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Secondary metabolites of soil Bacillus spp

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Biotechnol Lett (2011) 33:1523–1538
DOI 10.1007/s10529-011-0617-5
REVIEW
Secondary metabolites of soil Bacillus spp.
Estibaliz Sansinenea • Aurelio Ortiz
Received: 31 January 2011 / Accepted: 24 March 2011 / Published online: 29 April 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Bacillus species produce secondary
metabolites that are the object of natural product
chemistry studies. The wide structural variability of
these compounds has attracted the curiosity of
chemists and their biological activities have inspired
the pharmaceutical industry to search for lead struc-
tures in microbial extracts. Screening of microbial
extracts reveals the large structural diversity of
natural compounds with broad biological activities,
such as antimicrobial, antiviral, immunosuppressive,
and antitumor activities, that enable the bacterium to
survive in its natural environment. These findings
widen the potential industrial importance of Bacillus
spp., particularly of B. thuringiensis, beyond insec-
ticidal usage and may help explain the role of
Bacillus spp. in the soil ecosystem.
Keywords Antibiotics  Antimicrobials 
Bacillus spp.  Bio-control  Secondary metabolites
E. Sansinenea (&)  A. Ortiz
Facultad de Ciencias Quı́micas de la Benemérita
Universidad Autónoma de Puebla, 72570 Puebla,
PUE, México
e-mail: estisan@siu.buap.mx; estisan@yahoo.com
Introduction
In 1891, Kossel defined secondary metabolites by
exclusion (compounds that do not belong to primary
metabolites), provoking criticism which has never
ceased. The current, generally accepted concept, in
line with Kossel’s view, is that primary metabolites
are chemical components of living organisms that are
vital for their normal functioning, while secondary
metabolites are compounds which are dispensable.
Secondary metabolites are structurally highly diverse
and each compound is produced only by a small
number of species (Karlovsky 2008).
For many years, secondary metabolism was virtu-
ally ignored; study of this type of non-essential
metabolism was left to industrial scientists and
academic chemists and pharmacognocists. Today,
the situation is different. The wide structural variabil-
ity of these compounds has attracted the curiosity of
chemists and the biological activities possessed by
natural products have inspired the pharmaceutical
industry to search for lead structures in microbial
cultures and plant extracts. Many economically valu-
able microbial products are secondary metabolites
(Karlovsky 2008). Secondary metabolites serve: (i) as
competitive weapons used against other bacteria,
fungi, amoebae, plants, insects, and large animals; (ii)
as metal transporting agents; (iii) as agents of
symbiosis between microbes and plants, nematodes,
insects, and higher animals; (iv) as sexual hormones;
and (v) as differentiation effectors (Demain and Fang
123
1524
2000). The MALDI (Matrix-Assisted Laser Desorp-
tion Ionization) imaging can be used to observe
natural product production and chemical communi-
cation between microorganisms, providing an addi-
tional tool for exploration of interspecies interactions
and symbiotic or pathogen-host communication of
organisms grown in culture, and explaining the
biological effects of secondary metabolites (Yang
et al. 2009).
The genus Bacillus consists of a large number of
diverse, rod-shaped Gram-positive bacteria that are
motile by peritrichous flagella and are aerobic.
Members of the genus are capable of producing
endospores that are highly resistant to unfavorable
environment conditions (Claus and Berkeley 1986).
The Bacillus species subtilis, licheniformis, amylo-
liquefaciens, and pumilus are closely related. There
are several species of the genus that are known
pathogens. These include B. anthracis, which is
pathogenic to humans and other animals, and
B. cereus, which is a common cause of food poisoning
(Claus and Berkeley 1986). B. thuringiensis, and
some strains of B. sphaericus are pathogenic to certain
insects. Bacillus species have special characteristics
that make them good candidates as biological control
agents (Wulff et al. 2002). First, they are well known
as antibiotic producers with antagonistic activity
against fungal and some bacterial pathogens. Second,
they form spores that can be easily formulated, and
have high viability compared with vegetative cells.
Third, they are commonly found in soils.
B. thuringiensis is a Gram-positive, spore-forming,
soil bacterium and is the most successful biological
control agent that produces distinctly shaped crystals
during sporulation. These crystals are composed of
proteins, known as insecticidal crystal proteins
(ICPs), also called Cry proteins, which are selectively
toxic to different species of several invertebrate
phyla. Much of the technology developed to study
structure and function of Cry proteins has provided
the foundation for genetic engineering of this class
of biopesticides (Sansinenea et al. 2010). Formerly,
only the insecticidal properties of B. thuringiensis
attracted extensive attention. However, B. thuringi-
ensis has also been shown to produce other active
components that are secondary metabolites, some
only recently discovered.
Bacillus spp. secrete many secondary metabolites,
including antibiotics, antifungals and siderophores.
123
Biotechnol Lett (2011) 33:1523–1538
The metabolites produced by Bacillus spp. can also
affect the microflora in the rhizosphere, providing an
environment antagonistic to pathogens, or they can
trigger host defense responses (Velusamy and Gnana-
manickam 2008). There are two very recent reviews
about secondary metabolites of B. thuringiensis,
disappointingly only two secondary metabolites are
mentioned in these reviews (Bode 2009; Zhou et al.
2008). In this review we focus on the all secondary
metabolites produced by Bacillus spp. in soil and
B. thuringiensis reviewing all literature currently
available on this topic.
Secondary metabolites from Bacillus sp. in soil
Bacteriocins, lantibiotics and miscellaneous
antibiotics
The potential of B. subtilis to produce antibiotics has
been recognized for 50 years with peptide antibiotics
representing the predominant class. Bacteriocins are
proteins or ribosomal peptides with bactericidal
activity towards species that are often closely related
to the producer bacteria and display variable molec-
ular weights, biochemical properties, inhibitory spec-
tra, and mechanisms of action. Due to their potential
use as natural preservatives, bacteriocins produced by
lactic acid bacteria have been the subject of intensive
investigation in recent years. Bacillus spp. appears to
be a relatively abundant source of antimicrobials,
since many species of this genus synthesize antimi-
crobial peptides (Cherif et al. 2001; Lisboa et al.
2006). The possibility of screening for a new Bacillus
bacteriocin producer is considered to be one of the
major interests in bacteriocin research. B. subtilis, the
model system for Gram-positive organisms, is able to
produce more than two dozen antibiotics with a
variety of structures (Stein 2005).
Several bacteriocins have been reported such as
lichenin produced by the B. licheniformis 26-103RA
strain (Pattnaik et al. 2001), megacin produced by
strains of B. megaterium (Lisboa et al. 2006),
antilisterial coagulin produced by B. coagulans
(Le Marrec et al. 2000), polyfermenticin SCD pro-
duced by B. polyfermenticus (Lee et al. 2001), and
cerein produced by strains of B. cereus (Torkar and
Matijašić 2003; Bizani et al. 2005a, b). Moreover,
some of these bacteriocins have been indicated as
Biotechnol Lett (2011) 33:1523–1538
potential biopreservatives in food systems and bever-
ages, as agents for biological control of phytopatho-
gens (Bais et al. 2004), and as antibiotic precursors.
Bacteriocin production and secretion was observed
in stationary phase, after 10–16 h of bacterial pop-
ulation growth in BHI broth at 30°C. As is well
known, bacteriocins of lactic acid bacteria, particu-
larly lantibiotics, are usually produced in the mid-
growth phase. The production of bacteriocins takes
place successfully in both broth media and on solid
media (Khalil et al. 2009). The biopreservation
capacity of a bacteriocin could be achieved either
by using a bacteriocin producing starter culture or by
applying the bacteriocin itself as a food additive.
Therefore, a thorough study of the essential param-
eters affecting the inhibitory activity, followed by
optimization of production that is usually dependent
on multiple strain-specific factors is necessarily
required for introducing a bacteriocin into foods as
a potent biopreservative. For this purpose Khalil et al.
(2009) described the influence of cultural and phys-
ical conditions such as nutrients, nutrient concentra-
tions, nutrient combinations/interactions, aeration,
and other physical factors, including heat, UV, pH,
and storage conditions, to obtain better and more
stable bacteriocin activity from a newly isolated
strain of B. megaterium.
Peptide antibiotics with an inter-residual thioether
bonds as unique feature are called lantibiotics. The
best-characterized is subtilin, a 32-amino-acid penta-
cyclic lantibiotic of B. subtilis. Subtilin production is
controlled both by culture density in a quorum-
sensing mechanism in which subtilin plays a phero-
mone type role and in response to the growth phase
(Stein et al. 2002). There are some other lantibiotics,
such as ericin, mersacidin, sublancin and subtilolisin
A, that are produced by B. subtilis and are well
described in another review (Stein 2005).
Bacilysin 1 (Fig. 1), a non-ribosomally synthe-
sized dipeptide, is composed of L-alanine and the
unusual amino acid L-anticapsin and represents one
of the simplest peptide antibiotics known with
antifungal and antibacterial activities. Bacilysin 1 is
effective as a biocontrol agent, notably by inhibiting
the growth of Erwinia amylovora (Arguelles-Arias
et al. 2009). Its antibiotic activity depends on the
anticapsin moiety, which becomes released by pep-
tidases (Chmara et al. 1982) after bacilysin 1 uptake
into susceptible cells by a distinct peptide permease
1525
system. The genes encoding the biosynthesis of the
dipeptide bacilysin 1 and its antibiotic constituent
anticapsin were isolated from several strains of
B. subtilis as well as B. amyloliquefaciens and
B. pumilus (Steinborn et al. 2005). Bacilysin produc-
tion of B. subtilis 168 parallels active growth in a
synthetic medium and becomes inhibited by certain
growth conditions, i.e. supplements and above 30°C.
Its synthesis seems to be under transcription regula-
tion via the stringent response (Inaoka et al. 2003) as
well as under feedback regulation, likely by a
component of the global quorum-sensing control
system. In contrast to B. subtilis 168, over-production
of bacilysin in B. amyloliquefaciens GSB272 toler-
ated higher concentrations of yeast extract up to 0.2%
(vs 0.01%) and was achieved even at 37°C (vs 30°C)
(Steinborn et al. 2005).
There are some other miscellaneous antibiotics
produced by B. subtilis such as bacilysocin 2
(Tamehiro et al. 2002), 3,30 -neotrehalosadiamine
(NTD) 3 (Inaoka and Ochi 2007), and amicoumacin
4 (Pinchuk et al. 2002) (Fig. 1) among some others.
Bacilysocin 2 is an anti-microbial phospholipid that
can be isolated from B. subtilis 168 cells by
extraction with butanol, and is derived from the
major B. subtilis phospholipid phosphatidylglycerol
through YtpA-catalysed acyl ester hydrolysis (Tame-
hiro et al. 2002). The aminosugar antibiotic
3,30 -neotrehalosadiamine (NTD) 3, structurally 3,30 -
diamino-3,30 -dideoxy-a,b-trehalose, was originally
identified as an antibiotic produced by several
Bacillus species such as, B. pumilus and B. circulans
(Tsuno et al. 1986). Inaoka et al. (2004) showed that
the production of NTD, dormant in the wild-type
strain, can be induced by a rifampicin-resistant
phenotype of the RNA polymerase. The aminosugar
NTD 3 inhibits growth of Staphylococcus aureus and
Klebsiella pneumoniae and functions as an autoin-
ducer by activating its own biosynthetic operon
(Inaoka and Ochi 2007). NTD functions as a glucose
uptake modulator in B. subtilis and probably in other
NTD producing bacteria, with a gene arrangement
identical to that in B. subtilis. Amicoumacins 4 are
produced by several B. subtilis strains excluding the
168 strain (Pinchuk et al. 2002). Their anti-bacterial
and anti-inflammatory activities, as well as their
action on Heliobacter pylori make the amicoumacins
attractive for the treatment of chronic gastritis and
peptic ulcer in humans.
123
1526
Fig. 1 Chemical structures
of miscellaneous antibiotics
and surfactin (lipopeptide)
and its isoforms
Lipopeptides
The lipoheptapeptide surfactin 5 (Fig. 1) is a power-
ful biosurfactant; it exerts a detergent-like action on
biological membranes (Carrillo et al. 2003), and is
distinguished by its exceptional emulsifying, foam-
ing, anti-viral and anti-mycoplasma activities. A host
of interesting features of surfactin has led to a wide
range of applications such as antibacterial, antiviral,
anti-adhesive and anti-inflammatory uses. In spite of
the immense potential for commercial, therapeutic
and environmental applications, its use is still limited
123
Biotechnol Lett (2011) 33:1523–1538
due to its high cost of production and recovery (Das
et al. 2008).
Various strategies, such as strain improvement,
medium optimization, bioreactor design or using
agroindustrial wastes for fermentation to reduce
the raw material cost, have been implemented to
achieve improved biosurfactant production. Several
approaches have been tried to improve surfactin 5
yields both at the flask and fermentor level by
changing environmental parameters, and optimizing
medium components or trace elements. Both sub-
merged (SMF) and solid-state fermentation (SSF)
Biotechnol Lett (2011) 33:1523–1538
have been tried for surfactin 5 production. Batch
fermentation for surfactin 5 must be terminated at an
appropriate time in order to avoid the utilization of
surfactin as a carbon source, suggesting that the
supply of glucose is critical since it acts as a major
carbon source. Surfactin production is the highest in a
defined medium with ammonium nitrate as nitrogen
source. The recovery and concentration of biosurfac-
tants account for a major portion of the total
production cost. The separation strategies for biosur-
factants vary according to the fermentation process
and the physicochemical properties of the surfactant
in question (Shaligram and Singhal 2010).
Some biosurfactants may be used as alternatives to
synthetic medicines and antimicrobial agents (Shali-
gram and Singhal 2010). Their production is widely
distributed among B. subtilis, B. pumilus, B. lichen-
iformis (Tendulkar et al. 2007) and B. amylolique-
faciens strains (Wulff et al. 2002) and thus, a variety
of surfactin 5 isoforms have been described under
different synonyms such as bacircine 5a, halo- and
isohalobacillin 5b, lichenysin A/G 5c, daitocidin 5d
and pumilacidin 5e (Fig. 1) (Kalinovskaya et al.
2002). The variations are due to changes in the lipid
portion and/or the amino acid composition. Licheny-
sin 5c belongs to the family of the lipopeptide
biosurfactants produced by Bacillus spp. In addition
to its surface activity, this compound exhibits various
interesting biological properties making it attractive
for both industrial and biological applications (Gran-
gemard et al. 2001). The antibiotic pumilacidins 5e
A, B, C, D, E, F and G were isolated from the culture
broth of a strain of B. pumilus. They are cyclic
acylheptapeptide composed of a b-hydroxy fatty acid,
two L-leucine, two D-leucine, L-glutamic acid, L-
aspartic acid and L-isoleucine (or L-valine). Pumilac-
idin components were inhibitory to herpes simplex
virus type 1 and H?, K?-ATPase and demonstrated
antiulcer activity in rat (Naruse et al. 1990).
B. amyloliquefaciens is closely related to B.
subtilis so many lipopeptides are produced by both
microorganisms (Lisboa et al. 2006; Chen et al. 2007;
2009) among them are iturin A 7 (Fig. 2) (Yu et al.
2002; Pyoung et al. 2010) and Fengycin 8 (Jacques
et al. 1999) that are cyclic lipopeptides (CLP). These
surfactants may also play different roles in the
development and survival of Bacillus strains in their
natural habitat: increasing bioavailability of hydro-
phobic water-insoluble substrates, heavy metal
1527
binding, bacterial pathogenesis, quorum sensing,
motility, biofilm formation etc. The iturin family
(Fig. 2) encompasses the closely related cyclic lipo-
heptapeptides mycosubtilin 6 (Moyne et al. 2004), the
iturines 7 and the bacillomycins 9, that contains one
b-amino fatty acid and seven a-amino acids, and
exhibit strong antifungal and hemolytic activities and
a limited antibacterial activity (Stein 2005). Biolog-
ical effects of the iturin family peptides are due to
their capability of forming ion-conducting pores
(Maget-Dana and Peypoux 1994). Iturin A 7 and
bacillomycin L 9 provoked hemolysis and released
potassium from erythrocytes (Aranda et al. 2005).
Iturin A 7 contains the heptapeptide Asn1-Tyr2-
Asn3-Gln4-Pro5-Asn6-Ser7, whereas in the other
members the amino acid residues in the heptapeptides
vary slightly; e.g. mycosubtilin 6, that was isolated
from B. subtilis, has Asn1-Tyr2-Asn3-Gln4-Pro5-
Ser6-Asn7. Iturin A 7 was antagonistic to Fusarium
oxysporum, which cause potato diseases (Han et al.
2005). B. amyloliquefaciens strains B94 and FZB42,
produce iturins that also inhibit fungal plant patho-
gens (Yu et al. 2002; Kilian et al. 2000). In
B. amyloliquefaciens FZB42, bacillomycin D 9 is
responsible for the main antifungal activity exerted
by this bacterium and was shown to suppress growth
of such phytopathogenic fungi as Fusarium oxyspo-
rum (Ramarathnam et al. 2007; Koumoutsi et al.
2004). Fengycin 8 (synonymous to plipastatin) com-
bines several exceptional structural properties: cycli-
zation, branching and unusual constituents. Fengycin
8 is specifically active against filamentous fungi
(Stein 2005). Having in hand the whole genome
sequences of environmental Bacillus strains, we can
see that the gene clusters of many of these surfactants
are found in some Bacillus genomes (Chen et al.
2007, 2009), and we are in position to use the
accumulated knowledge of Bacillus molecular biol-
ogy to investigate the different aspects of lifestyle,
including biofilm formation, of a motile low GC-soil
bacterium in its natural environment.
Often the amount and type of a raw material can
contribute considerably to the production cost; it is
estimated that raw materials account for 10–30% of
the total production cost in most biotechnological
processes. Thus to reduce this cost it is desirable to
use low-cost raw materials for the production of
biosurfactants. One possibility explored extensively
is the use of cheap and agro-based raw materials as
123
1528
Fig. 2 Chemical structures
of lipopeptides and
polyketides
substrates for biosurfactant production. A variety of
cheap raw materials, including plant-derived oils, oil
wastes, starchy substances, lactic whey and distillery
wastes have been reported to support biosurfactant
production. An efficient and economical bioprocess is
the foundation for every profit-making biotechnology
industry. Hence bioprocess development is the
primary step towards commercialization of all bio-
technological products, including biosurfactants. Any
attempt to increase the yield of a biosurfactant
demands optimal addition of media components and
selection of the optimal culture conditions that will
123
Biotechnol Lett (2011) 33:1523–1538
induce the maximum or optimum productivity.
Similarly, efficient downstream processing tech-
niques and methods are needed for maximum product
recovery. Novel recombinant varieties of these
microorganisms, which can grow on a wide range
of cheap substrates and produce biosurfactants at high
yields, can potentially bring the required break-
through in the biosurfactant production process
(Muthusamy et al. 2008). This is possible only if
the genetics of the microbial surfactant production is
known in details. It is therefore desirable that the
future research on biosurfactants be focused on the
Biotechnol Lett (2011) 33:1523–1538
development and use of hyperproducers. A detailed
knowledge of the genetics of microbial surfactant
production should be used to produce organisms
giving higher production with better product charac-
teristics (Das et al. 2008).
Polyketides
In addition to peptides, polyketides are the other
dominant family of secondary metabolites having
relevant bioactivities. They are synthesized on
modularly organized assembly lines starting from
acyl-CoA precursors by decarboxylative Claisen
condensations. In general their biosynthetic pathway
follows the same logic as in non-ribosomally synthe-
sized peptides and requires at least three domains.
Essential domains of the modules harbored in bacte-
rial type I polyketide synthases are acyl transferase
(AT), ketosynthase (KS) and an acyl carrier protein
(ACP) which needs to be activated by Ppant-trans-
ferase (Chen et al. 2009).
Three functional gene clusters directing the syn-
thesis of difficidin 10, bacillaene 11 and macrolactin
12 (Fig. 2) were identified in FZB42 and GA1 strains
of B. amyloliquefaciens. Difficidin 10 is an unsatu-
rated 22-membered macrocyclic polyene lactone
phosphate ester with broad spectrum antibacterial
activity. It inhibits protein biosynthesis and was
recently shown promising in its suppressive action
against Erwinia amylovara, a devasting plant patho-
gen causing necrotrophic fire blight disease of apple,
pear and other rosaceous plants (Arguelles-Arias
et al. 2009). The bae gene cluster was assigned to
synthesis of the bacillaene polyketide 11 (Chen et al.
2009), an inhibitor of prokaryotic protein synthesis
with the empiric formula C35H48O7. The third
polyketide with macrolid-like structure, macrolactin
12, was originally detected in an unclassified deep-
sea marine bacterium (Jaruchoktaweechai et al.
2000). Macrolactins 12, contain three separated diene
structure elements in a 24- membered lactone ring. At
least 17 macrolactins have been described and one of
them, 7-O-malonyl macrolactin A, was effective
against Gram-positive bacterial pathogens (Romero-
Tabarez et al. 2006). Bacitracin is a mixture of related
cyclic polypeptides produced by organisms of the
licheniformis group of Bacillus. Bacitracin inter-
feres with bacterial cell wall synthesis and is
primarily active against the Gram-positive bacteria
1529
Streptococcus aureus and Streptococcus spp. Most
Gram-negative organisms and yeasts are resistant.
The maximum activity of bacitracin was observed for
S. aureus during 24 and 48 h of incubation at 30°C at
an alkaline pH of 8.0–9.0 with 5% (w/v) glucose
(Awais et al. 2008).
Secondary metabolites of Bacillus thuringiensis
Among Bacillus species, B. thuringiensis is the best-
known and best-studied entomopathogenic bacterium
that produces parasporal protein crystals, which are
selectively toxic to different species of several
invertebrate phyla (Bode 2009).
Many isolates of B. thuringiensis also produce an
assortment of various other virulence factors that are
secreted into the culture medium. These factors
include the vegetative insecticidal proteins (Vips),
and b-exotoxin I 13, also called thuringiensin 13
(Fig. 3), a nonproteinaceous toxin (Espinasse et al.
2002). Unlike the Vip and Cry toxins, b -exotoxin I
13 is not specific and thus, may have detrimental
effects on nontarget organisms; it is particularly
active against dipteran species, but it is also active
against coleopteran, lepidopteran, and some nema-
tode species. This toxin inhibits the synthesis of RNA
by competing with ATP for binding sites, thereby
affecting insect molting and pupation and causing
teratogenic effects at sublethal doses (Espinasse et al.
2002, 2004). The genetic determinants responsible
for b-exotoxin I production found on Cry-dependent
plasmids are likely to be regulatory elements and
large amounts of b-exotoxin I can be produced in the
absence of such plasmids. A putative ABC trans-
porter, encoded by berAB, is essential for b-exotoxin
I production. b-exotoxin I production can be acti-
vated in response to particular environmental condi-
tions (Espinasse et al. 2002, 2004).
The parasporal crystal proteins and spores, which
are the primary toxic substances to insects, are
inactivated quickly after exposure to sunlight. It was
shown that melanin is an excellent photoprotective
agent and because of this photoprotection of the
mosquitocidal activity of B. thuringiensis israelensis,
using melanin, has been studied. Several research
groups have obtained B. thuringiensis mutants pro-
ducing melanin from B. thuringiensis (Espinasse
et al. 2002; Saxena et al. 2002) but there are wild-
type strains of B. thuringiensis that produces melanin
123
1530
Fig. 3 Bacillus
thuringiensis some
secondary metabolites
as a secondary metabolite (Che et al. 2004). Despite
the appearance of melanin together with b-exotoxin
I during growth, regulation of the production of pig-
ment is probably independent of that of b-exotoxin I.
B. thuringiensis is also a producer of zwittermicin
A 14 (Fig. 3), a potent antibiotic and antifungal
compound. This compound was firstly isolated in B.
cereus for its ability to suppress damping-off disease
in alfalfa caused by Phytophthora medicaginis and its
structure is a linear aminopolyol antibiotic (Silo-Suh
et al. 1998). More importantly, zwittermicin A
enhances the activity of the endotoxin from
B. thuringiensis against insects (Zhou et al. 2008).
123
Biotechnol Lett (2011) 33:1523–1538
Zwittermicin A 14 has an unusual chemical structure
that includes a D-amino acid and ethanolamine and
glycolyl moieties, as well as having an unusual
terminal amide that is generated from the modifica-
tion of the nonproteinogenic amino acid ureidoala-
nine. The diverse biological activities and unusual
structure of Zwittermicin A 14 have stimulated
interest in understanding how this antibiotic is
biosynthesized.
The identification of the complete Zwittermicin A
14 biosynthesis gene cluster from B. cereus UW85
has been recently reported (Kevany et al. 2009). The
biosynthesis of Zwittermicin A 14 does not originate
Biotechnol Lett (2011) 33:1523–1538
in carbohydrate metabolism, but from a nonriboso-
mal peptide synthetase/polyketide synthase pathway
(NRPS/PKS) involving two newly described type 1
PKS extender units: hydroxymalonyl-acyl carrier
protein (ACP) and aminomalonyl-ACP. For industrial
purposes a rapid method for producing the antibiotic
is needed, and thus a large-scale purification method
was developed based on ion-exchange chromatogra-
phy and HPLC. Zwittermicin A 14 was difficult to
isolate in substantial quantities due to its highly polar,
charged nature at physiological pH, and its sensitivity
to alkaline conditions. The difficulty in obtaining
the compound from natural sources underscores an
outstanding need for practical syntheses of zwitter-
micin A 14, which has been recently reported (Rogers
and Molinski 2007, 2009).
One of the most widely utilized mechanisms of
microbial iron acquisition is the production and
secretion of siderophores, low molecular weight iron
chelators that bind ferric iron in the environment with
extremely high affinity and shuttle it into the cells.
Thus, the presence of siderophore-producing micro-
organisms in the rhizosphere contributes to plant
health by complexing iron and making it less
available to phytophathogens that are generally not
able to produce comparable Fe-transport systems
(Arguelles-Arias et al. 2009; Chen et al. 2009). All
members of the B. cereus group, including B. subtilis
and B. licheniformis, secrete bacillibactin 15 (Fig. 3),
a 2,3-dihydroxybenzoyl-Gly-Thr trilactone sidero-
phore and is also produced by B. thuringiensis. In
addition, B. anthracis and B. cereus primarily
produce petrobactin 16 and 3,4-dihydroxybenzoic
acid (3,4-DHB) 17 (Fig. 3) which were considered
virulence factors for these bacteria (Zawadzka et al.
2009). Although it was recently shown that both are
also produced by innocuous B. thuringiensis isolates,
indicating that by themselves are not indicative of
virulence (Koppisch et al. 2008a). Petrobactin 16,
which contains two 3,4-catecholate moieties and a
citrate-based backbone, was first isolated from the
marine bacterium Marinobacter hydrocarbonoclasti-
cus. The photoreactivity of ferric complexes formed
with citrate-based siderophores produced by marine
bacteria, including petrobactin and aerobactin, was
suggested to be a mechanism for the production of
biologically available ferrous iron. However, the
possible role of such photoreactivity in siderophores
1531
produced by soil or pathogenic bacteria such as
Bacilli remains unclear. In addition to petrobactin 16,
B. anthracis, B. thuringiensis and B. cereus also
secrete large quantities of the petrobactin precursor,
3,4-DHB 17. Although the role of 3,4-DHB 17 as a
siderophore was suggested for a magnetotactic bac-
terium, Magnetospirillum magneticum (Calugay et al.
2006), it also appears to be able to serve as a
siderophore in Bacillus.
Bacillibactin 15 synthesis is dependent on func-
tional Ppant-transferase (Sfp) (Chen et al. 2009). The
enzymes responsible for petrobactin 16 biosynthesis
are members of a family of proteins termed nonrib-
osomal peptide synthetase-independent siderophore
(NIS) synthases (Koppisch et al. 2008a). Biosynthe-
sis of DHB 17 through early shikimate intermediates
is favored over a mechanism involving late inter-
mediates or aromatic amino acids (Koppisch et al.
2008b).
As above described, the genus Bacillus encom-
passes a number of bacteriocinogenic species. Sev-
eral bacteriocins have been partially characterized
from B. thuringiensis: thuricin from strain HD2
(Favret and Yousten 1989), tochicin from strain
HD868 (Paik et al. 1997), kurstakin 18 (Fig. 3)
(Hathout et al. 2000), thuricin 7 from strain BMG1.7
(Cherif et al. 2001), thuricin 439A and thuricin 439B
from strain B439 (Ahern et al. 2003), entomocin from
B. thuringiensis subsp. entomocidus HD9 (Cherif
et al. 2003), bacthuricin F4 from B. thuringiensis
subsp. kurstaki strain BUPM4 (Kamoun et al. 2005),
thuricin S from B. thuringiensis subsp. entomocidus
HD198 strain (Chehimi et al. 2007), and thuricin CD
19 (Fig. 3) from strain DPC 6431 (Rea et al. 2010).
Thuricin exhibited a bactericidal and bacteriolytic
effect on a sensitive strain, and was active against
several Bacillus species (B. thuringiensis, B. mega-
terium, B. polymyxa, and B. sphaericus), but was not
active against B. licheniformis and B. macerans strain
(Favret and Yousten 1989). Tochicin showed a
narrow antibacterial spectrum of activity against
most of 20 typical B. thuringiensis strains and a
strain of B. cereus, but not against other bacteria and
yeasts tested (Paik et al. 1997). The kurstakin 18
structure is closely related to the structure of other
lipopeptides synthesized by Bacillus species, fungi,
and yeasts. However, its amino acid composition
incorporates a His residue, which is rare in the
123
1532
bacterial lipopeptides known today. Its antifungal
activity was demonstrated against Stachybotrys cha-
ratum (Hathout et al. 2000). Thuricin 7, which is
bactericidal and bacteriolytic (Cherif et al. 2001), was
active against several species of the genus Bacillus,
including three of the four known B. thuringiensis/
B. cereus bacteriocin producers, as well as against
Streptococcus pyogenes and Listeria monocytogenes
strains. The large spectrum of activity, biochemical
and physical properties, molecular mass, and the
kinetics of production differentiate entomocin
from the other bacteriocins produced either by the
B. cereus group or by lactic acid bacteria (Cherif
et al. 2003). Thuricin S exhibits a large inhibitory
spectrum, which includes Listeria monocytogenes,
Salmonella enterica, and Pseudomonas aeruginosa
(Chehimi et al. 2007). Thuricin CD 19 consists of two
distinct peptides, Trn-a and Trn-b, that act synergis-
tically to kill a wide range of clinical Clostridium
difficile isolates. However, this bacteriocin thuricin
CD 19 has little impact on most other genera,
including many gastrointestinal commensals (Rea
et al. 2010).
Bacteriocins are generally isolated from cultures
under laboratory conditions. During the long extrac-
tion, isolation, and purification process, they are
potentially subject to contamination by other bacteria
and related microorganisms. Like other compounds
used in research studies, they are stored prior to their
use in research applications. Nevertheless, these
bacteriocins must be free of other contaminating
bacteria when used for research activities. There is a
lack of information regarding their stability and
activity following sterilization procedures. Bacterio-
cins are completely degraded by autoclaving and
were similarly affected by the tested filter mem-
branes. Polyvinylidene fluoride (PVDF), polyester-
sulfone (PES), and cellulose acetate (CA) are
suitable for filter sterilization of these bacteriocins
(Woo-Jin et al. 2008). Unlike the previously
described B. cereus group bacteriocins, which show
a decrease in their antibacterial activities after the
induction of sporulation, entomocin bactericidal
activity was constant for more than 50 h of incuba-
tion indicating its high stability and resistance to
inactivation or degradation (Cherif et al. 2003). In
Table 1 secondary metabolites discussed in this
review have been summarized collecting some
information about them.
123
Biotechnol Lett (2011) 33:1523–1538
Conclusions and perspectives
Bacillus spp. are a relatively abundant source of
antimicrobials since many species of this genus
synthesize antimicrobial peptides. These bacteria in
general represent a new and rich source of secondary
metabolites that need to be explored. These second-
ary metabolites exhibit strong antifungal and anti-
bacterial activities and enable the bacterium to
survive in its natural environment (Stein 2005).
These findings widen the potential industrial impor-
tance of Bacillus spp. beyond just insecticidal usage
and may help explain the role of Bacillus spp. in the
soil ecosystem. The view that secondary metabolites
act by improving the survival of the producer in
competition with other living species has been
expressed more and more in recent years in terms
of having important biological effects (Demain and
Fang 2000), as MALDI imaging demonstrates (Yang
et al. 2009).
Environmental factors affect microbial secondary
metabolism. Stress, starvation or supplementation
with nutrients such as amino acids, phosphate,
nitrogen, etc., have a plethora of effects on the
production of secondary metabolites. It has long been
standard practice in the pharmaceutical industry to
develop specific and often complex culture media to
maximize the production yields of secondary metab-
olites such as antibiotics. Many mutations have been
introduced into so-called production strains to
enhance the yield of a particular antibiotic and in
the majority of cases the genetic and biochemical
nature of these strains is unknown. The growth of
antibiotic-producing organisms in the presence of low
concentrations of organic compounds (such as
DMSO) results in qualitative and quantitative
changes in secondary metabolite production. This
could have value in natural product drug discovery
programs, since solvent-induced effects may reveal
the presence of secondary metabolites that are
otherwise present in low concentrations under normal
growth conditions (Chen et al. 2000).
Lipopeptide antibiotics are among the most fre-
quently produced Bacillus antibiotics that have
well-recognized potential biotechnology and biophar-
maceutical applications due notably to their surface-
active properties. Some possible roles for such
bioemulsifiers have been proposed in pollution and
environmental control (Muthusamy et al. 2008).
Biotechnol Lett (2011) 33:1523–1538 1533

Table 1 Secondary metabolites of soil Bacillus spp.

Bacteria Compound Structure class Biological effects Reference

B. licheniformis Lichenin Bacteriocin Bactericidal, bacteriolytic Pattnaik et al. (2001)

B. megaterium Megacin Bacteriocin Bactericidal, bacteriolytic Lisboa et al. (2006)
B. coagulans Coagulin Bacteriocin Bactericidal, bacteriolytic Le Marrec et al. (2000)
B. polyfermenticus Polyfermenticin Bacteriocin Bactericidal, bacteriolytic Lee et al. (2001)
B. cereus Cerein Bacteriocin Bactericidal, bacteriolytic Bizani et al. (2005)
B. subtilis Subtilin Lantibiotic Antibacterial Stein (2005)
B. subtilis Ericin Lantibiotic Antibacterial Stein (2005)
B. subtilis Mersacidin Lantibiotic Antibacterial Stein (2005)
B. subtilis Sublancin Lantibiotic Antibacterial Stein (2005)
B. subtilis Subtilolysin Lantibiotic Antibacterial Stein (2005)
B. subtilis, Bacilysin 1 Dipeptide Antifungal antibacterial Chmara et al. (1982)
B. amyloliquefaciens,
B.pumilus
B. subtilis Bacilysocin 2 Phospholipid Fungicidal antibacterial Tamehiro et al. (2002)
B. subtilis, B. pumilus, NTD 3 Aminosugar Antibacterial Tsuno et al. (1986)
B. circulans
B. subtilis, B. pumilus Amicoumacin 4 Isocoumarin Antibacterial, anti- Pinchuk et al. (2002)
inflammatory
B. subtilis Surfactin 5 Cyclic lipoheptapeptide Hemolytic, cytotoxic Carrillo et al. (2003)
B. subtilis, Bacircine 5a Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
B. amyloliquefaciens,
B.pumilus
B. licheniformis Halobacillin 5b Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
B. licheniformis Isohalobacillin Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
5b
B. licheniformis Lichenysin 5c Cyclic lipoheptapeptide Hemolytic, cytotoxic Grangemard et al. (2001)
Bacillus sp. Daitocidin 5d Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
B. pumilus Pumilacidin 5e Cyclic lipoheptapeptide Antiulcer activity in rat Naruse et al.(1990)
B. subtilis Mycosubtilin 6 Cyclic lipoheptapeptide Hemolytic, fungicidal Moyne et al. (2004)
B. subtilis, Iturin 7 Cyclic lipoheptapeptide Hemolytic, fungicidal Aranda et al. (2005)
B. amyloliquefaciens
B. subtilis, Fengycin 8 Cyclic lipoheptapeptide Antifungal Koumoutsi et al.( 2005)
B. amyloliquefaciens
B. subtilis, Bacillomycin 9 Cyclic lipoheptapeptide Hemolytic, fungicidal Aranda et al. (2005)
B. amyloliquefaciens
B. amyloliquefaciens Difficidin 10 Polyketides Antibacterial Arguelles-Arias et al. (2009)
macrolactone
B. amyloliquefaciens Bacillaene 11 Polyketides Antibacterial Chen et al. (2009)
macrolactone
B. amyloliquefaciens Macrolactin 12 Polyketides Antibacterial Jaruchoktaweechai et al.
macrolactone (2009)
B. thuringiensis b-exotoxin 13 Adenine nucleotide Insecticidal Espinasse et al. (2002)
analog
B. thuringiensis Melanin Polyacetilene derivative Photoprotective Espinasse et al. (2002)
B. thuringiensis, Zwittermicin 14 Aminopolyol Antibiotic, antifungal Silo-Suh et al. (1998)
B. cereus

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1534 Biotechnol Lett (2011) 33:1523–1538

Table 1 continued
Bacteria Compound Structure class Biological effects Reference

B. subtilis, Bacillibactin 15 Catecholate siderophore Iron acquisition Arguelles-Arias et al. (2009)

B. licheniformis,
B. thuringiensis,
B. cereus,
B. anthracis
B. thuringiensis, Petrobactin 16 Catecholate siderophore Iron acquisition Zawadzka et al. (2009)
B. cereus,
B. anthracis
B. thuringiensis, DHB 17 Catecholate siderophore Iron acquisition Zawadzka et al. (2009)
B. cereus,
B. anthracis
B. thuringiensis Thuricin Bacteriocin Bactericidal Favret and Yousten (1989)
Bacteriolytic
B. thuringiensis Tochicin Bacteriocin Bactericidal Paik et al. (1997)
B. thuringiensis Kurstakin 18 Bacteriocin Fungicidal Hathout et al. (2000)
B. thuringiensis Thuricin 7 Bacteriocin Bactericidal Cherif et al. 2001
Bacteriolytic
B. thuringiensis, Thuricin 439A/ Bacteriocin Bactericidal Ahern et al. (2003)
anthracis B Bacteriolytic
B. thuringiensis Entomocin Bacteriocin Bactericidal Cherif et al. (2003)
B. thuringiensis Bacthuricin Bacteriocin Fungicidal Kamoun et al. (2005)
B. thuringiensis Thuricin S Bacteriocin Bactericidal Chehimi et al. (2007)
Bacteriolytic
B. thuringiensis, Thuricin CD 19 Bacteriocin Bactericidal Rea et al. (2010)
anthracis Bacteriolytic

These surfactants may also play different roles in the
development and survival of Bacillus strains in their
natural habitat (Das et al. 2008). One vital factor
determining their biosynthesis is the genetic makeup
of the producer organisms. Studies on molecular
genetics and biochemistry of the synthesis of several
biosurfactants have revealed the operons, the
enzymes and the metabolic pathways required for
their extracellular production.
Many of the therapeutically most useful and best
understood natural products are synthesized princi-
pally, by two different biosynthetic pathways for
peptides which allow the incorporation of unusual
(non-proteinaceous) constituents: (i) the non-ribo-
somal synthesis of peptides by large megaenzymes,
the non-ribosomal peptide synthetases (NRPSs) and
the polyketide synthases (PKSs) and (ii) the ribo-
somal synthesis of linear precursor peptides that are
subjected to post-translational modification and pro-
teolytic processing. Both molecular systems result in
the synthesis of a diverse array of elaborate chemical
structures, which have remarkable biological activi-
ties (Stein 2005).
Due to the importance of PKS and NRPS derived
compounds, the next frontier was to investigate the
corresponding gene clusters. As evidenced by
genome sequencing, bacteria dedicate up to 20% of
their genome to the biosynthesis of secondary
metabolites, underscoring the importance of these
small molecules to the fitness of the organism in its
native environment (Gross 2007). B. subtilis produces
more than two dozen antibiotics. If all pathways are
considered, their production requires more than
350 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

123
Biotechnol Lett (2011) 33:1523–1538
B. subtilis strain for antibiotic syntheses is compara-
ble with B. amyloliquefaciens (six operons of 340 kb,
8.5% of the genome; Chen et al. 2009). The marked
differences of B. subtilis strains with regards to their
produced antibiotic spectra suggest that the antibiotic
specifying loci must have been either recent acqui-
sitions or lost due to lack of selective advantage.
Horizontal exchange of genetic material facilitated
via uptake of phage, plasmid or naked DNA by
genetically competent cells is a plausible explanation
for this divergence (Tsuge et al. 2005). Presumably
also NRPSs specifying genes might be interchange-
able among different B. subtilis strains.
While analyzing specific gene clusters and their
functions, many researchers discovered additional
gene clusters belonging to the same class, leading to
the hypothesis that most of the investigated micro-
organisms have a broader genetic capacity to produce
natural products than suggested by the number of
compounds isolated from a particular strain. Such
orphan biosynthetic pathways are gene clusters for
which the encoded natural product is unknown (Gross
2007).
For many years, basic biologists were uninterested
in secondary metabolism. Today, the situation is
different. The frontier of expanding knowledge is
now secondary metabolism which poses many ques-
tions of considerable interest to science: What are the
functions of secondary metabolites in nature? How
are the pathways controlled? What are the origins of
secondary metabolism genes? What are the origins of
the resistance genes which producing organisms use
to protect themselves from suicide? Are these the
same genes as those found in clinically-resistant
bacteria? Fortunately, molecular biology has pro-
duced tools with which to answer these questions. It
is clear that basic mechanisms controlling secondary
metabolism are now of great interest to many
academic (and industrial) laboratories throughout
the world. We hope that this review can stimulate
the development of new secondary metabolites
producing strains.
Acknowledgments We thank VIEP (project) and CONACyT
(project No 80915) for financial support. We thank to the
referees who revised the manuscript and made a great effort to
improve it. We want to give a special acknowledgement to
Dr. Ernest Schnepf for proofreading the manuscript improving
it.
1535
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