duška vujaklija laboratory for molecular genetics zagreb, may 10, 2013
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Duška VujaklijaLaboratory for Molecular Genetics
Zagreb, May 10, 2013
INSTITUT RUĐER BOŠKOVIĆ
Molecular study of dominant soil bacteria: streptomycetes in nature and application to biotechnology
The 2nd International Symposium “Vera Johanides”
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
Antibiotics Other Total
Actinomycetes* 7900 1220 9120
Other bacteria 1400 240 1640
Fungi 2600 1540 4140
Total 11,900 3000 14,900
*70% from StreptomycesCourtesy of D.A.
Hopwood
F. Marinelli: isolation of novel species A. Mikoč: isolation of novel speciesA. Mikoč: cave Tounjčica
Sporulating colonies(Courtesy of D. Hopwood)
S. coelicolor
S. rimosus colonies(Zagreb group)
from liquid media
Model systems
The best studied model systemAntibiotic producer
LH arm = 1.5 Mb
RH arm = 2.3 Mb
Core = 4.9 Mb
7825 ORFs(55 pseudogenes)
63 tRNA genes6 rRNA operons
72.12% G+C
Genetic adaptability to a wide range of environments is evident in the genome of S. coelicolor
Sporulating colonies
Spiral aerial hyphae
whiE sigF whiD
whiI
whiH
whiA whiB
whiG whiJ
bldA,B,C,D,G,H,I,K...
Spore formation
Spores
Substrate
mycelium
Complex life cycle
phylum Actinobacteria
order Actinomycetales
family Streptomycetaceae
S. coelicolor
Elliot MA et al. Multicellular Development in Streptomyces
Reproductive stage of S. coelicolor growth
repoductive stage
Courtesy of D.Hopwood
SSB
http://www.pdbj.org/eprots/index_en.cgi?PDB%3A3BEP
Molecular study of streptomycetes: Implication of SSB in chromosome segregation
S coelicolor possesses two ssb genes
SSBs- primary structures
What is the biological role of SSBs ?
ssbA is essential
ssbB exibits Whi phenotype
“knock out” eksperiments
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.
▪ 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
ssbB mutant has defect in chromosome segregation
▪ Abberant distribution od chromosome in ssbB mutant , 30% of spores lacked DNA(n=2200)
A
B
C
1 2
18h 24h 48h 96h - 18h 24h 48h 96h -
18h 24h 48h 96h -
18h 24h 48h 96h -
16s-RT
16s-RT
1
2
3
1
2
3
RMMM
18h 24h 48h 96h -
Expression profiles of ssb genes
Manteca A et al. J. Proteome Res. 2011
T. Paradzik, et al. Nucleic Acids Res. 2013.
▪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)
Promoter regions of two ssb genes
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
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.
TSB01 TSB03
M145 TSB02
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.
Flardth K and Buttner M, Nature Reviews/Microbiology 2009
Cell processes during sporulation; role of SSB-B?
DivIVA
ParA
ParB
FtsZ
FtsK
Collaboration with D. Jakimowicz from Wroclav, Poland started.
Streptomyces: still represent an excellent source for genome mining
John Innes Center
Some Genes that Adapt for Life in the Soil
01112Chitinase,
cellulase
92122135Secreted hydrolase
807732141ABC transporter
881344Ser/Thr PK
323411852-comp sensor
7 (2)17 (7)14 (11)65 (45)Sigma (ECF)
E. coliB. subt.M. tub.S. coel.
Courtesy of D.A. Hopwood
Lipolytic activity of various Streptomyces isolated from soil
A.Mikočtricaprylin/TSB medium
Number of retrieved
sequences
Species/strain of genus Streptomyces
17S. clavuligerus ATCC 27064S. scabiei 87.22
16S. hygroscopicus ATCC 53653S. violaceusniger Tu 4113
15S. roseosporusS. sp. AA4
14S. ghanaensis ATCC 14672S. pristinaespiralis ATCC 25486
13S. albus J1074S. avermitilis MA-4680S. viridochromogenes DSM 40736
12S. sp. ACTES. sp. C
11S. flavogriseus ATCC 33331S. sviceus ATCC 29083
10S. bingchenggensis BCW-1S. griseoflavus Tu4000
9
S. griseus subsp. griseus NBRC 13350S. sp. ACT-1S. sp. Mg1S. sp. SPB78
8 S. sp. SA3_actG
7
S. coelicolor A3(2)S. lividans TK24S. sp. e14S. sp. SPB74
6 S. sp. SA3_actF
3 S. ambofaciens ATCC 23877
2 S. rochei
1S. diastatochromogenesS. fradiaeS. rimosus R6
organism lipase esterase
Streptomyces coelicolor 20 55
Streptomyces avermitilis 29 76
Streptomyces griseus 12 39
Streptomyces scabies 22 69
Genome mining
GDSL lipolytic family
► Multifunctionality
EC number Activity
2.3.1.43phosphatidylcholine:sterol O-acyltransferase
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
3.1.1.471-alkyl-2-acetylglycerophosphocholine 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
► Activity and Stability (Temp., pH, and organic solvents)
► Potential for application in biotechnology/bioremediation
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
0,00%
0,02%
0,04%
0,06%
0,08%
0,10%
0,12%
0,14%
Arc
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Bac
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Eum
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Fun
gi
Viri
dipl
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Taxonomic groups
Pro
port
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of S
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H h
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Uni
Pro
tKB
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taba
se (
%)
GDSL lipolytic enzymes are abundant in Actinobacteria
Scanning for genes encoding GDS(L) hydrolases in Actinobacteria from wide diversity of ecological niches
AnaAna BielenBielen
The 2The 2ndnd International Symposium “VERA JOHANIDES”, 2013International Symposium “VERA JOHANIDES”, 2013 Zagreb, May 11, 10,40 am
Taxonomic distribution...
IDENTIFICATION
Metagenomics
•
Babu A. ManjasettyEMBL, Grenoble
Ivo Piantanida, IRB
Paul Herron
Meri Luic & Zoran Stefanic
Nives Ivic
University of Strathclyde, Glasgow
Tina Paradžik, Želimira Filić i Ana Bielen
CIM-IRB
Pau University
Christine Cagnon, Robert Duran
Emina Durmiši
Bojan Hamer
Marija AbramićJ.Pigac
Adris Group - donation
Senka Džidić
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