DNA uptake during transformation of Bacillus subtilis. Public Health Research Institute and Department of Microbiology and Molecular Genetics University of Medicine and Dentistry Newark, New Jersey. Look here throughout for the names of the people who did the work!.
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DNA uptake during transformation of Bacillus subtilis Public Health Research Institute and Department of Microbiology and Molecular Genetics University of Medicine and Dentistry Newark, New Jersey Look here throughout for the names of the people who did the work!
Not another transformation talk! Edvard Munch
+ + Genes for DNA uptake ComK is the Master Regulator of Competence Pr comK
Competence Develops in Stationary Phase T 0 Transformation Growth Time
comK Synthesis Begins in Stationary Phase T 0 ComK Growth Time
Competence gene expression exhibits heterogeneity This shows a culture with a GFP fusion to a competence protein (ComK-GFP) Only 10-20% of the cells express competence! Jeanette Hahn
DNA Uptake: take home lessons 1. DNA uptake is mediated by two subsets of proteins that probably form complexes. 2. The first subset provides for access across the wall. It involves a pilus-like system. 3. The second subset includes three proteins that mediate membrane transport. These proteins may constitute an atypical ABC transporter. 4. DNA uptake probably takes place preferentially at the cell poles and at least some competence proteins are localized at the poles. (All the proteins to be discussed are under ComK control).
Binding Fragmentation Transport and degradation Integration TRANSFORMATION PATHWAY
comK comK comK comE operon (EC) EA DNA binding protein Major pilin-like protein GA GB GC GG GE comG operon GD GF ATP binding protein Integral membrane protein comC C Type 4 pilin-like proteins peptidase Proteins needed for DNA binding: Mark Albano, Jeanette Hahn, Sandhya Mohan
ComG ComC ComEA ComEA is a DNA receptor. It is an integral membrane protein. NucA is the fragmentation nuclease. It is an integral membrane protein. NucA But why are the ComG/ComC proteins needed for DNA binding? Roberta Provvedi, Young Sook Chung
The ComG/ComC proteins are needed to provide access to ComEA OUT Wall ComG proteins ComEA NucA Hypothesis: The ComG proteins form a complex (a pseudopilus) that may traverse the wall. Roberta Provvedi
Type IV pilins and pilin-like proteins hydrophobic core F prepilin peptidase cleavage Neisseria pili Klebsiella pseudopilus (Sauvonnet et al. EMBO J. (2000)19:2221)
The protoplast supernatant lysozyme osmoprotectant Gram positive cell (B. subilis)
s-s NH 2 ComGC monomer is oxidized, cleaved and translocated In protoplast supernatant COOH BdbDC S-S HS ComC OUT SH Integral membrane protein What are the roles of ComC, BdbD and BdbC? Does ComGC form a higher order structure? Roberta Provvedi, Ines Chen, Young Sook Chung & Sierd Bron lab
ComC is an integral membrane peptidase that converts pre-ComGC, pre-ComGD, pre-ComGE and pre-ComGG to their mature forms Young Sook Chung
ComC is needed for the translocation of ComGC Young Sook Chung
ComGC has an intramolecular disulfide bond MNEKGFTLVEMLIVLFIISILLLITIPNVTKHNQTIQKKGCEGLQNMVKAQMTAFELDHEGQTPSLADLQSEGYVKKDAVCPNGKRIIITGGEVKVEH Young Sook Chung
BdbD and BdbC are thiol-disulfide oxidoreductases required for competence bdbD bdbC ComK Sierd Bron lab, Mark Albano
BdbD and C are needed to stabilize ComGC, probably by introducing the disulfide bond bdbDxbdbC bdbCxbdbC comGA12 Protoplast supernatant bdbD bdbC WT bdbDxbdbC bdbCxbdbC Membrane comGA12 bdbD bdbC WT Ines Chen, Roberta Provvedi, Sierd Bron Lab
S BdbD S S BdbD S BdbDC act as oxidoreductase partners to introduce a disulfide bond in ComGC Oxidizing agents Added oxidizing agents (GSSG or cystine) S BdbD S OUT BdbC Reduced ComGC (unstable) Oxidized ComGC (stable)
ComGC forms a multimeric complex stabilized by disulfide bonds ComGC in protoplast supernatant + bME - bME kDa 46 29 20 15 5 comG comG Ines Chen, Roberta Provvedi
All the comG genes are necessary for ComGC complex formation wt DA DB DC DD DE DF DG GA(ATP) ComGC in protoplast supernatant Ines Chen
- + - + IPTG - + - + - + - + ßSH Strain has Pspac-comG, Pspac-bdbDC, Pspac-comC and is comK. The ComC, BdbDC and ComG proteins are sufficient as well as necessary for complex formation comGC monomer Ines Chen, Petrina Boucher
s-s BdbDC S-S HS ComG proteins ComC SH s-s s-s Processing and Assembly of ComGC multimer
Wall ComGC ComEA NucA ComGC does form a multimeric complex, providing access to ComEA OUT What is the composition and structure of the complex? Does the ComGC multimer traverse the wall and provide a passageway for DNA, as shown? Is the structure dynamic? Does its disassembly draw DNA into contact with ComEA?
Three membrane proteins needed for DNA transport DNA binding protein Out EA EC FA In ATP binding protein Putative channel protein Gordon Inamine, Roberta Provvedi, Arturo Londoño, Irena Draskovic, Jeanette Hahn
DNA binding may induce bending and presentation of DNA to the channel “QQGGGGSVQSDGG” linker (When linker is deleted, DNA binding still occurs, but no transport. So ComEA is needed for both steps) Dual role of ComEA Out EA EC FA In Putative channel protein Gordon Inamine, Roberta Provvedi
Role of ComFA 1. Required for transport, not binding. 2. Resembles DEAD family helicases and PriA. 3. Integral membrane protein, accessible to protease only from inside. 4. Walker A site essential for function. Out EA EC FA In ATP binding protein ComFA may be needed to drive DNA translocation, for gating the channel or as a helicase. Arturo Londoño
Topology of ComEC: absolutely required for transport N-loop OUT 1 2 3 4 5 phoA lacZ Both Irena Draskovic
ComEC has an intramolecular disulfide bond WT WT comEC comEC + - + - DTT Irena Draskovic
Oligomerization of ComEC + NEM 0’ 10’ 30’ 60’ 0’ 10’ 30’ 60’ Dimer EC monomer Cys is used here as a natural crosslinker. This disulfide bond does not occur in vivo. Irena Draskovic
Which cysteine residues are responsible for the in vivo intramolecular disulfide bond and for the in vitro cross-linking? Irena Draskovic
C C C 7 6 8 ComEC has 8 cysteine residues Each of the Cys residues has been converted to Ser. C2 C3 N-loop OUT C1 C4 C5 Irena Draskovic
The intramolecular -S-S- bond is in the N-loop C2 and C3 form an internal disulfide bond wt C2/C3 EC+N KO+N - + - + - + - + DTT EC(-SH) EC(-S-S-) ECN ECC2/C3 is degraded ECN is stable; no shift in mobility Irena Draskovic
Introduced by BdbDC C S mutations in the remaining 6 Cys residues do not affect stability, transformation or the mobility shift. ComEC Disulfide Bond S S N-loop 1 2 3 4 5 Irena Draskovic
BdbD and C are needed to introduce the N-loop disulfide bond wt bdbDC comG comC - + - + - + - + DTT (-SH) EC (-S-S-) EC Irena Draskovic
wt C6/7S C8S C6/7/8S 0 10 20 60 0 10 20 60 0 10 20 60 0 10 20 60 Dimerization assay Conclusion: ComEC dimerizes using contacts near cysteines (6), 7 and 8. Irena Draskovic
C C C 6 7 8 Disulfide bond (in N-loop) and the oligomerization helix S S 482 483 494 N-loop xxxxCCxxxxxxxxxxCx 1 2 3 4 5 Irena Draskovic
Protein motifs in ComEC S S N-loop 1 2 3 4 5 BPD Metallo-ß-lactamase domain Irena Draskovic
What is the BPD (Binding Protein Domain) motif? 1. Present in the permease component of ABC transporters. 2. Probably involved in protein-protein interactions, most likely with the ATPase component. 3. Several point mutations in the BPD of comEC inactivate function without affecting protein stability. Irena Draskovic
BPD mutants ComEC RAA - - - G- - - - -S- - - - -RV E -D- - - P L - S wt RE RL AD AS GP - + - + - + - + - + - + DTT 100% 2% 4% 36% 88% 6% Transformation frequencies Irena Draskovic
What is a metallo-ß-lactamase domain? 1. Present in a large family of proteins. 2. Includes enzymes that use water for nucleophilic attacks on covalent bonds. Usually with dinuclear Zn(II) centers. 3. Substrates often esters with associated negative charges. 4. Artemis: Involved in V(D)J recombination and double strand break repair. Processively degrades ssDNA (5’3’)!!! 5. Several point mutations, introduced in this domain of comEC, lose function without affecting stability. 6. Possible roles in ComEC: Cell-wall remodeling? Non-transforming strand nuclease? Irena Draskovic
2% 3% 4% 4% ß-lactamase domain mutants LhDsG HxHxDHxG C/H LIDTG Motif 1 2 HADQDHIG 3 H N A A A hhxsGD hhxxH WILTGD Motif 4 KVGHH 5 Irena Draskovic
Cartoon of ComEC channel N-loop OUT 4 3 5 5 3 1 1 2 2 BPD c c c ß-lactamase domain Irena Draskovic
ComEC-10His Contacts ComEA Competent cells +/- DNA - - + + His tag - + - + DNA Crosslink (DSP) EC Isolate membranes unbound EC Solubilize membrane proteins EA unbound EA Pull-down ComEC (Ni2+-resin) Irena Draskovic
DNA binding protein Out EA EC FA In ATP binding protein Putative channel protein Could these three proteins constitute an (atypical) ABC transporter? 1. Three components: ligand binding (ComEA), polytopic membrane permease (ComEC) and ATPase (ComFA). 2. ComEC is a dimer (so is ComEA). 3. ComEC has 5 transmembrane segments per monomer. 4. The BPD domain plays a role. 5. The ATP binding site of ComFA is essential for transport. 6. ComEA and ComEC contact one another.
But, transport may not be driven directly by ATP hydrolysis. Inhibitor data from the Konings lab (Groningen) suggests that transport is driven by the pH component of PMF. Perhaps ComFA plays a signaling role (channel gating?) or is needed as a helicase, while PMF drives transport.
receptor (EA) DNA uptake permease (EC) ABC cassette (FA) Irena Draskovic
receptor (EA) DNA uptake NucA permease (EC) ABC cassette (FA) Irena Draskovic