Download S. coelicolor

INSTITUT RUĐER BOŠKOVIĆ
The 2nd International Symposium
“Vera Johanides”
Molecular study of dominant soil bacteria:
streptomycetes in nature and application to biotechnology
Duška Vujaklija
Laboratory for Molecular Genetics
Zagreb, May 10, 2013
Actinobacteria - one of the major communities of the microbial population present in soil
responsible for the peculiar soil - smell after rain
• inhabit a wide range of environmental niches;
soil, freshwater, marine sediments
• Gram-positive bacteria
• produce a number of enzymes that help
degrade organic plant material, lignin, and
chitin…
•the best known known as secondary
metabolite producers; Streptomyces as
antibiotic producers
Most Actinobacteria of medical significance belong to order of Actinomycetales
F. Marinelli: isolation of novel species
A. Mikoč: cave Tounjčica
Antibiotics
A. Mikoč: isolation of novel species
Other
Total
Actinomycetes* 7900
1220
9120
Other bacteria
1400
240
1640
Fungi
2600
1540
4140
Total
11,900
3000
14,900
*70% from Streptomyces
Courtesy of D.A. Hopwood
Model systems
The best studied model system
Antibiotic producer
S. rimosus colonies
(Zagreb group)
from liquid media
S. coelicolor
Sporulating colonies
(Courtesy of D. Hopwood)
Genetic adaptability to a wide range of environments is evident in the genome of S. coelicolor
LH arm = 1.5 Mb
RH arm = 2.3 Mb
Core = 4.9 Mb
7825 ORFs
(55 pseudogenes)
63 tRNA genes
6 rRNA operons
72.12% G+C
Complex life cycle
S. coelicolor
Spores
Sporulating
colonies
whiE
sigF
whiD
bldA,B,C,D,G,H,I,K...
whiI
whiH
Spore formation
Spiral aerial hyphae
whiA
whiB
whiG
whiJ
Substrate
mycelium
phylum Actinobacteria
order Actinomycetales
family Streptomycetaceae
Reproductive stage of S. coelicolor growth
repoductive stage
Elliot MA et al. Multicellular Development in Streptomyces
Courtesy of D.Hopwood
Molecular study of streptomycetes:
Implication of SSB in chromosome segregation
SSB
http://www.pdbj.org/eprots/index_en.cgi?PDB%3A3BEP
 S coelicolor possesses two ssb genes
DEPPF
SSB-A
SSBs- primary structures
SSB-B
OB fold
 What is the biological role of SSBs ?
C-tail
“knock out” eksperiments
ssbA is essential
ssbB exibits Whi phenotype
Tina Paradzik, et al. Structure-function relationships of two paralogous single-stranded DNA
binding proteins from Streptomyces coelicolor: implication of SSB-B in chromosome segregation
during sporulation Nucleic Acids Res. 2013.
ssbB mutant has defect in chromosome segregation
▪ Abberant distribution od chromosome in ssbB mutant , 30% of spores lacked DNA(n=2200)
▪ Statistical analyses of spore length and number in S. coelicolor M145 and ssbB mutant
showes slightly increased spore length and number of spores in spore chain ssbB
Expression profiles of ssb genes
Manteca A et al. J. Proteome Res. 2011
A
C
1
18h
18h
24h
48h
96h
-
18h
24h
48h
96h
-
2
24h
48h
96h
-
18h
1
1
2
2
3
3
24h
48h
96h
-
16s
-RT
16s
-RT
B
18h
24h
48h
96h
-
RM
MM
T. Paradzik, et al. Nucleic Acids Res. 2013.
Promoter regions of two ssb genes
▪Two transcription start of ssbA gene is75 bp and 163 bp upstream of rpsF gene
the transcription start of ssbB gene to be 73 bp upstream of start codon
▪Promoter region of ssbB (79 % GC, a palindromic sequence, DnaA box two long
imperfect direct repeats)
Binding of SSB proteins to ФX174 DNA (EMSA)
- NaCl
100 mM NaCl
Tryptophane fluorescent quenching of SSB-A (1) and SSB-B (2) while binding to (dT)35
Stefanic et al (2009) Acta Crystallogr D Biol Crystallogr. 2009
T. Paradzik, et al. Nucleic Acids Res. 2013.
Detection of disulphide bonds in SsbB
(A) S. coelicolor SsbB isolated from E. coli .
(B) Western blot analysis: SsbB isolated from S. coelicolor
(C) Binding of SSB proteins to ФX174 DNA in a presence of DTT
SSB-B
OB fold
TSB01
TSB03
M145
TSB02
C-tail
Fluorescence microscopy after in vivo staining by DAPI
(A) the strain lacking ssbB (TSB01) or (C) only C-terminus of ssbB (ssbB∆C ,TSB03),
(B) M145, wild type strain, and (D) TSB02, ssbB mutant complemented with ssbB.
Cell processes during sporulation; role of SSB-B?
FtsZ
DivIVA
ParB
ParA
FtsK
A - Arial hyphae grow by tip extension; FtsZ helical filaments which are remodelled into Z rings
After septation, MreB localizes to closing septa and spread around developing spore.
B- Chromosome segregation, ParA / ParB binds near oriC, its distribution is driven by ParA.
Collaboration with D. Jakimowicz from
Wroclav, Poland started.
Flardth K and Buttner M, Nature Reviews/Microbiology 2009
Streptomyces: still represent an excellent source for genome mining
John Innes Center
Some Genes that Adapt for Life in the Soil
Sigma (ECF)
2-comp sensor
Ser/Thr PK
ABC transporter
Secreted hydrolase
Chitinase,
cellulase
S. coel.
65 (45)
85
44
141
135
M. tub.
14 (11)
11
13
32
22
B. subt.
17 (7)
34
8
77
21
E. coli
7 (2)
32
8
80
9
12
1
1
0
Courtesy of D.A. Hopwood
Lipolytic activity of various Streptomyces isolated from soil
Number of
retrieved
sequences
17
16
15
14
13
A.Mikoč
tricaprylin/TSB medium
12
11
Genome mining
organism
lipase
esterase
Streptomyces coelicolor
20
55
Streptomyces avermitilis
29
76
Streptomyces griseus
12
39
Streptomyces scabies
22
69
10
9
8
7
GDSL lipolytic family
6
3
2
1
Species/strain of genus
Streptomyces
S. clavuligerus ATCC 27064
S. scabiei 87.22
S. hygroscopicus ATCC 53653
S. violaceusniger Tu 4113
S. roseosporus
S. sp. AA4
S. ghanaensis ATCC 14672
S. pristinaespiralis ATCC 25486
S. albus J1074
S. avermitilis MA-4680
S. viridochromogenes DSM 40736
S. sp. ACTE
S. sp. C
S. flavogriseus ATCC 33331
S. sviceus ATCC 29083
S. bingchenggensis BCW-1
S. griseoflavus Tu4000
S. griseus subsp. griseus NBRC 13350
S. sp. ACT-1
S. sp. Mg1
S. sp. SPB78
S. sp. SA3_actG
S. coelicolor A3(2)
S. lividans TK24
S. sp. e14
S. sp. SPB74
S. sp. SA3_actF
S. ambofaciens ATCC 23877
S. rochei
S. diastatochromogenes
S. fradiae
S. rimosus R6
► Multifunctionality
► Activity and Stability (Temp., pH, and organic solvents)
► Potential for application in biotechnology/bioremediation
EC number
Activity
2.3.1.43
phosphatidylcholine:sterol Oacyltransferase
3.1.1.1
esterase
3.1.1.2
arilesterase
3.1.1.3
lipase
3.1.1.4
phospholipase A(2)
3.1.1.5
lysophospholipase
3.1.1.6
acetylesterase
3.1.1.47
1-alkyl-2acetylglycerophosphocholine
esterase
3.1.1.53
sialate O-acetylesterase
3.1.1.72
acetylxylan esterase
3.1.1.77
acyloxyacyl hydrolase
3.1.2.2
palmitoyl-CoA hydrolase
Prediction of SrL 3D structure
• Abramić et al, Enzyme Microb Technol, 1999
• Vujaklija et al, Arch Microbiol , 2002
• Vujaklija et al, Food Technol Biotechnol, 2003
• Leščić et al, Biological Chemistry, 2004
• Zehl et al, J Mass Spectrom, 2004
• Leščić Ašler et al, BBA, 2006
• Bielen et al, Biochimie, 2009
0,14%
0,12%
0,10%
0,08%
0,06%
0,04%
0,02%
Viridiplantae
Fungi
Eumetazoa
Eukaryota
Actinobacteria
Bacteria
0,00%
Archaea
Taxonomic distribution...
Proportion of SGNH hydrolases in UniProtKB
database (%)
GDSL lipolytic enzymes are abundant in Actinobacteria
Taxonomic groups
Scanning for genes encoding GDS(L) hydrolases in Actinobacteria from wide
diversity of ecological niches
Ana Bielen
The 2nd International Symposium “VERA JOHANIDES”, 2013
Zagreb, May 11, 10,40 am
Metagenomics
•
IDENTIFICATION
PCR
• Sampling and DNA isolation
• 16S rRNA analysis
• Sequencing, TRLFP analysis,
CLONE cloning of PCR products
IDENTIFI • Phylogenetic analysis
CATION
Tina Paradžik, Želimira Filić i Ana Bielen
Nives Ivic
Meri Luic &
Zoran Stefanic
Babu A. Manjasetty
EMBL, Grenoble
Marija Abramić
J.Pigac
Adris Group - donation
Ivo Piantanida, IRB
CIM-IRB
Senka Džidić
Christine Cagnon, Robert Duran
Bojan Hamer
Paul Herron
University of Strathclyde,
Glasgow
Emina Durmiši
Pau University