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Structure and function of the divisome in E. coli – the factory of cell division Course:Physics of the living cell Author:Guy Alis Lecturer:Prof. Mario Feingold BGU 2016 Prokaryotes – simplest cells – no nucleus, only ribosomes, cell wall - bacteria, archaea ()חיידקים קדומים E. coli - Cw – cell wall, N – nucleoid (DNA), R - ribosomes The Prokaryotic Cell Bilayer (Membrane) E coli is Gram-Negative Scanning electron micrographs of gram-positive and gram-negative bacteria,respectively. note differences in surface texture. Each cell in the TEMs is about 1µm wide Nutrition and cell chemistry C-carbon; O-oxygen; N-nitrogen; H-hydrogen; P-phosphorus (;)זרחן Se-selenium (;)מינרל Introduction • The cell cycle includes 3 stages • 1)lag • 2)constriction • 3)generation The Microbial Growth Cycle Death Phase If incubation continues after a population reaches the stationary phase, the cells may remain alive and continue to metabolize, but they will eventually die. The rate of cell death is much slower than the rate of exponential growth. The Mathematics of Exponential Growth N N0 2 n g=100minutes t g n N N0 2 t g N-final cell number N0-the initial cell number N-number of generations during the period of exponential growth g=107minutes A photomicrograph of cells of E.Coli treated in such a way as to make the nucleoid visible.A single cell is about 3µm and the nucleoid about 1µm long Bacterial Cell Division-who cares? •Designing methods to control microbial growth,in particular, bacterial growth. This methods are used to treat a lifethreatening infectious disease or simply to disinfect a surface.Cancer is uncontrolled cell growth. Cell Growth and Binary Fission Cells elongate to approximately twice their original length and then form a partition that constricts the cell ito two doughter cells. Cell Growth and Binary Fission This partition is called septum and results from the inward growth of the cytoplasmic membrane and cell wall from opposing directions; The Zring The Zring • Fission ( )בקועis coordinated by a protein complex called the divisome • The assembly of which is directed by the conserved tubulin (cytoskelton component) homologue FtsZ. Before The Zring Elongation mode of peptidoglycan synthesis. Insertion of peptidoglycan precursors in to the existing peptidoglycan layers. Needed for efficient invagination ( התקפלות )כלפי פנים Of the outer membrane Forms the Z ring plane and attracts other divisome proteins The Zring • First, FtsZ undergoes polymerization into filaments that form a defined ring- like structure (Z ring) at the site of cytokinesis. • Membrane-associated FtsZ-interacting proteins, such as FtsA and ZipA in E.Coli,tether the Z ring to the cell envelope, which results in an initial complex called the proto-ring. ZipA(pink) and FtsA(blue) tether FtsZ(green) protofilament and polymer bundles to the membranes using flexible linkers Overlay of the cytoplasmic membrane (red) and the Zring (green-yellow) (b)FtsZ - membrane tethering (c)Cyro-electron tomography of FtsZ and FtsA (d)Flouorescence microscopy and differential interference contrast microscopy of FtsZ and FtsA The Zring After about 40% of generation time, the FtsZring is formed. Connects the FtsZ ring to the cytoplasmic membrane and stabilizes it Same as FtsA but has a relatively stronger bound to the membrane and is less concentrate d The Zring-Role of FtsA in Formation of the Divisome • It tethers the FtsZ filaments to the membrane • Along with ZipA, it is required for recruitment of all the downstream division proteins. • FtsA is much more conserved in evolution than ZipA,it has more direct role in the recruitment of downstream proteins. The Zring-Role of FtsA in Formation of the Divisome • Two FtsA molecules arranged in a filament • MreB is similar to conventional actin The Zring Appearance and breakdown of the FtsZ ring during the cell cycle of E.Coli. Phase contrast Stained cells Ftsz Ring iPALM images of vertical E.coli not yet formed start to forms as segregate cell elongates breakdown, division The Zring iPALM images of horizontal E.coli Trapped cell , GFP images of FtsZ Fts Proteins and Cell Division •In a cell of E.coli about 10,000 FtsZ molecules polymerize to form the ring. •The ring attracts other divisome proteins •ZipA is an anchor that connects the FtsZ ring to the cytoplasmic membranes The cytoskeletal protein MreB is an actin analog that winds as a coil through the long axis of a rod-shaped cell, making contact with the cytoplasmic membrane in several locations . These are sites of new cell wall synthesis. MreB protein flourescence The Zring • During the seconds stage,the proto-ring recruits enzymes that are involved in cell wall (septum) synthesis, such as FtsI, and proteins that probably coordinate signalling between these enzymes and the proto-ring, such as FtsN and FtsBLQ, thereby forming the mature divisome. FtsA(blue) recruits FtsN(purple).The periplasmic domain of FtsN is targeted to peptidoglycan,which reinforces its loclazation and potentially stimulates Ftsl and the synthesis of septal peptidoglycan After The Zring About one fifth of a generation time later (overall 60% generation time since the beginning), the proteins Ftsk up to AmiC assemble onto the ring, and constriction of the cell is initiated . Last division proteins to arrive at the ring,triggers septation,forming the mature divisome Encodes the membrane components of theABC transporter Encodes the ATPase components of the ABC transporter Dedicated to peptidoglycan synthesis during cell elongation only Localize to the ring and promotes the integrity of the Z ring The Zring • The divisome then constricts the cytoplasmic membrane, which in concert with the synthesis of new peptidoglycan( קיפול כלפי )פניםat the division septum and invagination of the outer membrane achieves cytokinesis and cell separation. Translocates DNA away from the septum. May play a role in fusion of the invaginating membrane to complate cytokinesis The Zring In the absence of AmiB and AmiC, AmiA is still able to carry out the seplitting of the septum The Z ring is targeted to the site of division by related but distinct mechanisms. Phase Constrast For M9 (poor media) Ƭg≈100 minutes For LB (rich media) Ƭg ≈20 minutes Double Stranded DNA From DNA to proteins – information flow • when the cell divides DNA is replicated – DNA polymerase • - DNA contains genes that code for proteins • - when activated, RNA polymerase builds an RNA copy, messenger RNA (mRNA) – transcription • - mRNA goes to the ribosome – there transfer RNA (tRNA) helps copying the mRNA into a protein – translation. Each 3 mRNA bases code for 1 amino acid • - polypeptide folds into 3D structure, sometimes chaperones help DNA Replication DNA replication is a semi conservative process in all cells – the two resulting double helices have consist of one new strand and one parental strand Events at the DNA replication fork Synthesized a short strech of RNA (11-12 nucleotides Unwinds a double helix Catalyze ( )מזרזיםthe addition of nucleotides Nucleic acid molecule Extension of a DNA chain by adding a deoxyribonucleoside triphosphate at the 3’ end. DNA helicase unwinding a double helix Events at the DNA replication fork Descrete 3’-OH Keeps the double helix locally open while the replication fork continues Continueous 3’-OH Why is a primer required for DNA replication? Bidirectional Replication and the Replisome Circular DNA. The origin of the replication is also known as OriC the origin of the end of replication is also known as TerC. Notice the change of the inner and outer color of the circumference of the replicated DNA strainds ? How can Escherichia coli carry out cell division in less time than it takes to duplicate its chromosome? Answer: superposition of replication forks References QUIZ