Dr. G. Gottschalk. Dr. H. Liesegang. CeBiTec University Bielefeld. Prof. Alfred Pühler. Dr. Christian Rueckert. Dr. Jochen Blom. Prof. Qi Wang, CAU Beijing China.
Structural and functional genomics of plant-associated Bacillus strains used for biocontrol of plant diseases Rainer Borriss, ABiTEP GmbH and Humboldt University Berlin KSPP International Conference 2012
CDS: 3693, 8.5 % are devoted to non-ribosomal synthesis of secondary metabolites
gb: CP000560
1
Secondary metabolites with biocontrol and plant growth promoting activities OH
O N H
I-Y-Q-P O
HN
fengycin
C10-12CHCH2CO=E-L-L-V-D-L-L
O
C11-13CHCH2CO=N-Y-N-P-E-S-T
C13-14CHCH2CO=E-O-Y-T-E-A
surfactin
O
bacillomycinD
OH
OH
bacilysin O H2N
Srf-NRPS O
Fen-NRPS
OH
H2C-C-COO-
1
H2C-C-COO-
Bae-MS
OH
H3C-C-COO- 2
H3C-CH
H3C-C
H3C-C
O
O
2x Pyruvat
Sfp
O
2-Acetolactat
OH
Trp
Dif-M S
OH
Acetoin
?
CH2COOH
Dbe-NRPS
DHB
O
O HO
macrolactin
CH3 O
DHB-Gly-N H
HO
Chen et al. 2007: Nature Biotechnology
H3C
O CH3
R
CH2COOH
Gly-DHB HN
difficidin
2,3-butandiol
IAA
H
OH
H3C-CH OH
3
N
Mln-MS
O
?
OH H3C-CH
IAA P
COOH
H
O
NRS-NRPS
O
N
Bac-NRPS Bmy-NRPS
NH
HO
O
O
bacillaene
O O
O
O N-Gly-DHB
CH3
OH
HO
N H
O OH
H
bacillibactin
1. Antimicrobial secondary metabolites 2
Giant gene clusters are devoted to non-ribosomal peptide synthesis. More than 8.5% of the whole genome are occupied by gene clusters involved in non-ribosomal synthesis of secondary metabolites.
1. Antimicrobial secondary metabolites
3
Giant gene clusters devoted to non-ribosomal polyketide synthesis
1. Antimicrobial secondary metabolites
4
Gene clusters devoted to synthesis of plantazolicin in FZB42 and closely related strains
Plantazolicin is a ribosomally synthesized thiazol/oxazol peptide suppressing closely related Bacillus sp. including Bacillus anthracis 1. Antimicrobial secondary metabolites 5
Gene clusters devoted to mersacidin synthesis in FZB42 and closely related strains. FZB42contains only a fragmentary part of the whole gene cluster and is unable to synthesize mersacidin.
1. Antimicrobial secondary metabolites
6
B. amyloliquefaciens type strain DSM7
2. Comparative Genomics 7
Venn diagram: DSM7 (1), FZB42 (2), B. subtilis (3): 3049 genes are shared by all three strains. core genome
2. Comparative Genomics 8
Chromosomal locations of genome plasticity regions identified by different methods.
A, B, C: putative horizontally transferred genomic islands are depicted as predicted by IslanViewer (red spots), IslandPath (blue spots), SIGI-HMM (yellow spots) and SeqWord Sniffer (green spots). D graph shows a global alignment of bacterial chromo-somes built by the M-GCAT program. Indels are depicted by red rectangles. 9
Whole genome sequences of Bacillus amyloliquefaciens
Strain
note
NCBI
gene bank
Size bp
GC protein
rrna
tRNA
gene
Source
Bacillus amyloliquefaciens subsp.amyloliquefaciens DSM7
type strain
NC_014551 FN597644
3,980,199
46.1 %
3893
30
94
4044 DSMZ
LL3
chromosome NC_017190 CP002634
3,995,227
45.7 %
4219
22
72
4346 Nankai Univ.
plasmid
NC_017189 CP002635
6758
42.0 %
9
TA208
guanosine
NC_017188 CP002627
3,937,511
45.8 %
4089
18
70
4177 CAS Beijing
XH7
purine
NC_017191 CP002927
3939203
45.8 %
4190
21
75
4286 Guangzhou
NC_009725 CP000560
3,918,589 46.5 %
3693
30
87
3813 ABiTEP
30
86
4014 Lallemand
9
Bacillus amyloliquefaciens subsp. plantarum FZB42
type strain
IT-45
chromosome
CM001433
3,925,087 46.6 %
3898
plasmid
CM001434
8,009 40.5 %
9
CAU B946 rice isolate
NC_016784 HE617159
4,019,861 46.5 %
3823
30
95
3948 CAU Beijing
YAU - Y2
CeBiTec
NC_017061 HE774679
4,242,774
45.9 %
3989
30
91
4110 YAU Kunming
BGI
NC_017912 CP003332
4,238,624
45.9 %
4238
29
85
4352
419617 bp
46.0 %
4193
30
92
4315 NAU Nanjing
NZ_AJST01000 3899657 bp 46.6 %
3898
30
86
KRIBB
NAU B3
draft genome unpublished
9
Bacillus 5_B6
cherry tree
10
Phylogenetic tree based on core genomes
2. Comparative Genomics
11
2. Comparative Genomics
Within the „plantarum“ group ,13 gene clusters are involved in synthesis of antibiotics (polyketides, lipopeptides, bacteriocins, and lantibiotics)
12
Conclusions part 1 1. Comparative genomics has been proven as a valuable mean for improving bacterial taxonomy. Discriminating between the plant-associated and the non-plant- associated representatives of B. amyloliquefaciens has revealed two subspecies: „plantarum“ and „amyloliquefaciens“ (Borriss et al. 2011.IJSEM 61, 1786-1801). Subspecies plantarum is distinguished by a higher genomic capacity to synthesize nonribosomally antimicrobial metabolites. 130 genes have been identified as being unique for the subspecies. Besides comparative genomics, other methods of functional genomics have to apply for further elucidating molecular factors important for rhizosphere competence of those bacteria. 2. Due to the importance of the „plantarum“ subspecies, we propose: a) to choose Bacillus amyloliquefaciens FZB42 as a model strain for PGP bacilli. The strain is freely available for basic research from BGSC (strain number 10A6) and DSMZ (strain number DSM 23117) b) to intensify research concerning factors affecting root colonization and to screen for bacilli with improved ability to colonize plant roots c) In order to reduce the costs for registration, it would be highly desirable to permit generally use of representatives of this species as bicontrol agents without performing the whole set of investigations necessary today. 13
Colonization of Arabidopsis roots by FB01mut
Tips of the primary root [C], and of root hairs (D) are colonized by the bacteria 3. Rhizosphere competence 14
gfp-insertion into FZB42
•
Stable integration of gfp into the chromosome of FZB42
•
All tested colonies are emitting green light about 30 generations without selective pressure
•
No differences in growth between gfp-labeled FZB42 and wild type was observed
Fan et al. 2011, JBT Vol 151, Issue 4
3. Rhizosphere competence 15
Colonization of Lemna minor One day: Colonization mainly at root tip.
Five days: Increasing number of microcolonies at roots.
Nine days: Sunken spaces are surrounded by Chloroplast bearing parenchyma cells. GFP labeled cells are colonizing sunken space aerea of the Lemna fronds. 16
Colonization of Arabidopsis by FZB42 and mutant strains
A) Root tip of the primary root and B) primary root surface with a emerging lateral root colonized by FB01mut
G) Border cells of primary root tip and H) emerging lateral root colonized by ΔdegU-mutant.
I) Border cells of primary root tip and J) emerging lateral root colonized by ΔpznA-mutant.
M) Border cells of primary root tip and N) emerging lateral root colonized by ΔRBAM017410-mutant
3. Rhizosphere competence 17
GFP-labeled FZB42 colonizing maize roots
A: Junction between primary root and lateral roots is heavily colonized.
B: Presence of FZB42 in a concavity of maize root surface.
C,D: Bacteria colonizing root hairs. Bacteria grow along a root hair.
Right: Preferred sites of growth in maize (A) and (B), Arabidopsis [C], and Lemna minor (D)
3. Rhizosphere competence 18
FZB42 (FB01mut) coloizing lettuce roots
1.5 cm from main root tip
2 cm from main root tip
4 cm from main root tip
Root hairs in vicinity of main root tip
3. Rhizosphere competence
Suppression of Rhizoctonia solani by FZB42 in a gnotobiotic system
R.solani
R.solani FZB42
Left: Cocultivation of FZB42 and R. solani in Petri dishes. Right: Gnotobiotic system in quartz sand (lettuce, R. solani and FZB42) established and used by IGZ, Helmholtz, and ABiTEP 4. Biocontrol 20
Detection of secondary metabolites by FZB42 in presence of Rhizoctonia
1054.0
441.2 459.4
1084.0
Bacillomycin D
1119.3
1086.0
1.5
1098.0
1.0
461.2
656.3
441.2
x10 4
2.0
1045.2
Bacillomycin D, Surfactin
2.0
Intens. [a.u.]
1084.2 1114.3
x10 4 2.5
1108.0
997.9 1032.0
861.5
905.6
690.3 709.2
603.2
550.5
423.2
459.4
0.0 400
600
800
1000
1200
1400
1600
m/z
Fengycin
1.0
FZB42 (LA + P) in presence of R. solani Extract of agar in vicinity of the mycelium
1544.8
1502.7 1464.6
1355.3 1375.2
1135.3
1010.0
893.5
843.7
721.7
666.3 621.5
525.2
704.3
477.2
1032.1
Plantazolicin
0.5
495.4
0.5
1.5
411.2
Intens. [a.u.]
(LA + P) by mass spectroscopy after 4 days growth
0.0
4. Biocontrol 400
600
800
1000
1200
1400
m/z
21
Inhibiting effect of FZB42 on growth of phytopathogenic fungus Rhizoctonia solani in a gnotobiotic quartz sand system
GFP-tagged FZB42 (FB01, green fluorescence) growing at lettuce roots suppress completely R. solani (left). Development of Rhizoctonia solani on lettuce roots only in absence of FZB42 (right).
4. Biocontrol 22
The impact of B. amyloliquefaciens FZB42 on lettuce growth in the field natural infested with the bottom rot pathogen R. solani 8
Shoot dry mass [g/plant] a
a
6 4 b 2
0 Control
R. solani
FZB42 +Rs
Under biotic stress (presence of the pathogen R. solani, +Rs) and in respect of applied spore numbers (106, 107, 108 spores/ml) of FZB42-Rif. Plants were cultivated at 22/15°C for 4 weeks. Dry mass followed by the same letter are not significantly different according to Dunnett’s test (P=0.1).
Golzow, Brandenburg Size 100 x 25 square meters, Alluvial loam
Chowdhury et al. submitted
4. Biocontrol 23
Conclusions: part 2 A chemically defined axenic system was established with Lactuca sativa grown on quartz sand Inoculation studies with GFP-tagged B. amyloliquefaciens FZB42 showed long chain-like colonies and after 2-4 weeks biofilm like appearance in the oldest part of the roots Co-inoculation with R. solani in model quartz sand showed that FZB 42 was able to inhibit fungal infection for up-to 2 weeks, with no visible signs of infection
First mutant studies revealed that Surfactin is necessary for root colonization Field trials revealed that FZB42 enhances yield of lettuce in presence of Rhizoctonia solani without affecting rhizosphere community.
24
Thanks to.... Humboldt University Berlin, Germany Dr. Oliwia Makarewicz Dr. Xiaohua Chen Dr. Alexandra Koumoutsi Dr. Lilia Carvailhis Dr. Kristin Dietel Dr.Romy Scholz Dr. Ben Fan and many others
Prof. Qi Wang, CAU Beijing China Prof. Yuewen Gao, NAU Nanjing China Prof. Xueqiu He, YAU, Kunming China Dr. ChongMing Ryu, KRIBB, South Korea This researcht was mainly supported by the BMBF Germany
Technical University Berlin, Germany Dr. Joachim Vater Prof. Dr. Roderich Süßmuth
Rheinische Friedrich-Wilhelms-University Bonn, Germany Prof. Dr. Jörn Piel Jana Moldenhauer Goettingen Genomics Laboratory, Germany Prof. Dr. G. Gottschalk Dr. H. Liesegang CeBiTec University Bielefeld Prof. Alfred Pühler Dr. Christian Rueckert Dr. Jochen Blom
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