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83
Comparative Analysis of the Complete
Genome Sequence of the Plant
Growth-Promoting Bacterium Bacillus
amyloliquefaciens FZB42
Rainer Borriss
ABiTEP GmbH, Germany
83.1
INTRODUCTION
Bacteria that are associated with plant roots and exert
beneficial effects on plant development are referred
to as plant growth-promoting rhizobacteria (PGPR)
(Kloepper et al., 1980; see Chapter 53). They competitively colonize plant roots and can simultaneously act
as biofertilizers and as antagonists of plant pathogens,
including bacteria, fungi, viruses, and nematodes (Biocontrol; see Chapter 54). The aerobic, endo-spore-forming
rhizobacteria belonging to Bacillus amyloliquefaciens
subsp. plantarum are known as being especially suitable
for commercial use in enhancing yield of crop plants
(“biofertilizer” function) and to suppress microbial plant
pathogens (“biocontrol” function) (Borriss et al., 2011a;
Borriss, 2011). The type strain FZB42T (=BGSC 10A6,
DSM23117) was isolated from plant-pathogen-infested
soil of a sugar beet field in Brandenburg, Germany
(Krebs et al., 1998). FZB42 has been shown to act as
an efficient PGPR (Bochow et al., 2001; Grosch et al.,
1999; Idriss et al., 2002; Idris et al., 2007) and biocontrol
bacterium (Koumoutsi et al., 2004; Chen et al., 2006;
Chen et al., 2009a; Schmiedeknecht et al., 2001). Analysis of the whole genome of the plant root-associated
bacterium FZB42 revealed an impressive capability to
produce a vast array of secondary metabolites aimed
to suppress competitive bacteria and fungi within the
plant rhizosphere (Chen et al., 2007). Notably, it ruled
out that more than 8.5% of the whole genome capacity
was devoted to synthesis of antimicrobial secondary
compounds. Recently, genome sequences from nonplantassociated B. amyloliquefaciens subsp. amyloliquefaciens
strains (Geng et al., 2011; Yang et al., 2011; Zhang et al.,
2011), including type strain DSM7T (Rueckert et al.,
2011), have become available. In addition, we have
completed the sequencing and annotation of the genomes
of two further PGPR strains belonging to B. amyloliquefaciens subsp. plantarum, isolated from Chinese soil
(Blom et al., 2012; HE617159; HE774679). We discuss
here the novel genomic data, available for the two B.
amyloliquefaciens subspecies, with their implications
for our recent knowledge of this important group of
rhizobacteria. Exploiting genomics for further research to
identify genes important for plant–bacteria interactions,
transposon mutagenesis, and a first proteomics approach
are other topics of this chapter.
83.2 COMPARATIVE GENOME
ANALYSIS OF ROOT-ASSOCIATED
AND NONROOT-ASSOCIATED
B. amyloliquefaciens
The whole genome sequence of the type strain of
plant-associated B. amyloliquefaciens subsp. plantarum,
Molecular Microbial Ecology of the Rhizosphere, Volume 2, First Edition. Edited by Frans J. de Bruijn.
© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.
883
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Comparative Analysis of the Complete Genome Sequence of the Plant Growth
FZB42T , has been determined in 2007, as the first
representative of Gram-positive, plant growth-promoting
bacteria (Chen et al., 2007). Its 3918-kb genome, containing an estimated 3695 protein-coding sequences (CDS),
lacks extended phage insertions, which occur ubiquitously
in the related B. subtilis 168 genome (Kunst et al., 1997;
Barbe et al., 2009). The B. amyloliquefaciens genome
reveals a huge potential to produce secondary metabolites,
including the polyketides bacillaene and difficidin. More
than 8.5% of the genome is devoted to synthesizing
antibiotics and siderophores by pathways not involving
ribosomes. A first comparison of its genomic sequence
with that of the B. amyloliquefaciens-type strain DSM7T
revealed significant differences in the genomic sequences
of both strains (Rueckert et al., 2011). The strains have
in common 3345 CDS residing in their core genomes
(Borriss et al., 2011b), while 547 and 344 CDS were
found to be unique in FZB42T and DSM7T , respectively.
The core genome shared by both strains exhibited 97.89%
identity on the amino acid level. Notably, the gene clusters
encoding nonribosomal synthesis of antibacterial polyketides difficidin and macrolactin are absent in DSM7T . For
comparison, B. subtilis 168T has a similar number of CDS
in common with B. amyloliquefaciens strains DSM7T and
FZB42T (3222 and 3182 CDS, respectively) (Borriss et al.,
2011a). Recently, besides FZB42T , the genomes of three
other B. amyloliquefaciens plantarum strains have
become available (Blom et al., 2012; Borriss et al.,
2011a). Moreover, except DSM7T , the genomes of three
other representatives of the B. amyloliquefaciens amyloliquefaciens subsp. have been published (Geng et al.,
2011; Yang et al., 2011; Zhang et al., 2011), enabling a
comparative genome analysis of plant root-associated and
free-living soil B. amyloliquefaciens strains (Table 83.1).
The genome size of all nonplant-associated B. amyloliquefaciens strains (subsp. “amyloliquefaciens”) does
not vary significantly and ranges from 3937 kb (TA208)
to 3995 kb (LL3). However, there are considerable differences in the sizes of the genomes of plant-associated
B. amyloliquefaciens subsp. plantarum strains, ranging
from 3918 kb (FZB42T ) to 4242 kb (YAU Y2), respectively. As in DSM7T , these differences are mainly due
to phage insertions, which also occupy large regions
of the genomes of the plant-associated strains YAU
Y2 and NAU B3. A global alignment was performed,
and synteny plots of the Bacillus core genomes were
generated using software EDGAR (Blom et al., 2009;
Borriss et al., 2011b). The synteny plots of FZB42T with
the core genomes of representatives of B. amyloliquefaciens plantarum (CAU B946), B. amyloliquefaciens
amyloliquefaciens (DSM7T ), B. subtilis (strain 168),
and Bacillus licheniformis (DSM13T ) reveal that despite
increasing phylogenetic distance, no large genomic
rearrangements occurred (Fig. 83.1).
The core genome formed by the orthologous genes
present in the four B. amyloliquefaciens plantarum
genomes presently available consists of 3351 CDS. Their
number is reduced when the four representatives of
the B. amyloliquefaciens amyloliquefaciens subspecies
are included: 3156 orthologous genes are shared by all
members of the genus B. amyloliquefaciens. Besides the
core genome, regions of plasticity in which exchanges
of DNA often occur are present in the FZB42T genome.
Fifteen genomic islands (GIs) and large indels interrupting the core regions of close synteny occur in the
Table 83.1 Whole genomes from plant-associated and nonplant-associated Bacillus amyloliqefaciens strains
Strain
Accession (gb) Size (bp)
GC content (%) Protein tRNAs rRNAs
B. amyloliquefaciens subsp. amyloliquefaciens
DSM7T
TA208
FN597644
CP002627
3,980,199
3,937,511
46.08
45.83
3,924
4,089
94
70
30
18
XH7
LL3
CP002927
CP002634
3,939,203
3,995,227
45.82
45.71 %
4,190
4,219
75
72
21
22
B. amyloliquefaciens subsp. plantarum
FZB42T CP000560
3,918,589
CAU 946 HE617159
4,019,861
YAU Y2 HE 7774679.1 4,242,774
46.48
46.51
45.85
3,693
3,823
3,991
87
95
91
30
30
30
IT-45
NAU B3
46.6
45.99
3,898
4,250
86
92
30
30
CM001433
Not submitted
3,925,087
4,196,170
Remarks
Industrial strains used for the production of
enzymes and metabolites
Type strain
Industrial strain for the production of
guanosine and ribavirin
Production of purine nucleosides
Glutamic acid independent production of
poly-γ-glutamic acid
Plant growth-promoting rhizobacteria
Type strain
Synthesis of iturinA instead of bacillomycinD
Synthesis of mersacidin, presence of an orphan
NRSP gene cluster
Contains plasmid pBA45-1
Draft genome, annotation not finished
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83.3 Genes Involved in Plant–Bacteria Interactions
(a)
(b)
(c)
(d)
885
Figure 83.1 Synteny plots generated by EDGAR comparing the core genome of FZB42T with Bacillus amyloliquefaciens plantarum CAU
B946 (a), B. amyloliquefaciens amyloliquefaciens DSM7T (b), Bacillus subtilis subtilis 168 (c), and Bacillus licheniformis DSM13T (d). Despite
increasing phylogenetic distances, synteny of the core genomes still remained, and no significant rearrangements were observed.
B. amyloliquefaciens-type strains DSM7T and FZB42T
(Rueckert et al., 2011). These GIs are defined by local
deviations in the tetranucleotide usage patterns from the
signature of the whole genome (Reva and Tuemmler,
2005). In some of the FZB42T islands, additional features,
such as adjacent tRNAs, remnants of phages, transposase
like sequences, and direct repeats, are indicative of
horizontal gene transfer (Chen et al., 2007). The approximately 150 genes only occurring in the plant-associated
subspecies “plantarum” were found mainly clustered in
the GIs. Generally, such genes are termed singletons,
meaning that no orthologous genes can be identified in any
other strain of the comparison set (Borriss et al., 2011b).
A comparative analysis between the core genome
sequences of B. amyloliquefaciens plantarum and B.
amyloliquefaciens amyloliquefaciens strains revealed that
the plant-associated strains represent a distinct group
and are discernable by their potential to synthesize a
huge spectrum of different secondary metabolites. The
phylogram based on computing of the Bacillus core
genomes underlines this finding (Fig. 83.2). The genomic
data separating plant-associated and nonplant-associated
strains of B. amyloliquefaciens on the subspecies level
are supported by the results of in silico DNA–DNA
hybridization (DDH) (Borriss et al., 2011b) and MALDITOF MS analysis of cellular components, as well as the
spectra of secondary metabolites present in culture fluids
(Borriss et al., 2011a).
83.3 GENES INVOLVED IN
PLANT–BACTERIA INTERACTIONS
The ability of B. amyloliquefaciens subsp. plantarum
to colonize surfaces of plant roots is a prerequisite for
phytostimulation. Rhizosphere competence is linked to
the capability to form sessile, multicellular communities
(biofilms; see Chapter 66). In liquid culture without
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Figure 83.3 Confocal laser scanning microscopy of GFP-tagged
FZB42 colonizing root hairs of lettuce. Source: The image was taken
seven days after inoculation (with courtesy of Dr. Kristin Dietel).
Figure 83.2 Phylogenetic tree reconstructed using the core
genomes of several representatives of the B. subtilis species
complex: B. amyloliquefaciens subsp. plantarum,
B. amyloliquefaciens subsp. amyloliquefaciens, Bacillus atrophaeus,
B. subtilis subsp. subtilis, and B. licheniformis. Bar, 0.1 substitutions
per amino acids within the coding regions of the core genomes.
shaking, FZB42T forms robust pellicles at the liquid–air
interface, where as domesticated B. subtilis strains usually
form thin, fragile pellicles (Chu et al., 2006). Using
a green fluorescent protein (GFP)-tagged derivative of
FZB42, we recently studied the fate of bacterial root
colonization in three different plant species. The studies
were performed in a gnotobiotic system, in which FZB42
was applied to seedlings, axenically grown in soft agar
containing Murashige–Skoog (MS) medium. They ruled
out that the bacterium behaves distinctly in colonizing
surfaces of plant roots of different species. In contrast to
maize, FZB42 colonizes preferentially the root tips when
colonizing Arabidopsis roots, for example (Fan et al.,
2011; 2012). Root hairs of lettuce plantlets are also a
preferred site of colonization (Fig. 83.3).
An important precondition for root colonization is
motility. FZB42T displays a robust swarming phenotype.
The proteins encoded by swrA and swrB (yvjD), and the
cyclic lipopeptide surfactin present in B. amyloliquefaciens plantarum are thought to be essential for swarming
motility. These proteins permit colonization of surfaces
and nutrient acquisition through their surface wetting and
detergent properties (Kearns et al., 2005). The genome
of FZB42T contains the complete set of genes implicated
in biofilm- and fruiting-body formation in B. subtilis,
including the 15-gene exopolysaccharide operon epsA-O,
apparently required for producing an exopolysaccharide
that holds chains of cells together in bundles (Kearns et al.,
2005). The unique genes RBAM007750, RBAM007760,
and RBAM007770 encode proteins with a collagenrelated GXT structural motif and are probably involved
in surface adhesion or biofilm formation (Chen et al.,
2007). Notably, triple helix repeat-containing collagen
proteins were not detected in other representatives of the
B. subtilis species complex, except for B. atrophaeus and
B. pumilus. However, in the plant-associated B. amyloliquefaciens YAU Y2 and NAU B3, genes homologous to
RBAM007760 and RBAM007770, were present, while at
the same genomic region a gene with putative cell wall
anchor function was located in B. amyloliquefaciens CAU
B946 and DSM7T . CAU B946 possesses a mucin-17-like
protein bearing a repetitive PVX motif, unique in all
Bacillus genomes presently known.
Several mechanisms are involved in plant-beneficiary
activities of PGPR; they include synthesizing phytohormones (Idris et al., 2007; see Chapter 27) and volatile
organic compounds (Ryu et al., 2003; see Chapter 63)
and producing available nutrients for plants (van Loon,
2007). Some PGPR also act beneficially by eliciting
plant response reactions directed against biotic (“induced
systemic resistance, ISR”) and abiotic stress (“induced
systemic tolerance, IST”) (van Loon, 2007; Yang et al.,
2008; see Chapter 54).
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Analysis by high performance liquid chromatography (HPLC) and gas-chromatography-mass spectrometry
(GC-MS) demonstrated the presence of indole-3-acetic
acid (IAA) in culture filtrates of FZB42T . (trpBA and
trpED) and two other strains bearing knock-out mutations in genes probably involved in IAA synthesis, ysnE
(putative IAA transacetylase) and yhcX (putative nitrilase), produce smaller amounts of IAA than wild type.
Three of these mutant strains are less efficient in promoting plant growth (Idris et al., 2007).
A blend of volatile compounds (VOCs) is released by
several PGPR Bacillus strains, including FZB42T (Borriss, 2011). The volatiles 3-hydroxy-2-butanone (acetoin)
and 2,3 butandiol trigger enhanced plant growth. To synthesize 2,3-butanediol, pyruvate is firstly converted into
acetolactate by acetolactate synthase (AlsS) under conditions of low pH and oxygen starvation. The next step of
this alternative pathway of pyruvate catabolism, conversion of acetolactate to acetoin, is catalyzed by acetolactate
decarboxylase (AlsD). The final step, from acetoin to 2,3butandiol, is catalyzed by the bdhA gene product, acetoin reductase/2,3-butanediol dehydrogenase (Nicholson
2008). The FZB42T genome contains all the three genes
encoding this pathway. FZB42T mutant strains, incapable
of producing volatiles due to knock-out mutations introduced into the alsS and alsD genes, are unable to support
growth of Arabidopsis seedlings (Borriss, 2011).
As other environmental members of the B. subtilis
species complex, B. amyloliquefaciens plantarum secrete
different hydrolases, enabling them to use external cellulosic and hemicellulosic substrates present in plant cell
walls. Microbe-associated hydrolytic enzymes digesting
plant cell wall structures, resulting in free oligosaccharides, have been shown to act as elicitors of plant defense
(Ebel and Scheel, 1997). A wide range of extracellular depolymerases, including -glucan-, ß-glucan-, and
protein hydrolyzing enzymes are possibly involved in beneficial plant–bacteria interactions, making low molecular
weight hydrolysis products of macromolecules for plant
uptake and nutrition available. Two of these enzymes,
endo-1,4-ß-glucanase (“cellulase,” EC 3.2.1.4) and endo
1,4-ß-xylanase (“xylanase,” EC 3.2.1.8) are encoded in
B. subtilis 168 by the genes eglS and xynA, respectively
(Wolf et al., 1995). The same genes were also present
in the genomes of B. amyloliquefaciens plantarum but
not in the B. amyloliquefaciens amyloliquefaciens strains.
Similarly, the genes xylA, involved in xylose degradation
(EC 5.3.1.5), xynB, encoding 1,4 ß-xylan xylosidase (EC
3.2.37), and bglC, encoding an endo -1,4-ß-glucanase (EC
3.2.1.4), are present in B. subtilis and B. amyloliquefaciens
plantarum but missing in the B. amyloliquefaciens amyloliquefaciens genomes. Unlike DSM7T , the genomes of
the B. amyloliquefaciens plantarum strains do not contain
the amyA gene encoding liquefying amylase but instead
887
an amyE-like sequence encoding a saccharifying amylase
common in B. subtilis (Chen et al., 2007). About 262
genes encoding proteins with putative secretion signals
recognized by signal peptidases type I (150 gene products), type II (98 gene products), and the Tat system (14
gene products) have been found (Chen et al., 2007).
83.3.1 Proteomics: Response to
Plant Root Exudate
The analysis of the effects of root exudates on expression
of extracellular and cytosolic proteins by two-dimensional
(2-D) gel electrophoresis revealed 17 and 21 proteins,
respectively, as being significantly altered (Kierul,
2012). Most of the differentially expressed proteins were
involved in nutrient acquisition, uptake, and utilization.
AlsS involved in synthesis of volatiles (see above), was
10-fold up-regulated in the presence of root exudate
during transitional growth stage. Chitin-binding protein
RBAM 1754 and malate dehydrogenase (MdH) are
greatly enhanced during stationary growth phase in the
presence of root exudate. A cytosolic protein involved in
the conversion of proline to glutamate is induced in B.
amyloliquefaciens. Phytase is a prominent member of the
FZB42T secretome and its concentration increases in the
presence of plant root exudates. By contrast, a promoter
mutation prevents phytase expression in the domesticated
B. subtilis (Makarewicz et al., 2006). The bacterial
phytate-degrading activity could generate phytate phosphorous to nourish plants during phosphate starvation
and thus promote their growth (Idriss et al., 2002).
Root exudates also stimulated up-regulation of
enzymes probably involved in response to oxidative
stress generated in plant roots by thiol peroxidase or
enzymes catabolizing compounds secreted by plant
roots. Enzymes of this type include bacillopeptidase F,
δ-glutamyl transpeptidase, and phosphotransacetylase
(Chen et al., 2007).
Our proteome analysis suggested that the hookassociated flagellar proteins HAP1 and HAP2, and
the HAG flagellin are differently affected by exudates
secreted by maize roots. Although the level of the flgK
gene product HAP1 was reduced in the presence of
root exudates, expression of fliD and hag gene products
was up-regulated. Flagellin proteins are thought to elicit
a general host plant basal defense against potential
pathogens (Abramovitch et al., 2006).
83.3.2 Identification of Functional
Genes by Transposon Mutagenesis
More genes, involved in plant growth promotion, were
identified by transposon mutagenesis performed with the
mariner tranposon HIMAR 1 introduced into FZB42T
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wild-type cells (Budiharjo, 2011). Using a miniaturized
test system based on L. minor plantlets (Idris et al., 2007),
three genes were shown to be efficient in plant growth
promotion: nfrA (ywcH), abrB, and RBAM_017410.
The nfrA mutant exhibited a reduction in plant
growth-promoting activity either in the Lemna system
(26%) or Arabidopsis thaliana (40%). Complementation
with the wild-type gene restored the growth-promoting
activity in the mutant strain. The nrfA gene encodes
an oxidoreductase that is induced under heat shock and
oxidative stress. Disruption of the abrB gene by the
TnYLB-1 transposon reduced the growth-promoting
activity in Lemna (27%) and A. thaliana (17%). AbrB
is a DNA-binding global regulator protein expressed
during transition state and under conditions of limited
growth. It is involved in the regulation of various cellular
functions, such as antibiotic production, competence
development, and expression of degradative enzymes,
and it is likely that AbrB controls a pathway involved
in plant–bacteria interactions. The mutant impaired in
the unique RBAM_017410 gene showed a reduction of
plant growth-promoting activity in both Lemna (42%)
and Arabidopsis (33%). The deduced RBAM_017410
gene product is a small peptide with unknown function,
similar to the small subunit of ribonucleoside-diphosphate
reductase (NDP reductase). The enzyme catalyzes reduction of ribonucleotides to deoxyribonucleotides. Cells
carrying that mutation were found to be not only altered
in their morphology but also unable to colonize plant
roots (Budiharjo, 2011).
83.4 THIRTEEN GENE CLUSTERS
ARE INVOLVED IN BIOCONTROL
Representatives of B. amyloliquefaciens subsp. plantarum possess at least 13 gene clusters involved in
nonribosomal and ribosomal synthesis of secondary
metabolites with antimicrobial function. Two of them are
encoding for nonribosomal peptide synthetases (NRPS,
see 3.1) with hitherto unknown products. The cyclic
lipopeptides, such as surfactin, and antifungal iturin-like
compounds, including bacillomycin D and fengycin,
are encoded by large gene clusters (Koumoutsi et al.,
2004). Nonribosomal synthesis of the Fe-siderophore
bacillibactin is also common in other members of the
B. subtilis species complex. Three giant clusters encode
polyketide synthases (PKSs) involved in nonribosomal
synthesis of the antibacterial polyketides, bacillaene,
difficidin, and macrolactin. Synthesis of difficidin and
macrolactin is unique for B. amyloliquefaciens plantarum
(Borriss et al., 2011b). Sfp-independent nonribosomal
synthesis of bacilysin is common in B. amyloliquefaciens
plantarum and other representatives of the B. subtilis
species complex (Chen et al., 2009a). During the last
two years or so, gene clusters were identified, mainly in
FZB42T , that are involved in ribosomal synthesis of several bacteriocins, modified peptides with specific action
against Gram-positive bacteria. Examples are mersacidin
(Herzner et al., 20011), plantazolicin (Scholz et al.,
2011), and amylocyclizin (Scholz, 2011). Figure 83.4
demonstrates that most of the 13 gene clusters mentioned
above occur in the genomes of B. amyloliquefaciens
plantarum, but with some notable exceptions. Gene clusters involved in plantazolicin and mersacidin synthesis
and self-immunity are located within GIs acquired by
horizontal gene transfer. The same is true for the orphan
nrps gene clusters detected in FZB42T and YAU B9601.
83.4.1 Nonribosomal Synthesis of
Antimicrobial Lipopeptides and
Polyketides
Polyketides and nonribosomal peptides comprise two families of natural products biosynthesized in a similar manner by multimodular enzymes acting in assembly line
arrays. The monomeric building blocks are organic acids
or amino acids, respectively (Walsh, 2004).
FZB42T genome analysis revealed the presence of
numerous gene clusters involved in synthesis of cyclic
lipopeptides (Koumoutsi et al., 2004) and polyketides
(Chen et al., 2006; Schneider et al., 2007) with distinct
antimicrobial action. Besides the five-gene clusters
known in B. subtilis to mediate the nonribosomal synthesis of secondary metabolites, four additional giant
clusters, bmyD, mln, dfn, and nrs, have been identified
in FZB42T (Chen et al., 2009b). While the function of
the two orphan nrs gene clusters found in FZB42T and
NAU-Y2 remains still undetermined, the functions of
the bmyD (bacillomycin D), mln (macrolactin), and dfn
(difficidin) genes are known (Koumoutsi et al., 2004;
Chen et al., 2006; Schneider et al., 2007). As B. subtilis
168T , the genomes of B. amyloliquefaciens DSM7T
and the other representatives of B. amyloliquefaciens
amyloliquefaciens harbor a significantly lower number
of gene clusters involved in nonribosomal synthesis of
secondary metabolites than strain FZB42T . Synthesis of
lipopeptides and polyketides depends on Sfp, an enzyme
that transfers 4’-phosphopantheine from coenzyme A
to the carrier proteins of nascent peptide or polyketide
chains. An exception is the antibacterial dipeptide
bacilysin (Chen et al., 2009a).
Nonribosomal synthesis of lipopeptides is governed
by very large protein templates called peptide synthetases
(NRPS) that exhibit a modular organization, allowing
polymerization of monomers in an assembly-line-like
mechanism. The core of each module of a peptide
synthetase is the adenylation (A) domain that catalyzes
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Figure 83.4 Maps of the circular genomes of four B. amyloliquefaciens strains. Only gene clusters for surfactin, bacillaene, bacillomycin D or
iturinA, bacillibactin siderophore, and bacilysin synthesis are present in all strains. The giant gene clusters involved in nonribosomal synthesis of
secondary metabolites are located in close vicinity of the termination site ter in which the bidirectional replication fork meets.
substrate recognition and activation of amino acids to
acyl adenylates. The adenylation domain that specifies
the amino acids used for peptide synthesis is flanked
by two other essential components, the condensation
(C) and the thiolation (T) domain, also named peptidyl
carrier protein (PCP). Further domains can chemically
modify the incorporated residues (e.g., epimerization
and N-methylation). The PCP is equipped with cofactor
P-pant (4’-phosphopantetheine) that serves as a swinging
arm to mediate the peptide elongation reaction between
adjacent modular units. It can be positioned at three sites
necessary for amino acid loading and peptide elongation.
A 4’-phosphopantetheine-transferase (PPTase) converts
the apo-form of the PCP into its holo-form by loading the
P-pan cofactor to an active serine. B. subtilis Sfp is
a prototype of PPTases with wide substrate tolerance.
The formation of a new peptide bond is catalyzed by
the C-domain. A termination domain at the end of the
module that incorporates the last amino acid catalyzes
liberation or cyclization of the linear intermediate (Mootz
and Marahiel, 1997). The assembly of the multifunctional proteins of the peptide synthetases is reflected in
its genetic organization following the colinearity rule
(Duitman et al., 1999).
Besides direct antagonism of phytopathogens, cyclic
lipopeptides, such as surfactin and fengycin, can also
interact with plant cells as bacterial determinants for
turning on an immune response through the stimulation of the induced systemic resistance phenomenon
(Ongena et al., 2007).
Bacillomycin D is a member of the iturin family that
comprises iturin A, C, D, and E, bacillomycin F and L,
bacillopeptin, and mycosubtilin. Members of the iturin
family contain one ß-amino fatty acid and seven d-amino
acids (Chen et al., 2009b). The peptide moiety of the iturin
lipopeptides contains a tyrosine in the d-configuration at
the second amino acid position and two additional damino acids at positions 3 and 6 (Fig. 83.5). The members
of the iturin family exhibit strong fungicidal activity and
bacillomycin D has been identified as the main antifungal activity directed against fungal plant pathogens in
B. amyloliquefaciens plantarum strains FZB42 and C06.
Mycelium growth and spore germination are suppressed in
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Figure 83.5 Gene clusters involved in
nonribosomal synthesis of lipopeptides and
bacilysin in FZB42T . Amino acids specified by the
NRPS are indicated. From top to bottom: bmyD
gene cluster involved in synthesis of bacillomycin
D. The bmyA gene product is a NRPS/PKS hybrid
protein. The fen gene cluster is involved in
synthesis of fengycin. The fenE gene product
contains a thioesterase domain. The srf gene cluster
is involved in the synthesis of surfactin. The srfAC
gene product contains a thioesterase. The dhb gene
cluster is involved in the synthesis of the dipeptide
bacillibactin consisting of l-alanine and
l-anticapsin.
Fusarium oxysporum, Rhizoctonia solani, and Monilinia
fructicola (Koumoutsi et al., 2004; Liu et al., 2011).
The gene cluster involved in bacillomycin D synthesis covers 37,293 bp and consists of four genes
(Koumoutsi et al., 2004). The first open reading frame
(ORF), bmyD, encodes a putative malonyl coenzyme A
transacylase, which participates in fatty acid synthesis.
The ORFs encoding the hybrid PKS/NRPS enzyme,
BmyA, and the NRPS enzymes, BmyB and BmyC, are
organized like their respective counterparts in the iturin
A and mycosubtilin operons. Seven amino acid-activating
modules are distributed in BmyA (A1), BmyB (B1-4),
and BmyC (C1-2). In addition to the canonical A, C, and
T domains, modules B1 (d-Tyr), B2 (d-Asn), and C1
(d-Ser) contain epimerization domains, directing conversion of the respective amino acids in a d-configuration.
The last domain of this multienzyme system is a
thioesterase domain, which is required for release and
circularization of the synthesized lipopeptide molecule.
Transcription of the bacillomycin D gene cluster is
directly controlled by global regulator DegU. A transmembrane protein of unknown function, YczE, is
also necessary for the synthesis of bacillomycin D
(Koumoutsi et al., 2007). While the majority of B. amyloliquefaciens plantarum strains were found to contain a
gene cluster encoding bacillomycin D, strain CAU B946
was found to synthesize iturin A, which is reflected by
its ituA operon located at the same site as the bmyD gene
cluster in FZB42 (Blom et al., 2012).
Fengycin (synonymous to plipastatin) is a cyclic
lipodecapeptide containing a ß-hydroxy fatty acid with a
side change of 16–19 carbon atoms. Four d-amino acids
and one nonproteinogenic ornithine residue have been
identified in the peptide portion of fengycin. Fengycin
is active against filamentous fungi and is known for
inhibiting phospholipase A2 . Similar to bacillomycin
D, toxicity against pathogenic fungi relies mainly on
their membrane permeabilization properties. Owing to
its high productivity in synthesizing fengycin, biocontrol
exerted by strain C06 relies rather on fengycin than on
bacillomycin D (Liu et al., 2011).
The fen five-gene cluster occurring in FZB42 is
related to the pps operon in B. subtilis 168 and is situated
at the same locus as this, about 25 kb downstream
from the bmyD gene cluster. It is detectable in the
genomes of the B. amyloliquefaciens plantarum strains
but occurs only fragmentarily in B. amyloliquefaciens
amyloliquefaciens-type strain DSM7T (Rueckert et al.,
2011). The fen gene cluster covers the same size as the
bmyD operon, although the five NRPS enzymes encoded
by fenA-E contain 10 modules specifying a dekapeptide.
Modules A2, B2, C2, and D3 contain additional epimerization domains responsible for conversion to D-amino
acids. FenE contains a thioesterase protein liable for
release and cyclization of the lipopeptide.
Surfactin is a heptapeptide with an LLDLLDL chiral
sequence linked by a ß-hydroxy fatty acid consisting
of 13–15 carbon atoms to form a cyclic lactone ring
structure. Surfactin is surface active (biotenside) and acts
hemolytic, antiviral, and antibacterial by altering membrane integrity (Peipoux et al., 1999). The biological role
of surfactin is thought to be as supporting colonization
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of surfaces and acquisition of nutrients through its
surface wetting and detergent properties. Mutants of
B. amyloliquefaciens, blocked in surfactin biosynthesis,
have been shown to be impaired in biofilm formation
(Chen et al., 2007).
The giant gene cluster involved in the synthesis of
surfactin consists of four genes covering 26 kb. Three of
them encode the NRPS SrfAA, SrfAB, and SrfAC, while
the terminal SrfAD is highly homologous to external
thioesterases of type II. Modules present in SrfAA
assemble the first three amino acids Glu, Leu, and Leu
within the nascent peptide. The third module converts
l-Leu into the d-configurated isomer by its C-terminal
epimerization domain. SrfAB incorporates Val, Asp, and
d-Leu, while SrfAC is responsible for the activation and
incorporation of the last leucine residue and catalysis of
product release by cyclization.
While nonribosomal synthesis of the lipopeptides mentioned above does strictly depend on
P-pant transferase, Sfp, synthesis of the antimicrobial
dipeptide bacilysin does not. Bacilysin [l-alanyl-(2,3epoxycyclohexanone-4)-l-alanine] contains an l-alanine
residue at the N-terminus and nonproteinogenic lanticapsin, at the C-terminus. The peptide bond with
l-alanine proceeds in a nonribosomal mode, catalyzed
by amino acid ligase DhbE. Bacilysin is active in a
wide range of bacteria and against the yeast, Candida
albicans, due to the anticapsin moiety, which is released
after uptake into susceptible cells. Culture fluids of a
bacilysin producing mutant strain of FZB42 suppress
Erwinia amylovora, the causative agent of fire blight in
orchard trees (Chen et al., 2009a).
The bacilysin gene cluster, detected in FZB42, displays the same gene organization as the one known for
B. subtilis 168, in which the genes bacABCDE and ywfG
have been shown necessary for bacilysin synthesis (Steinborn et al., 2005).
Polyketides are a large family of secondary metabolites that include many bioactive compounds with
antibacterial, immunosuppressive, antitumor, or other
physiologically relevant properties. Bacterial polyketides
are synthesized by type I PKSs, modularly organized
assembly lines, starting from acyl CoA precursors by
decarboxylative Claisen condensations. In general, their
biosynthetic pathway follows the same logic as in
nonribosomally synthesized peptides and requires at least
three domains: an acyltransferase (AT), a ketosynthase
(KS), and an acyl-carrier-protein (ACP) corresponding to
PCP in NRSP. Usually the order of the modules dictates
the sequence of biosynthetic events (Chen et al., 2009c).
A few years ago, a special class of PKSs that lack the
cognate AT domain and require a separate AT enzyme
acting iteratively in trans was detected in bacilli and
other bacteria (Chen et al., 2007; Shen, 2003).
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In the genome of FZB42T , three giant PKS gene
clusters are located at sites approximately 1.4 Mbp (pks2,
mln), 1.7 Mbp (pks1, bae), and 2.3 Mbp (pks3, dfn)
clockwise distant from the origin of replication. Notably,
none of the PKSs encoded by the three pks gene clusters
harbor modules with cognate AT domains. Instead, one
or more genes encoding single AT proteins were detected
upstream from the megasynthases encoding genes in
all three pks gene clusters of FZB42. Discrete ATs
act iteratively by loading malonyl CoA onto all PKS
modules during polyketide synthesis. Another notable
feature observed in analysis of Bacillus polyketide
gene clusters is the presence of noncanonical bimodular
sequence KS KR ACP KS ◦ DH ACP (KS ◦ being a
nonelongating KS) with a split downstream module on
two proteins (Moldenhauer et al., 2007). With the help of
cassette mutagenesis in combination with advanced mass
spectrometric techniques, two polyketides, bacillaene
and difficidin/oxydifficidin, have been identified, which
are encoded by gene clusters pks1 (bae) and pks3 (dfn)
(Chen et al., 2006). Gene cluster pks2 was assigned
for the synthesis of macrolactin and the cluster has
been renamed mln. Bacillaene, but not difficidin, and
macrolactin are produced in B. subtilis and B. mojavensis.
The bacillaene gene cluster is also present in the closely
related B. amyloliquefaciens subsp. amyloliquefaciens
(Schneider et al., 2007).
Bacillaene is, due to its molecular structure, a highly
unstable inhibitor of prokaryotic protein synthesis and
does not have effects on eukaryotic organisms (Patel et al.,
1995). NMR studies of partially purified extracts from
B. subtilis revealed bacillaene as an open-chain, unsaturated enamine acid with an extended polyene system
(Butcher et al., 2007). Recently, special features of bacillaene synthesis, the archetype of trans-AT PKS, have been
uncovered in FZB42, and bacillaene B bearing a glucosyl
moiety has been identified as the final product of the bae
pathway (Moldenhauer et al., 2007, 2010).
Organization and genomic localization of the bae
gene cluster is very similar to that of the pksX gene
cluster of B. subtilis 168 (Fig. 83.6), but a pksA-like
sequence corresponding to a putative transcriptional regulator is missing in the operon. In contrast to B. subtilis,
a putative regulator PksR, a homolog of B. subtilis PksA
was detected as a discrete gene in a region far upstream
from bae (Chen et al., 2007). Using strain OKB105, a
derivative of B. subtilis 168 harboring an intact copy of
sfp encoding P-pant-transferase, we assigned bacillaene
as the synthesis product of the pksX gene cluster in B.
subtilis 168 (Chen et al., 2006). The observation that the
baeM gene of FZB42T does not contain the superfluous
last module present in the homologous pksM gene of B.
subtilis suggests that this module is skipped during evolution. The presence of three discrete ATs that are iteratively
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Figure 83.6 Gene clusters involved in nonribosomal synthesis of polyketides in B. amyloliquefaciens plantarum FZB42T .
acting in trans, BaeC, BaeD, and BaeE is an interesting
trait unique for the bae gene cluster. Our unpublished results performed with knock-out mutant strains
(Chen XH and Borriss R) have revealed that the action of
those trans-ATs is not restricted on bacillaene synthesis
but has also some impact on synthesis of the two other
polyketides produced by FZB42T , difficidin and macrolactin, respectively. The final gene of the operon, baeS,
was shown to encode a P450-like enzyme involved in
the desaturation of bacillaene (Moldenhauer et al., 2007).
Difficidin and oxydifficidin were identified as
products of the dfn gene cluster in FZB42T (Chen et al.,
2006). Difficidin has been shown to inhibit protein
biosynthesis (Zweerink and Edison, 1987), but the exact
molecular target remains unknown. The polyketides are
highly unsaturated 22-membered macrocyclic polyene
lactone phosphate esters (Wilson et al., 1987) and are by
far the most effective antibacterial compounds produced
by FZB42T . Notably, difficidin is efficient in suppressing
plant pathogenic bacterium Erwinia amylovora, which
causes fire blight disease in orchard trees (Chen et al.,
2009a). Our studies have revealed that the production
of difficidin is restricted to the representatives of B.
amyloliquefaciens plantarum and the giant gene cluster
encoding the respective megasynthases occurs only in
that plant-associated subspecies of B. amyloliquefaciens
(Schneider et al., 2007; Borriss et al., 2011b).
Unlike bae, but similar to the third pks gene cluster
encoding macrolactin synthesis, the dfn operon harbors
only one trans-AT, which presumably acts iteratively in
transfer of the acyl moiety. A model for the biosynthesis
of difficidin following largely the collinearity rule of KS
module structure and chain elongation has been established (Chen et al., 2006). A candidate for the likely
final step in difficidin biosynthesis, phosphorylation, is
DifB, which resembles a hypothetical bacterial protein
kinase and contains typical kinase-like sequence profiles
(smart00220, COG0661).
Macrolactins are the biosynthesis product of the
pks2 (mln) gene cluster in FZB42T and have been
characterized as an inhibitor of peptide deformylase
(Yoo et al., 2006). Macrolactins, originally detected in an
unclassified deep-sea bacterium, contain three separate
diene structure elements in a 24-membered lactone ring
(Gustafson et al., 1989). 7-O-malonyl macrolactin induces
disruptions of cell division, thereby inhibiting the growth
of multiresistent enterococci (Romero-Tabarez et al.,
2006). Macrolactin A and 7-O-succinyl macrolactin
displays antibacterial properties against vancomycinresistant enterococci and methicillin-resistant S. aureus
(Kim et al., 2011). In the culture fluid of FZB42T, four
macrolactins were identified—macrolactin A and D
as well as 7-O-malonyl- and 7-O-succinyl-macrolactin
(Schneider et al., 2007).
The domain structure of the mln gene cluster of
FZB42T reflects the pathway, whereby the macrolactin
skeleton is synthesized by extension of an acetyl starter
unit by successive Claisen condensations with malonyl
CoA. The polyketide megasynthase encoded by mlnB to
mlnH comprises 11 modules, each containing at least the
three basic domains—a KS, a ketoreductase (KR), and
an ACP—but lacking an integrated AT domain. MlnA
displays striking similarity to malonyl-CoA-specific
trans-ATs and its activity should lead to incorporation of
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the same extender unit in all modules. The completed
polyketide is released and cyclized by the thioesterase
at the C-terminus of the last module present in MlnH
(Chen et al., 2007).
83.4.2 Ribosomal Synthesis of
Bacteriocins
There are two different pathways for the synthesis of
bioactive peptides, the nonribosomal mechanism (see 4.1)
and the ribosomal synthesis of linear precursor peptides
that are post-translationally modified and processed (lantibiotics and lantibiotic-like peptides). While numerous
secondary metabolites (lipopeptides and polyketides), synthesized independently from ribosomes in plant associated
B. amyloliquefaciens, are known (see above), antimicrobial peptides (bacteriocins) conventionally synthesized by
ribosomes remained unknown for a long time in B. amyloliquefaciens plantarum. However, during the last years,
lantibiotics and highly modified peptides with microcinlike structure have been revealed as being synthesized by
B. amyloliquefaciens plantarum.
Lantibiotics (lanthionine containing antibiotics), a
special group of bacteriocins, are amphiphilic peptides
of bacterial origin and are nearly exclusively produced
by Gram-positive bacteria. They contain unusual constituents such as nonproteinogenic didehydroamino acids
and lanthionines (Sahl and Bierbaum, 1998). Interresidual
thioether bonds are unique features of lantibiotics (Stein,
2005). Lanthionine Subtilin was the first lantibiotic
isolated from B. subtilis ATCC6633, and a corresponding
gene cluster involved in synthesis of the lantibiotic-like
peptide ericin was found in plant-associated B. subtilis
A1/3 (Stein et al., 2002). We characterized strain A1/3
as a member of the B. amyloliquefaciens plantarum
group (Borriss et al., 2011b) and therefore, ericin can be
considered as an example of a lantibiotic produced by
plant-associated B. amyloliquefaciens. However, in the B.
amyloliquefaciens plantarum-type strain FZB42T , the spa
(subtilin) gene cluster is fragmentary, containing only the
immunity genes of the operon (Borriss, 2011a).
The same is true for mersacidin, a type B lantibiotic
and at first detected in Bacillus sp. HIL Y-85 (Chatterjee et al., 1992). The peptide consists of 20 amino
acids and exerts its antibacterial activity by inhibition
of cell wall synthesis. The producer strain Bacillus sp.
HIL Y-85 has recently been reclassified as B. amyloliquefaciens plantarum (Herzner et al., 2011). Again, the
genome of FZB42T contains a fragment of the mersacidin
operon, presumably directing immunity against the lantibiotic (Borriss et al., 2011a). MIC determinations of
HIL Y-85 (25 mg/l) and FZB42T (25 mg/l) demonstrated
that FZB42T was at least as resistant to mersacidin as the
producer strain. Moreover, it was possible to reconstitute
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synthesis of heterologous mersacidin in FZB42T by introducing the respective biosynthetic genes cloned from HIL
Y-85 (Herzner et al., 2011).
Recently, we detected a complete set of genes encoding mersacidin, including the genes for synthesis and modification in the genome of B. amyloliquefaciens plantarum
Y2, isolated from Chinese soil (Borriss et al., 2011b).
The biosynthetic gene cluster of mersacidin consists of
10 genes (Altena et al., 2000); it is located in a variable
region of the genome, as a part of GI 13. In addition to
the structural gene mrsA, the gene cluster comprises genes
encoding proteins involved in post-translational modification of the prepeptide, mrsM, and mrsD, a gene coding for
a transporter with an associated protease domain, mrsT.
Genes encoding proteins for self-protection, mrsF, mrsE,
and mrsG, were also found in the chromosome of FZB42T .
Likewise, the two-component system MrsR2/K2 involved
in ensuring “immunity” is present in FZB42T , while the
single regulator MrsR1, essential for mersacidin biosynthesis, is without counterpart in FZB42T . Notably, the
mersacidin operon detected in YAU B946 did mirror completely the gene cluster of strain HIL Y-85 (Fig. 83.7).
Well-known examples for the group of bacteriocins,
which are not lantibiotics, are colicins produced by E. coli
and microcins, a heterogenous group of small peptides
(<10 kDa) produced by enterobacteria. Those compounds
act antagonistically against closely related taxa. However,
many of the prophage sequences common in B. subtilis
168 and often harboring biosynthetic gene clusters of
ribosomally synthesized peptides were found missing in
FZB42T (Chen et al., 2007). A breakthrough in our search
for bacteriocins in FZB42 was achieved by using mutant
strain RS6, deficient in the Sfp-dependent synthesis of
lipopeptides and polyketides and in Sfp-independent
bacilysin production (Chen et al., 2009a). Analysis of
RS6 revealed that the strain still produced an antibacterial substance efficient against B. subtilis and other
closely related Gram-positive bacteria. Simultaneously,
an independent analysis of the FZB42T genome revealed
a ribosomal encoded biosynthetic gene cluster that is
conserved among many species across two domains
of life (Lee et al., 2008). This cluster encodes a small
precursor peptide that is post-translationally modified to
contain thiazole and oxazole heterocycles. These rings
are derived from Cys and Ser/Thr residues through the
action of a trimeric “BCD” synthetase complex, which
consists of a cyclodehydratase (C), a dehydrogenase (B),
and a docking Protein (D) (Molohon et al., 2011). This
mechanism of modification is used in the biosynthetic
pathways for streptolysin S from Streptococcus pyogenes (Datta et al., 2005) and microcin B17 (Li et al.,
1996), for example. The products of these clusters have
been collectively classified as thiazole/oxazole-modified
microcins (TOMM) due to their genetic and chemical
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Figure 83.7 Complete mersacidin operons (mrs) in
B. amyloliquefaciens plantarum strains HIL-85 and YAU
Y2. The incomplete gene cluster directing immunity
against mersacidin in FZB42T is shown at the bottom.
structure conservation (Haft et al., 2010). The novel
TOMM discovered from FZB42T has a molecular mass
of 1335 Da and has been named plantazolicin (PZN)
(Scholz et al., 2011). The chemical structure of plantazolicin A (R=CH) and B (R=H) has been elucidated
by resolving ESI-MSMS, 2D 1 H-13 C-correlated NMR
spectroscopy, as well as 1 H-15 N-HMQC/1 H-15 N-HMBC
NMR experiments. 15 N-labeling before the experiments
facilitated the structure determination, unveiling a hitherto
unusual number of thiazoles and oxazoles formed from a
linear 14mer precursor peptide (Kalyon et al., 2011). PZN
exhibited a highly selective antibiotic activity toward
Bacillus anthracis, but no other tested human pathogen
(Molohon et al., 2011).
As in the case of mersacidin, biosynthetic pzn genes
are located in a variable part of the genome within GI 3,
together with unique genes involved in restriction and
modification of DNA. They are transcribed into two polycistronic mRNAs (pznFKGHI and pznJCDBEL) and a
monocistronic mRNA for pznA (Fig. 83.8) as revealed by
reverse transcriptase polymerase chain reaction (RT-PCR)
(Scholz et al., 2011). It is assumed that the leader peptide of PznA serves as recognition site for the PznBCD
synthetase complex (see above). Methyltransferase PznL
performs N,N-dimethylation presumably after proteolytic
processing by the protease PznE. The complete pzn gene
cluster was also detected in the genome of B. amyloliquefaciens plantarum CAU B946 but not in NAU B3. Strain
YAU B9601 contained only a small part of the gene cluster
directing immunity against plantazolicin in this nonproducer strain. While the plantazolicin gene cluster was not
detected in B. amyloliquefaciens amyloliquefaciens or B.
subtilis, a highly conserved homolog of the pzn gene cluster was detected in the more distantly related B. pumilus.
Last but not the least a very recent finding obtained
after mariner transposon mutagenesis of the FZB42
mutant strain RS06 should be mentioned here. Besides
mersacidin and plantazolicin, a circular bacteriocin with
similarity to uberolysin, named amylocyclizin, AZN, was
identified (Scholz, 2011). The peptide suppressed growth
of the plant pathogenic actinobacterium Clavibacter
michiganensis and of several Gram-positive bacteria.
An azn operon consisting of six genes was proven to
be responsible for the synthesis of the bacteriocin, but
further investigations are necessary to reveal its exact
molecular structure.
83.5 CONCLUSIONS AND
OUTLOOK
During the last 10 years, a considerable knowledge base
about Gram-positive PGPR has been accumulated (see
Chapter 53). Especially a group of root-surface associated
bacilli, closely related to industrial B. amyloliquefaciens strains, has gained increasing attention. These
bacteria have been recognized as members of subspecies
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Acknowledgments
Figure 83.8 Plantazolicin gene clusters in B.
amyloliquefaciens plantarum strains.
“plantarum” clearly distinct from the type strain B.
amyloliquefaciens DSM7T (Borriss et al., 2011b). B.
amyloliquefaciens FZB42T has been proposed as type
strain for the new subspecies. The strain is distinguished
by several features:
1. FZB42T and its genomic sequence (CP000560)
are freely available for scientific investigations
from the BGSC stock center (strain number 10A6)
and DSMZ (strain number DSM23117). It is
freely available despite the fact that the strain is
commercialized by ABiTEP GmbH Berlin and successfully used in agri- and horticulture applications
(http://www.abitep.de/en/Research.html; see also
Chapter 64).
2. In contrast to most environmental Bacillus strains,
B. amyloliquefaciens FZB42T is naturally competent
and amenable for genetic transformation. In order to
assign unknown gene functions, a large mutant strain
collection is available. Some are already deposited at
BGSC, and the others can be obtained after request
from the Research department of ABiTEP GmbH.
3. A GFP-labeled derivative of FZB42 useful for root
colonization studies is also available.
4. A transposon library of FZB42 using the mariner
transposon TnYLB-1 has been generated and can be
used to screen for genes involved in plant growth
promotion and biocontrol.
5. Microarrays with oligonucleotides, representing
the complete set of FZB42 genes and numerous
intergenic sequences harboring candidate small
(regulatory) RNAs for transcriptomic studies, are
available.
6. B. amyloliquefaciens FZB42T is closely related
to other known PGP bacilli used commercially
(QST713, GB03, A1/3). Studies performed with
FZB42T are valuable for a general understanding
of the mode of action of that interesting group of
bacteria.
As already proposed (Borriss, 2011), it is hoped that
the community of scientists, interested on exploiting plant
growth-promoting (PGP) bacilli, will choose FZB42 as a
paradigm for research on that group of bacteria with a
high potential to substitute or at least to replace, in part,
harmful agrochemicals.
ACKNOWLEDGMENTS
The author thanks all the former members of his laboratory at Humboldt University and the many colleagues
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and friends with whom he worked together in the
exciting field of plant growth promotion and biocontrol
during the last decade. Their trustful collaboration and
innovative work made it possible to write this chapter.
Special thanks to the German Ministry of Education
and Research (BMBF) for financial support given in
the framework of the Chinese–German collaboration
program, the GenoMik and GenoMikPlus network, and
recently in the PATHCONTROL project.
REFERENCES
Abramovitch RB, Anderson JC, Martin GB. Bacterial elicitation and evasion of plant innate immunity. Nat Rev Mol Cell Biol
2006;7:601–611.
Altena K, Guder A, Cramer C, Bierbaum G. Biosynthesis of the lantibiotic mersacidin: organization of a type B lantibiotic gene cluster.
Appl Environ Microbiol 2000;66:2565–2571.
Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G,
Sekowska A, et al. From a consortium sequence to a unified
sequence: the Bacillus subtilis reference genome a decade later.
Microbiology 2009;155:1758–1775.
Blom J, Albaum SP, Doppmeier D, Pühler A, Vorhölter FJ,
Zakrzewski M, Goesmann A. EDGAR: a software framework for
the comparative analysis of prokaryotic genomes. BMC Informatics
2009;10:154.
Blom J, Rueckert C, Niu B, Wang Q, Borriss R. The complete
genome of Bacillus amyloliquefaciens subsp. plantarum CAU B946
contains a gene cluster for nonribosomal synthesis of iturin A. J Bacteriol 2012;194:1845–1846.
Bochow H, El Sayed SF, Junge H, Stavropoulos A,
Schmiedeknecht G. Use of Bacillus subtilis as biocontrol agent.
IV. salt-stress tolerance induction by Bacillus subtilis FZB24 seed
treatment in tropical field crops, and its mode of action. J Plant Dis
Prot 2001;108:21–30(in German).
Borriss R. Use of plant-associated Bacillus strains as biofertilizers and
biocontrol agents. In: Maheshwari DK, editor. Bacteria in Agrobiology: Plant Growth Responses. Heidelberg; Dordrecht; London; New
York: Springer; 2011. p 41–76.
Borriss R, Rueckert C, Blom J, Bezuidt O, Reva O, Klenk HP.
Whole genome sequence comparisons in taxonomy. In: Rainey F,
Oren A, editors. Methods in Microbiology. Taxonomy of Prokaryotes. Vol. 38. Amsterdam; Boston; Heidelberg; London; New York;
Oxford; San Diego; San Francisco; Singapore; Sydney; Tokyo: Elsevier; 2011a. p 409–436.
Borriss R, Chen XH, Rueckert C, Blom J, Becker A, Baumgarth B, et al. Relationship of Bacillus amyloliquefaciens clades
associated with strains DSM7T and FZB42T : a proposal for Bacillus
amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons. Int J Syst Evol Microbiol
2011b;61:1786–1801.
Budiharjo A. 2011. Plant-bacteria interactions: molecular mechanism
of phytostimulation by Bacillus amyloliquefaciens FZB42 [PhD thesis]. Berlin: Humboldt University.
Butcher RA, Schroeder FC, Fischbach MA, Straight PD,
Kolter R, WashD CJ. The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis. Proc Natl Acad
Sci USA 2007;104:1506–1509.
Chatterjee S, Chatterjee DK, Lad SJ, Phansalkar MS, Rupp RH,
Ganguli BN, Fehlhaber HW, Kogler H. Mersacidin, a new antibiotic from Bacillus: fermentation, isolation, purification and chemical
characterization. J Antibiot 1992;45:832–838.
Chen XH, Vater J, Piel J, Franke P, Scholz R, Schneider K, et al.
Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42. J Bacteriol
2006;188:4024–4036.
Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, et al. Comparative analysis of the complete genome sequence
of the plant growth–promoting bacterium Bacillus amyloliquefaciens
FZB42. Nat Biotechnol 2007;25:1007–1014.
Chen XH, Scholz R, Borriss M, Junge H, Mögel G, Kunz S, Borriss R. Difficidin and bacilysin produced by plant-associated Bacillus
amyloliquefaciens are efficient in controlling fire blight disease. J
Biotechnol 2009a;140:38–44.
Chen XH, Koumoutsi A, Scholz R, Borriss R. More than anticipated - production of antibiotics and other secondary metabolites
by Bacillus amyloliquefaciens FZB42. J Mol Microbiol Biotechnol
2009b;16:14–24.
Chen XH, Koumoutsi A, Scholz R, Schneider K, Vater J, Suessmuth R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42
reveals its potential for biocontrol of plant pathogens. J Biotechnol
2009c;140:27–37.
Chu F, Kearns DB, Branda SS, Kolter R, Losick R. Targets of the
master regulator of biofilm formation in B. subtilis. Mol Microbiol
2006;59:1216–1228.
Datta V, Myskowski SM, Kwinn LA, Chiem DN, Varki N,
Kansal RG, et al. Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive
infection. Mol Microbiol 2005;56:681–695.
Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H,
Saenger W, et al. The mycosubtilin synthetase of Bacillus subtilis
ATCC6633: a multifunctional hybrid between a peptide synthetase,
an amino transferase, and a fatty acid synthase. Proc Nat Acad Sci
USA 1999;96:13294–13299.
Ebel J, Scheel D. Signals in host–parasite interactions. Berlin, Heidelberg: Springer-Verlag; 1997.
Fan B, Chen XH, Budiharjo A, Bleiss W, Vater J, Borriss R.
Efficient colonization of plant roots by the plant growth promoting
bacterium Bacillus amyloliquefaciens FZB42, engineered to express
green fluorescent protein. J Biotechnol 2011;151:303–311.
Fan B, Borriss R, Bleiss W, Wu X. Gram-positive rhizobacterium
Bacillus amyloliquefaciens FZB42 colonizes three types of plants in
different patterns. J Microbiol 2012;50:38–44.
Geng W, Cao M, Song C, Xie H, Liu L, Yang C, et al. Complete
genome sequence of Bacillus amyloliquefaciens LL3, which exhibits
glutamic acid-independent production of poly-g-glutamic acid. J Bacteriol 2011;193:3393–3394.
Grosch R, Junge H, Krebs B, Bochow H. Use of Bacillus subtilis as biocontrol agent. III. Influence of Bacillus subtilis on fungal root diseases and on yield in soilless culture. J Plant Dis Prot
1999;106:568–580(in German).
Gustafson K, Roman M, Fenical W. The macrolactins, a novel class
of antiviral and cytotoxic macrolides from a deep-sea marine bacterium. J Am Chem Soc 1989;111:7519–7524.
Haft DH, Basu MK, Mitchell DA. Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family. BMC Biol 2010;8:70.
Herzner AM, Dischinger J, Szekat C, Josten M, Schmitz S,
Yakéléba A, et al. Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42. PLoS One 2011;6(7):e22389. DOI:
10.1371/journal.pone.0022389.
de Bruijn c83.tex
References
Idriss EES, Makarewicz O, Farouk A, Rosner K, Greiner R,
Bochow H, Richter T, Borriss R. Extracellular phytase activity
of Bacillus amyloliquefaciens FZB 45 contributes to its plant growthpromoting effect. Microbiology 2002;148:2097–2109.
Idris EES, Iglesias DJ, Talon M, Borriss R. Tryptophan dependent
production of indole-3-acetic acid (IAA) affects level of plant growth
promotion by Bacillus amyloliquefaciens FZB42. Mol Plant-Microbe
Interact 2007;20:619–626.
Kalyon B, Helaly SE, Scholz R, Nachtigall J, Vater J, Borriss R,
Süssmuth RD. Plantazolicin A and B: structure of ribosomally synthesized thiazole/oxazole peptides from Bacillus amyloliquefaciens
FZB42. Org Lett 2011;13:2996–2999.
Kearns DB, Chu F, Rudner R, Losick R. A master regulator for biofilm formation by Bacillus subtilis. Mol Microbiol
2005;55:739–749.
Kierul K. 2012. Comprehensive proteomic study of Bacillus amyloliquefaciens strain FZB42 and its response to plant root exudates [PhD
thesis]. Berlin: Humboldt University.
Kim DH, Kim HK, Kim KM, Kim CK, Jeong MH, Ko CY, et al. Antibacterial activities of macrolactin A and 7-O-succinyl macrolactin A from
Bacillus polyfermenticus KJS-2 against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. Arch Pharm
Res 2011;34:147–52.
Kloepper JW, Leong J, Teintze M, Schroth MN. Enhancing plant
growth by siderophores produced by plant-growth promoting rhizobacteria. Nature 1980;286:885–886.
Koumoutsi A, Chen XH, Henne A, Liesegang H, Hitzeroth G,
Franke P, Vater J, Borriss R. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive
cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. J Bacteriol 2004;186:1084–1096.
Koumoutsi A, Chen XH, Vater J, Borriss R. DegU and
YczE positively regulate the synthesis of bacillomycin D by
Bacillus amyloliquefaciens strain FZB42. Appl Environ Microbiol
2007;73:6953–6964.
Krebs B, Höding B, Kübart S, Workie MA, Junge H,
Schmiedeknecht G, Bochow H, Hevesi M. Use of Bacillus subtilis as biocontrol agent. I. Activities and characterization of Bacillus
subtilis strains. J Plant Dis Prot 1998;105:181–197(in German).
Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G,
Azevedo V, et al. The complete genome sequence of the grampositive bacterium Bacillus subtilis. Nature 1997;390:249–256.
Lee SW, Mitchell DA, Markley AL, Hensler ME, Gonzalez D,
Wohlrab A, et al. Discovery of a widely distributed toxin biosynthetic gene cluster. Proc Nat Acad Sci USA 2008;105:5879–5884.
Li YM, Milne JC, Madison LL, Kolter R, Walsh CT. From peptide precursors to oxazole and thiazole-containing peptide antibiotics:
microcin B17 synthase. Science 1996;274:1188–1193.
Liu J, Zhou T, He D, Li XZ, Wu H, Liu W, Gao X. Functions of
lipopeptides bacillomycin D and fengycin in antagonism of Bacillus
amyloliquefaciens C06 towards Monilinia fructicola. J Mol Microbiol
Biotechnol 2011;20:43–52.
Makarewicz O, Dubrac S, Msadek T, Borriss R. Dual role of the
PhoP∼P response regulator: Bacillus amyloliquefacien FZB45 phytase gene transcription is directed by positive and negative interactions
with the phyC promoter. J Bacteriol 2006;188:6953–6965.
Moldenhauer J, Chen XH, Borriss R, Piel J. Biosynthesis
of the antibiotic bacillaene produced by a giant polyketide synthase complex of the trans-AT family. Angew Chem Int Ed Engl
2007;46:8195–8197.
Moldenhauer J, Götz DC, Albert CR, Bischof SK, Schneider K,
Süssmuth RD, et al. The final steps of bacillaene biosynthesis in
Bacillus amyloliquefaciens FZB42: direct evidence for beta,gamma
dehydration by a trans-acyltransferase polyketide synthase. Angew
Chem Int Ed Engl 2010;49:4065–4067.
V3 - 02/20/2013
8:58 P.M. Page 897
897
Molohon KJ, Melby JO, Lee J, Evans BS, Dunbar KL, Bumpus SB, et al. Structure determination and interception of biosynthetic intermediates for the plantazolicin class of highly discriminating
antibiotics. ACS Chem Biol 2011;6:1307–13.
Mootz HD, Marahiel MA. Biosynthetic systems for nonribosomal
antibiotic assembly. Curr Opin Chem Biol 1997;1:543–551.
Nicholson WL. The Bacillus subtilis ydjL (bdhA) gene encodes acetoin reductase/ 2,3-butandiol dehydrogenase. Appl Environ Microbiol
2008;74:6832–6838.
Ongena M, Jourdan E, Adam A, Paquot M, Brans A,
Joris B, et al. Surfactin fengycin lipopeptides of Bacillus subtilis as
elicitors of induced systemic resistance in plants. Environ Microbiol
2007;9:1084–1090.
Patel PS, Huang S, Fisher S, Pirnik D, Aklonis C, Dean L, et al.
Bacillaene, a novel inhibitor of prokaryotic protein synthesis produced by Bacillus subtilis: production, taxonomy isolation, physicochemical characterization and biological activity. J Antibiot (Tokyo)
1995;48:997–1003.
Peipoux F, Bonmatin JM, Wallach J. Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 1999;51:553–563.
Romero-Tabarez M, Jansen B, Sylla M, Luensdorf H, Häussler S,
Santosa DA, et al. 7-O-Malonyl macrolactin A, a new macrolactin antibiotiic from Bacillus subtilis - active against methicillinresistent Staphylococcus aureus, vancomycin-resistent enterococci
and a small-colony variant of Burkholderia cepacia. Antimicrob
Agents Chemother 2006;50:1701–1709.
Rueckert C, Blom J, Chen XH, Reva O, Borriss R. Genome sequence
of Bacillus amyloliquefaciens type strain DSM7T reveals differences
to plant-associated Bacillus amyloliquefaciens FZB42. J Biotechnol
2011;155:78–85.
Ryu CM, Farag MA, Hu CH, Reddy M, Wei HX, Pare PW, Kloepper J. Bacterial volatiles promote growth in Arabidopsis. Proc Nat
Acad Sci USA 2003;100:4927–4932.
Sahl HG, Bierbaum G. Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria. Annu
Rev Microbiol 1998;52:41–79.
Schmiedeknecht G, Issoufou I, Junge H, Bochow H. Use of Bacillus
subtilis as biocontrol agent. V. Biological control of disease on maize
and sunflowers. J. Plant Dis Prot 2001;108:500–512(in German).
Schneider K, Chen XH, Vater J, Franke P, Nicholson G, Borriss R,
Suessmuth RD. Macrolactin is the polyketide biosynthesis product
of the pks2 cluster of Bacillus amyloliquefaciens FZB42. J Nat Prod
2007;70:1417–1423.
Scholz R, Molohon KJ, Nachtigall J, Vater J, Markley AL,
Süssmuth RD, et al. Plantazolicin, a novel microcin B17/streptolysin
S-like natural product from Bacillus amyloliquefaciens FZB42. J Bacteriol 2011;193:215–224.
Scholz R. 2011. Synthese der Bacteriocine Amylocyclicin A und Plantazolicin in Bacillus amyloliquefaciens FZB42 [PhD thesis]. Berlin
(in German): Humboldt University.
Shen B. Polyketide biosynthesis beyond the type I, II and III polyketide
synthase paradigms. Curr Opin Chem Biol 2003;7:285–295.
Stein T, Borchert S, Conrad B, Feesche J, Hofemeister B, Hofemeister J, Entian KD. Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3. J Bacteriol
2002;184:1703–1711.
Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific
functions. Mol Microbiol 2005;56:845–57.
Steinborn G, Hajirezaei MR, Hofemeister J. bac genes for recombinant bacilysin and anticapsin production in Bacillus host strains.
Arch Microbiol 2005;183:71–79.
Reva O, Tuemmler B. Differentiation of regions with atypical oligonucleotide composition in bacterial genomes. BMC Bioinformatics
2005;6:251.
de Bruijn c83.tex
898
Chapter 83
V3 - 02/20/2013
8:58 P.M. Page 898
Comparative Analysis of the Complete Genome Sequence of the Plant Growth
Van Loon L. Plant responses to plant-growth-promoting rhizobacteria.
Eur J Plant Pathol 2007;119:243–254.
Walsh CT. Polyketide and nonribosomal peptide antibiotics: modularity
and versatility. Science 2004;303:1805–1810.
Wilson KE, Flor JE, Schwartz RE, Joshua H, Smith JL,
Pelak BA, et al. Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis: II. isolation and physico-chemical characterization. J Antibiot (Tokyo)
1987;40:1682–1691.
Wolf M, Geczi A, Simon O, Borriss R. Genes encoding xylan- and
ß-glucan hydrolysing enzymes in Bacillus subtilis: characterization,
mapping and construction of strains deficient in lichenase, cellulase
and xylanase. Microbiology 1995;141:281–290.
Yang H, Liao Y, Wang B, Lin Y, Pan L. Complete genome sequence
of Bacillus amyloliquefaciens XH7, which exhibits production of
purine nucleosides. J Bacteriol 2011;193:5593–5594.
Yang Y, Kloeppper JW, Ryu CM. Rhizosphere bacteria help plants
tolerate abiotic stress. Trends Plant Sci 2008;14:1–4.
Zhang G, Deng A, Xu Q, Liang Y, Chen N, Wen T. Complete genome sequence of Bacillus amyloliquefaciens TA208, a strain
for industrial production of guanosine and ribavirin. J Bacteriol
2011;193:3142–3143.
Yoo JS, Zheng CJ, Lee S, Kwak JH, Kim WG. Macrolactin N, a new
peptide deformylase inhibitor produced by Bacillus subtilis. Bioorg
Med Chem Lett 2006;16:4889–4892.
Zweerink MM, Edison A. Difficidin and oxydifficidin: novel
broad spectrum antibacterial antibiotics produced by Bacillus subtilis. III. Mode of action of difficidin. J Antibiot (Tokyo) 1987;
40:1691–1692.