesting operons, namely skf (sporulation killing
factor) and sdp (sporulation delay protein) (Fawcett et al.,
© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 56, 845–857
2000; Molle et al., 2003). Early sporulating B. subtilis cells
(Spo0A-active) produce and export the antibiotic-like killing factor Skf, to which they are immune, and that causes
lysis of non-sporulating (Spo0A-inactive) sister cells – a
mechanism designated as ‘cannibalism of siblings’
(Gonzalez-Pastor et al., 2003). Remarkably, Skf (YbcO)
exhibits also anti-microbial activity, in particular against
the rice pathogen Xanthomonads (Lin et al., 2001). The
sporulation delay protein Sdp acts cooperatively with Skf
and effectuates programmed cell death in Spo0A-inactive
cells, and furthermore, Sdp holds up sporulation within
Spo0A producer cells (Gonzalez-Pastor et al., 2003). The
nutrient scavenge of lysed sister cells is beneficial for
Spo0A-active Skf/Sdp-producing cells, a mechanism that
allows them to keep growing rather than to complete the
energy-consuming last resort sporulation pathway.
We become increasingly aware that single-cell microorganisms display sophisticated social behaviours: prokaryotic B. subtilis cells live in complex communities where
they co-ordinate gene expression and group behaviour
through different quorum-sensing pathways (Shapiro,
1998). The collective cell death of a subpopulation can be
seen as ‘altruistic suicide’, as a consequence of developmental processes which would ensure the survival of the
remaining unharmed and/or better-adapted cells. Such a
mechanism might be one of the clues to understand the
classical question: why are antibiotic production and
sporulation so often related to one another (Katz and
Demain, 1977; Marahiel et al., 1993). Although antibiotics
are not obligatory for sporulation, the biosyntheses of a
couple of them are regulated by factors shared with the
sporulation process (Fig. 4). It is conceivable that AbrBregulated antibiotics that are consequently induced in
Spo0A-active cells (e.g. subtilin, subtilosin, bacilysin, surfactin) are also involved in the action against non-sporulating (Spo0A-inactive) sister cells. However, the direct
regulation of the skf cluster by Spo0A (Fawcett et al.,
2000; Gonzalez-Pastor et al., 2003) clearly distinguishes
Skf from other B. subtilis antibiotics. It is remarkable that
the B. subtilis lantibiotics subtilin (Stein et al., 2002a) and
ericin (J. Hofemeister, pers. comm.), both autoregulated
via two-component regulatory systems, function as pheromones for quorum sensing (Stein et al., 2002a; Kleerebezem, 2004). It has to be elucidated whether quorum
sensing via lantibiotics is restricted to only a handful B.
subtilis strains or whether it is wider distributed than actually known. Notably, we have begun to understand that
distinct B. subtilis antibiotics and antibiotic-like compounds play crucial roles in communal development and
contribute to the survival of B. subtilis in its natural habitat.
It is to be expected that future studies will give us a
detailed and more integrated understanding of the challenging biological functions of anti-microbial compounds
of Bacillus and other organisms.
854 T. Stein
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