Duška Vujaklija Laboratory for Molecular Genetics Zagreb, May 10, 2013

1. 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

2. 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

3. Other Total Antibiotics Actinomycetes* 7900 1220 9120 Other bacteria 1400 240 1640 Fungi 2600 1540 4140 Total 11,900 3000 14,900 Most Actinobacteria of medical significance belong to order of Actinomycetales A. Mikoč: cave Tounjčica F. Marinelli: isolation of novel species A. Mikoč: isolation of novel species *70% from Streptomyces Courtesy of D.A. Hopwood

4. Model systems Antibiotic producer The best studied model system S. rimosus colonies (Zagreb group) from liquid media S. coelicolor Sporulating colonies (Courtesy of D. Hopwood)

5. LH arm = 1.5 Mb RH arm = 2.3 Mb Core = 4.9 Mb Genetic adaptability to a wide range of environments is evident in the genome ofS. coelicolor 7825 ORFs (55 pseudogenes) 63 tRNA genes 6 rRNA operons 72.12% G+C

6. Spores Sporulating colonies Substrate mycelium Spore formation Spiral aerial hyphae phylum Actinobacteria Complex life cycle order Actinomycetales S. coelicolor family Streptomycetaceae

7. Reproductive stage of S. coelicolor growth repoductive stage Elliot MA et al. Multicellular Development in Streptomyces Courtesy of D.Hopwood

8. Molecular study of streptomycetes: Implication of SSB in chromosome segregation SSB http://www.pdbj.org/eprots/index_en.cgi?PDB%3A3BEP

9. S coelicolor possesses two ssb genes DEPPF • SSBs- primary structures SSB-A SSB-B • What is the biological role of SSBs ? OB fold C-tail

10. “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.

11. 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 M145and ssbB mutant showes slightly increased spore length and number of spores in spore chain ssbB

12. Expression profiles of ssb genes A C 18h 24h 48h 96h - 1 2 18h 24h 48h 96h - 18h 24h 48h 96h - 16s -RT Manteca A et al. J. Proteome Res. 2011 1 2 3 1 2 3 16s -RT B 18h 24h 48h 96h - RM MM 18h 24h 48h 96h - T. Paradzik, et al.Nucleic Acids Res. 2013.

13. Promoter regions of two ssb genes ▪Two transcription start of ssbAgene is75 bp and 163 bp upstream of rpsFgene the transcription start of ssbB gene to be 73bp upstream of start codon ▪Promoter region of ssbB(79 % GC, a palindromic sequence, DnaA box two long imperfect direct repeats)

14. 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

15. Stefanic et al (2009) Acta Crystallogr D Biol Crystallogr.2009 T. Paradzik, et al.Nucleic Acids Res. 2013.

16. 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

17. TSB01 TSB03 M145 TSB02 SSB-B • Fluorescence microscopy after in vivo staining by DAPI • the strain lacking ssbB(TSB01) or (C) only C-terminus of ssbB (ssbB∆C ,TSB03), • (B) M145, wild type strain, and (D) TSB02, ssbBmutant complemented with ssbB. OB fold C-tail

18. 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

19. Streptomyces: still represent an excellent source for genome mining John Innes Center

20. S. coel. M. tub. B. subt. E. coli Sigma (ECF) 65 (45) 14 (11) 17 (7) 7 (2) 2-compsensor 85 11 34 32 Ser/ThrPK 44 13 8 8 ABC transporter 141 32 77 80 Secreted hydrolase 135 22 21 9 Chitinase, cellulase 12 1 1 0 Some Genes that Adapt for Life in the Soil Courtesy of D.A. Hopwood

21. Lipolytic activity of various Streptomyces isolated from soil tricaprylin/TSB medium A.Mikoč Genome mining GDSL lipolytic family

22. ►Activity and Stability (Temp., pH, and organic solvents) ► Potential for application in biotechnology/bioremediation ►Multifunctionality 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

23. GDSL lipolytic enzymes are abundant in Actinobacteria Taxonomic distribution... Scanning for genes encoding GDS(L) hydrolases in Actinobacteria from wide diversity of ecological niches AnaBielen The 2ndInternational Symposium “VERA JOHANIDES”, 2013Zagreb, May 11, 10,40 am

24. Metagenomics IDENTIFICATION

25. Tina Paradžik, Želimira Filić i Ana Bielen Meri Luic & Zoran Stefanic Babu A. Manjasetty EMBL, Grenoble Ivo Piantanida, IRB Marija Abramić J.Pigac Adris Group - donation CIM-IRB Senka Džidić Christine Cagnon, Robert Duran Nives Ivic Bojan Hamer University of Strathclyde, Glasgow Emina Durmiši Pau University Paul Herron