The Ins and Outs of DNA Transfer in Thermus thermophilus

Transcription

1 Zur Anzeige wird der QuickTime Dekompressor benötigt. Campus Riedberg, Biocenter Molecular Microbiology & Bioenergetics The Ins and Outs of DNA Transfer in Thermus thermophilus Beate Averhoff Institute of Molecular Biosciences Goethe University Frankfurt Germany

2 Fractions of candidate interdomain horizontal gene transfer Archaea: Archaeoglobus fulgidus Methanococcus janaschii Thermoplasma acidophilum Halobacterium sp. 5.8 % 11.1 % 8.8 % 15.6 % Bacteria: Aquifex aeolicus Thermotoga maritima Escherichia coli Bacillus subtilis Haemophilus influenzae Mycoplasma pneumoniae Chlamydophyla pneumoniae Rickettsia prowazekii 8.5 % 14.0 % 0.4 % 0.1 % 3.0 % 3.6 % 2.4 % 0.9 % Koonin et al. (Annu. Rev. Microbiol. 2001) Protein coding sequence (Mbps)

3 Transformable Bacteria Photolithotrophic bacteria Anacystis nidulans Chlorobium limicola Synechocystis sp. strain 6803 Chemolithotrophic bacteria Thiobacillus thioparus Thiobacillus sp. strain Y Heterotrophic bacteria Acinetobacter sp. BD413 Bacillus subtilis Pseudomonas stutzeri Streptomyces spp. Methylotrophic bacteria Methylobacterium organophilum Clinical isolates Haemophilus influenzae Helicobacter pylori Neisseria gonorrhoeae Neisseria meningitidis Streptococcus pneumoniae Phytopathogens Ralstonia solanacearum Thermophilic bacteria Thermus thermophilus HB27 Thermus flavus Thermus caldophilus Thermus aquaticus

4 Naturally transformable model strain Thermus thermophilus HB27 Thermus thermophilus HB27 thermophilic (45-80 C) aerob, heterotroph gram-negative highly transformable Henne et al., Nature Biotechnol. (2004)

5 Zur Anzeige wird der QuickTime Dekompressor benötigt. Kinetics of the DNA transporter

6 uptake system ds DNA chromosome chromosome chromosome non-competent cell competent cell DNA binding acid soluble nucleotides ss DNA chromosome chromosome DNA uptake DNA integration or plasmid reconstitution Steps in natural transformation

7 DNA binding and uptake T. thermophilus cell suspension P 32 -labeled DNA cells +DNA silicone oil centrifugation DNA cells with bound and imported DNA cells +DNA silicone oil DNase DNasesensitive DNA cells with DNase-resistant DNA

8 v [µg DNA / mg protein min] /v [µg DNA/mg protein ml ] /v max /K M 1/S [µg DNA/ml] -1 V max = 1.5 µg DNA/mg protein min (40 kb/sec cell) K m = 50 µg DNA/ml DNA [µg / ml] Kinetics of DNA binding and uptake Schwarzenlander et al. (2006) FEBS J. 273,

9 DNA transport velocities T. thermophilus HB27 40 kb x s -1 x cell -1 B. subtilis 8 kb x s -1 x cell -1 H. influenzae 16 kb x s -1 x cell -1 S. pneumoniae 4 kb x s -1 x cell -1 Thermus exhibits a very high DNA transport velocity

10 µg DNA / mg protein time [min] DCCD 50 µm 100 µm 250 µm 500 µm 20 µm TCS DNA uptake by T. thermophilus is energy dependent DCCD, Dicyclohexylcarbodiimid; TCS, Tetrachlorosalicylanilid

11 Zur Anzeige wird der QuickTime Dekompressor benötigt. Function of acquired DNA? Carbon source? Nitrogen source? Phosphor source? Precurser for DNA-Synthesis?

12 Zur Anzeige wird der QuickTime Dekompressor benötigt. Specificity of DNA binding and transport

13 Transport of different DNA types? accumulation factor linear plasmid circular plasmid chromosomal DNA The DNA translocator does not discriminate between different DNA types

14 accumulation factor binding uptake The DNA translocator of Thermus binds and transports DNA from different domains Donor DNAs: 1, Thermus thermophilus HB27, 2, Acetobacterium woodii, 3, Escherichia coli DH5α, 4, Saccharomyces cerevisiae, 5, Nicotiana tabacum BY2, 6, Methanocaldococus jannaschii, 7, Methanosarcina mazei Gö1, 8, Pyrococcus furiosus, 9, Sulfolobus solfataricus P2. The total amount of accumulated Thermus-DNA (DNase-sensitive + DNAse-resistant) was set to 1. Schwarzenlander and Averhoff (2008), submitted

15 Zur Anzeige wird der QuickTime Dekompressor benötigt. Subunits of the DNA translocator?

16 Pili pilm piln pilo pilw pilq Pili n.d. n.d. - - Zur Anzeige wird der QuickTime Dekompressor TIFF (LZW) benötigt. comea comec pild pilc Pili n.d. pila1 A2 A3 comz A4 pilf dpra 1kb Organization of the Thermus thermophilus HB27 competence genes Averhoff (2004), J. Bioenerg. Biomembr. 36; Rumszauer et al., (2006), FEBS J. 273, Bettermann et al., in prep.; Rose et al., in prep.

17 SECRETIN outer layer assembly/ disassembly PREPILIN- PEPTIDASE PILINS periplasm inner membrane INNER MEMBRANE TRANSLOCATOR TRAFFIC NTPase ATP ADP + Pi ssdna The competence proteins belong to five distinct protein families

18 Zur Anzeige wird der QuickTime Dekompressor benötigt. Function of the competence proteins? Identification and subcellular localization

19 C CM OM WT PilQ - WT PilQ - WT PilQ - PilQ (82 kd) Subcellular localization of PilQ Rumzsauer et al., FEBS J. 2006

20 Subcellular localization of DNA translocator subunits S-layer outer layer PilA4, PilQ, PilW hydrophilic matrix peptidoglycan inner membrane 40 nm PilA4, PilQ PilF and ComEC PilM, PilN, PilO, PilW, PilC Thin section of Thermus cells in collaboration with W. Haase and W. Kühlbrandt, Max Planck Institut für Biophysik, Frankfurt

21 Zur Anzeige wird der QuickTime Dekompressor benötigt. Function of the competence proteins? Role of single subunits in DNA binding and transport?

22 transport The role of competence proteins in DNA binding and transport accumulation factor binding 0.0 wildtype pilq::kat pila1-3::kat pila4::kat pild::kat comea::kat pilf::kat comec::kat Schwarzenlander and Averhoff (2008), submitted

23 Conclusions 1. PilQ is essential for binding (and transport) of DNA. 2. PilA4 and PilF are important for the transport of DNA through the OM into a DNase-resistant state. 3. PilA1-3 are not essential for transport of DNA into a DNase-resistant state. 4. ComEC is essential for transport of DNA through the inner membrane. Thin section of a T. thermophilus cell

24 dsdna S-layer OM PilQ PilQ SCWPs PilW PilA4 PilW PG CM PilC PilA1-3 ssdna ComEC (channel protein) ComEA (putative DNA binding protein) PilN PilO PilM PilF (AAA + -ATPase) PilD (prepilin peptidase) Model of DNA transport in Thermus thermophilus Topologies, conserved motifs and cellular localization of DNA translocator proteins

25 Alexandra Friedrich Judit Rumszauer Cornelia Schwarzenlander Katrin Bettermann Janin Burghardt Ilona Rose Katharina Baatz Collaborations: J. Berenguer, Madrid W. Egge-Jacobsen, Oslo G. Grüber, Singapur W. Haase and W. Kühlbrandt, Frankfurt M. Karas, Frankfurt V. Müller, Frankfurt Funding: DFG CMP (Center of Membrane Proteomics)