Escherichia coli as a model organism model organism

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Escherichia coli as a model organism

Escherichia coli as a model organism

model organism Escherichia coli baker’s yeast Saccharomyces cerevisiae worm Caenorhabditis elegans thale cress Arabidopsis

model organism Escherichia coli baker’s yeast Saccharomyces cerevisiae worm Caenorhabditis elegans thale cress Arabidopsis thaliana fruitfly Drosphila melanogaster zebra fish Danio rerio mouse Mus musculus human genome length Mbp (106) number of genes (x 103) number of DNA molecules 5 5 1 12 6 16 100 19 6 115 28 5 180 13 4 25 1. 800 2. 600 30 20 3. 200 25 23

Escherichia coli intestinal bacteria, gram negative, nonsporulating rod shape, 1 x 2 µm, facultative

Escherichia coli intestinal bacteria, gram negative, nonsporulating rod shape, 1 x 2 µm, facultative anaerobic fermentation of glucose, arabinose and mannitol under anaerobic conditions "mixed acid fermentation": production of lactate, succinate, ethanol, acetate, CO 2 inhabits the intestinal tract several hours after birth

- the most studied organism - probably developed 120 -160 mil. y. , coevolved

- the most studied organism - probably developed 120 -160 mil. y. , coevolved with mammals as E. coli feeds on lactose from milk and can survive caustic conditions of bile salts - survives in different conditions: in gut microenvironment is stable, anaerobic, with constant temperature and rich in nutrients - with feces comes to the harsh environment, with fluctuations in temperature, with aerobic conditions and poor nutrient sources - the ability to survive different conditions made E. coli a model organism

E. coli as a model organism: prokaryote; one cell genetics: small genome of 4.

E. coli as a model organism: prokaryote; one cell genetics: small genome of 4. 6 million base pairs and 4 000 - 5000 genes simple multiplication: clones - all individuals are equal rapid growth: division 20 min genome was sequenced in 1997. a simple mechanism to control gene expression mutants can be easily obtained: haploid grow in defined laboratory conditions, selective media, different conditions in which it can live simple techniques to introduce genes into the bacterium a large set of data about gene regulation and protein functions gene mapping by simple methods most strains are not pathogenic

Cultivation: heterotroph, grows on synthetic media rich and minimal media liquid and agar plates

Cultivation: heterotroph, grows on synthetic media rich and minimal media liquid and agar plates selective media (with addition of antibiotics, without aminoacids. . ) phenotype: colony morphology

Binarny division

Binarny division

E. coli as a model basic life processes (synthesis of DNA, RNA, proteins), metabolism,

E. coli as a model basic life processes (synthesis of DNA, RNA, proteins), metabolism, cell structure. . infectious diseases of animals and plants host-pathogen interactions evolution ("mini-evolution") and adaptation to extreme environmental conditions signaling between cells "Mathematical" model microbial "community" and their "metagenomes" "Instrument" of molecular genetics biotechnology - production of insulin and other biopharmaceuticals

4 639 221 pb for 4200 genes 1500 genes of unknown function "Dense" genome,

4 639 221 pb for 4200 genes 1500 genes of unknown function "Dense" genome, only about 100 bp between genes -transposons, repetitive sequences, cryptic prophages, defective bacteriophages

GRAM - Bacteria Enterobacteriaceae K 12 strain first isolated from the stool in patients

GRAM - Bacteria Enterobacteriaceae K 12 strain first isolated from the stool in patients suffering from malaria laboratory strains: different from those in nature: removed plasmids, some genes. . . -marker of faecal contamination (type detection) lots of strains (only 20% of the genome common to all), so that the „pan genome" of 16 000 genes (introduced horizontally from other species) -probiotic and pathogen strains

Structure of E. coli: no nucleus, double membrane organels - "Naked" chromosome - haploid

Structure of E. coli: no nucleus, double membrane organels - "Naked" chromosome - haploid -can have a small circular DNA - plasmids and viruses - bacteriophages

Characteristic Prokaryote Eukaryote Nucleus Absent Present Diameter of a typical cell ≈1 μm 10–

Characteristic Prokaryote Eukaryote Nucleus Absent Present Diameter of a typical cell ≈1 μm 10– 100 μm Cytoskeleton Absent Present Cytoplasmic organelles Absent Present DNA content (base pairs) 1 × 106 to 5 × 106 1. 5 × 107 to 5 × 109 Chromosomes Single circular DNA molecule Multiple linear DNA molecules

-cocci -rod shaped -spiral cells Structure of cell wall and flagellum

-cocci -rod shaped -spiral cells Structure of cell wall and flagellum

-mycoplasms: without a cell wall, sterol layer -archebacteria: pseudo peptidoglycan -glycocalix and gelatinous substance;

-mycoplasms: without a cell wall, sterol layer -archebacteria: pseudo peptidoglycan -glycocalix and gelatinous substance; capsules or slimy substance for adherence to the surface endotoxins: toxic parts of the cell wall -lipopolisaccharide causes immunological reaction regardless of the bacteria strain -exotoxins: molecules that kill target cells or interfere with the metabolization secreted or released by cell lysis

Filamentous extensions: chemotaxis causes bacterial movement -pili: structure similar to flagellum, a number of

Filamentous extensions: chemotaxis causes bacterial movement -pili: structure similar to flagellum, a number of protein subunits arranged helically with a central cavity -fimbriae for adhesion with the adhesin protein (diarrheal E. coli) F pilus: slightly longer, mediates bacterial conjugation

Bacterial strains: subgroup within a species that has some unique features: differences may be

Bacterial strains: subgroup within a species that has some unique features: differences may be at the molecular level, in resistance to antimicrobial agents, specific biochemical reactions and life cycle, physiology, pathogenic capacity Eg. O: lipopolysaccharide H: flagellin K: antigen capsules

Pili and flagella

Pili and flagella

rigid fimbriae in diarrheagenic E. coli for adhesion bundle-forming pilus on a diarrheagenic E.

rigid fimbriae in diarrheagenic E. coli for adhesion bundle-forming pilus on a diarrheagenic E. coli cell

normal bowel flora and pathogen : non-pathogenic bacteria in certain tissues can cause infection

normal bowel flora and pathogen : non-pathogenic bacteria in certain tissues can cause infection in impaired and immunosuppressed patients pathogenic strains causing infection -colon (more strains of E. coli diarrheal : 0157 H 7 ) have specific antigens which adhere to the small intestine, produce enterotoxins and interfere with cell signaling pathways (c. GMP : loss of water and salts ) 6 types of interaction -urinary tract : adhesins (fimbriae and toxins ) -sepsis, meningitis. . .

Examples of pathogenic E. coli enterohemorrhagic E. coli (EHEC): (HUS: haemolytic uraemic syndrome): O

Examples of pathogenic E. coli enterohemorrhagic E. coli (EHEC): (HUS: haemolytic uraemic syndrome): O 157: H 7 Sudden loss of kidney function enteropathogenic (EPEC): adhesin; toxin can be similar to Shiga toxin (dysentery) enterotoxigenic (ETEC): enterotoxin similar to cholera-toxin diarrhea of children and passengers enteroinvasive (EIEC): like Shigellosis - dysentery enteroagregation (EAEC): fimbriae, hemolysin and enterotoxin Extra-intestinal (Ex. PEC) - Neonatal meningitis E. coli (NMEC) - Uropathogenic(UPEC) - Avian pathogens (APEC) - E. coli as a cause of hospital infections

Sites of pathogenic Escherichia coli colonization

Sites of pathogenic Escherichia coli colonization

production of different adhesive molecules specific fimbria injector system to introduce molecules in host

production of different adhesive molecules specific fimbria injector system to introduce molecules in host cell, receptor molecules modification of actin cytoskeleton production of toxins which inhibit signaling molecules, such as G proteins and regulation of salt intake and water Mechanisms of interference with host cells DAEC: difuse adherent and neonatal meningitis http: //cmgm. stanford. edu/theriot/movies. htm http: //www. hhmi. org/biointeractive/e-coli-infection-strategy

E. coli on kidney epithelial cells, P pili -specific microbiological tests for E. coli

E. coli on kidney epithelial cells, P pili -specific microbiological tests for E. coli -presence of enterotoxins: cytotoxicity in cell culture

šiga toksin Contribution of horizontal acquisition of mobile genetic elements to the evolution of

šiga toksin Contribution of horizontal acquisition of mobile genetic elements to the evolution of Escherichia coli pathotypes. - plasmids - transposons - phages • Bacterial pathogenicity is an ability of bacteria to induce and develop infectious diseases in multi-cellular organisms (human, animals and plants). • “Pathogenicity Islands” are the bacterial genome mobile elements that carry genes encoding the pathogenicity factors production. • PAI: „pathogenicity island”: mobile elements with genes for proteins – patogenicity factors • Virulence is a degree of pathogenicity measured by the in vivo (LD 50) and in vitro (ID 50) tests

-Shigella: dysentery - shiga toxin transferred to E. coli through a bacteriophage -extrachromosomal virulence

-Shigella: dysentery - shiga toxin transferred to E. coli through a bacteriophage -extrachromosomal virulence plasmid Genetic differences between pathogens and nonpathogens -similar genomes, differences in virulence genes (each insert many virulence genes) -each E. coli strain different

Aerobactin is a bacterial siderophore UPEC: uropathogenic E. coli ABU: asimptomatic bacterium

Aerobactin is a bacterial siderophore UPEC: uropathogenic E. coli ABU: asimptomatic bacterium

E. coli was a model organism in the discovery of the many basic processes

E. coli was a model organism in the discovery of the many basic processes that have the same principles as in eukaryotes -more is known about E. coli than about any other organism

Tatum i Beadle: one gene– one protein (1958 g. Nobel prize: “for their discovery

Tatum i Beadle: one gene– one protein (1958 g. Nobel prize: “for their discovery that genes act by regulating definite chemical events“) Tatum, nevertheless, tried to see if he could extend his nutritional studies to E. coli K 12. He isolated a large number of double mutant strains. This was a significant advance because he demonstrated the first real proof of heredity in bacteria -- the mutant phenotype persisted over generations. Some of the strains he obtained were: Strain Auxotroph for: 58 -161 methionine & biotin 58 -278 phenylalanine & biotin 58 -309 cysteine & biotin 58 -336 isoleucine & biotin 58 -580 thiamine & biotin 58 -741 histidine & biotin 58 -2651 proline & biotin 679 -183 proline & threonine 679 -662 glutamic acid & threonine 679 -680 leucine & threonine -genes determine proteins -mutations can be inherited genes define chemical processes (such as a growth on a selective medium)

Transcription and translation in procariotes and eucariotes

Transcription and translation in procariotes and eucariotes

-A. Kornberg: discovery of DNA polymerase (DNA pol I) in vitro polymerization of DNA

-A. Kornberg: discovery of DNA polymerase (DNA pol I) in vitro polymerization of DNA with nucleotides and bacterial enzyme for the first time

translation : ribosome isolation; 16 S , 23 S and 5 S DNA code

translation : ribosome isolation; 16 S , 23 S and 5 S DNA code for protein synthesis : base triplet → amino acids mechanisms of transcriptional control : operon and inducible system (Lac operon) -model of " the perception of the environment " : the first example of how cell metabolism adjusts to the presence or absence of certain sugar -reaction to other conditions : temperature ( Hsp ) -RNA polymerase sigma factors in certain conditions regulate the transcription of specific genes -Specific sigma factors regulate the response according to the level of nutrients or to the number of cells in the environment E. coli can even use the extracellular DNA as a source of carbon and energy

without lactose no genes for its metabolism are produced with lactose and glucose, small

without lactose no genes for its metabolism are produced with lactose and glucose, small quantity of Lac enzyme are produced, as glucose is prefered carbon source https: //academickids. com/encyclopedia/index. php/Lac_operon

Changing the genetic material of bacteria -mehanisms of horizontal gene transfer -transformation : "naked

Changing the genetic material of bacteria -mehanisms of horizontal gene transfer -transformation : "naked " DNA ( 1 : 107 ) -transduction : DNA bacteriophage E. coli as a model for studying the biology of the phage , genetic regulation of lytic and lysogenic cycle ( T 4 , lambda) -conjugation (transmission by direct contact between cells) It's not equivalent to sexual reproduction or pairing ) -transposons -plasmids

Pathways of foreign DNA introduction into E. coli

Pathways of foreign DNA introduction into E. coli

Transfer of DNA between bacterial cells. 1. transduction 2. conjugation 3. transposition – transposons

Transfer of DNA between bacterial cells. 1. transduction 2. conjugation 3. transposition – transposons are integrated into bacterial DNA or plasmids https: //www. youtube. com/watch? v=7 st. Zk 6 Tes. Kk

Discovery of conjugation 1946. Lederberg and Tatum statistically imposible to get so high frequency

Discovery of conjugation 1946. Lederberg and Tatum statistically imposible to get so high frequency of double mutants An Introduction to Genetic Analysis. 7 th edition. Griffiths AJF, Miller JH, Suzuki DT, et al. New York: W. H. Freeman; 2000.

Plasmids - extrachromosomal inheritance: circular DNA origin of replication Plasmid types: R: antibiotic resistance

Plasmids - extrachromosomal inheritance: circular DNA origin of replication Plasmid types: R: antibiotic resistance , -xenobiotics Col : colicins : toxins for strains without plasmids -inhibicija synthesis of DNA , RNA or protein -virulence genes (which enable bacteria to cause disease) -degradation of complex chemical compounds (use new metabolites ) -F plasmids (conjugability plasmid – plasmid for "fertility" ) Plasmids : "Bacterial endosimbionts " or parasites

(40 gena; pilin) ori. T ori. V ori S – one-direction replication F plasmid

(40 gena; pilin) ori. T ori. V ori S – one-direction replication F plasmid -conjugability plasmid prototype: F factor (fertility) F plasmid - episome of 100 kb and with 100 gena episome can be integrated in bacterial chromosome by integration -F plasmid is selftransferable plasmid because it carries all the genes needed for transfer(pilus synthesis and DNA mobilisation for transfer) -other plasmids are not selftransferable because do not code for proteins for both of these functions selftransferable plazmid can help the nontransferable plasmid to transfer to another cell

1. F + replicate and survive in the population 2. F + genes code

1. F + replicate and survive in the population 2. F + genes code for proteins, which allow the production of pilus and enable transfer of DNA 3. F + transfers newly synthesized copy of the circular plasmid the F- cell: F + remains F + and F- becomes F + 4. F + cells are inhibited to contact other F + 5. F is sometimes incorporated into the bacterial chromosome and can transfer part of the host's DNA to another bacterium

Bacterial conjugation donor produces pilus (pilin) pilus adheres to recipient cell and in plasmid,

Bacterial conjugation donor produces pilus (pilin) pilus adheres to recipient cell and in plasmid, relaxase makes singlestrand break on ori. T and single strand transfers to recipient cell -transfer of T chain 5’-3’ both cells recirculate their plasmids and polimeraze second strand both of them are now F+, https: //www. youtube. com/watch? v=Etxkc. SGU 698

Plasmid transfer gene tra : transfer operon which allows transfer by conjugation genes for

Plasmid transfer gene tra : transfer operon which allows transfer by conjugation genes for pilus proteins -transfer of resistance genes between different species of bacteria a spontaneous loss of plasmid F possible an episome is a plasmid of 100 kb and 100 genes ; 40 gene transfer

bacterial conjugation

bacterial conjugation

Bacterial population with non-integrated F plasmids usually has several cells that randomly integrate plasmid

Bacterial population with non-integrated F plasmids usually has several cells that randomly integrate plasmid and which are responsible for the low incidence of transmission of chromosomal genes between bacteria. If they are isolated and grown they have high efficiency of chromosomal genes transfer and are called Hfr strains (high frequency of recombination). If the F plasmid is incorporated into the host DNA , it can be transmitted in parallel with the plasmid DNA. The amount of DNA to be transferred depends on how long the contact lasts. The transferred DNA can be integrated into the genome of recipient recombination.

-HFR transfer of host genes: if transfer lasts enough time, the whole bacterial genome

-HFR transfer of host genes: if transfer lasts enough time, the whole bacterial genome will be transferred and recipient will be F+ if transfer is not complete, recipient will stay F-, as the end of plasmid is on the end of bacterial DNA which is transferred.

Mapping of the E. coli genome through Hfr strain conjugation -first genom mapping -F

Mapping of the E. coli genome through Hfr strain conjugation -first genom mapping -F plasmid: first cloning vector

different cases of transfer: if DNA stays linear, it will be degraded F’: episomal

different cases of transfer: if DNA stays linear, it will be degraded F’: episomal plasmid having bacterial gene because of abnormal excision from bacterial DNA: it could conjugate with F- and transfer bacterial gene to another cell

4 639 221 pb for 4000 genes 1500 genes of unknown function Gene map

4 639 221 pb for 4000 genes 1500 genes of unknown function Gene map of E. coli 50% GC 87, 8 % coding 0, 8 % RNA genes 0, 7% uncoding repetitive DNA 11% regulation DNA

Agrobacteria: conjugation between kingdoms: Ti i Ri plasmids can be transferred to plants mechanism

Agrobacteria: conjugation between kingdoms: Ti i Ri plasmids can be transferred to plants mechanism of transfer depends on genes for virulence – vir genes (between bacteria: tra operon) -plasmids stimulate plant cell to produce opins: molecules which serve them for retrieval of carbon and energy

Bacteria communicate by sending and receiving signals in the form of small molecules and

Bacteria communicate by sending and receiving signals in the form of small molecules and can share genetic information during conjugation. In this issue, Dubey and Ben-Yehuda (pp. 590– 600) show that bacteria interface directly with their neighbors through nanotubes. The tubes allow passage of proteins and plasmids and may represent a significant avenue for sharing of molecules and genetic information between individual bacteria of the same and different species. The cover shows B. subtilis cells grown on solid LB medium and visualized by highresolution scanning electron microscopy (HR-SEM). Nanotubes connecting bacterial cells are visible. Artificial colors were added.

E. coli placed fundaments of genetic engeneering and recombinant DNA Paul Berg received Nobel

E. coli placed fundaments of genetic engeneering and recombinant DNA Paul Berg received Nobel prize in chemistry 1980. "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA" similar did Cohen i Boyer (1972): introduction of plasmid obtained by ligation of two plasmids – carriers of resistance genes Berg: Circular Simian Virus 40 and lambda plasmid DNA were cleaved by restriction endonucleases, leaving them in a linear form. The two DNA molecules were annealed (blunt end ligation) E. coli DNA polymerase and DNA ligase sealed gaps in the structure (Jackson, Symons, Berg). This was the first time that anyone had spliced genes.

Specific strains for genetic engeneering: Genetic variant E. coli K-12 and improved strains: restriction

Specific strains for genetic engeneering: Genetic variant E. coli K-12 and improved strains: restriction modification mechanism removed to abrogate DNA rearangement, system of recombination modified - restriction enzymes metylation of bacterial own sequences to avoid restriction

Today: signaling chemotaxis and flagella regulation http: //www. weizmann. ac. il/Biomolecular_Sciences/Eisenbach/content/research-topics

Today: signaling chemotaxis and flagella regulation http: //www. weizmann. ac. il/Biomolecular_Sciences/Eisenbach/content/research-topics

Signaling in bacteria

Signaling in bacteria

„Quorum sensing”: communication among bacteria by small molecules - the higher number of cells,

„Quorum sensing”: communication among bacteria by small molecules - the higher number of cells, more signaling molecules, activation of the receptor and downstream answer (production of certain molecules which give benefit to the whole community Asfahl et al. FEMS MR, 2017 .

CRISPR: „clustered regulatory interspaced short palindromic repeats” DNA loci with multiple short repetitive sequences

CRISPR: „clustered regulatory interspaced short palindromic repeats” DNA loci with multiple short repetitive sequences originated from viruses with which the cell came in contact before -some sort of „memory” for „prokariotic immune system” to destroy exogenous genetic material new techniques in molecular biology

Overview of the CRISPR/Cas mechanism of action.

Overview of the CRISPR/Cas mechanism of action.

CRISPR: repetitive sequences in form of palindrome Cas 9 is nuclease which is guided

CRISPR: repetitive sequences in form of palindrome Cas 9 is nuclease which is guided to target DNA

ventralno tkivo C. elegans, zebra fish and fruitfly and rice (dwarf albino rice) genetically

ventralno tkivo C. elegans, zebra fish and fruitfly and rice (dwarf albino rice) genetically modified by CRISPR – Cas 9 -problem: specificity -positive side: simple method https: //www. youtube. com/watch? v=2 pp 17 E 4 E-O 8 tamne oči

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts Bourdeau, Raymond W.

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts Bourdeau, Raymond W. and Lee-Gosselin, Audrey and Lakshmanan, Anupama and Farhadi, Arash and Kumar, Sripriya Ravindra and Nety, Suchita P. and Shapiro, Mikhail G. (2018) Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature, 553. pp. 86 -90. ISSN 0028 -0836.

E. coli as a model biochemical processes bacterial viruses bacterial and viral genetics genetic

E. coli as a model biochemical processes bacterial viruses bacterial and viral genetics genetic code, mechanisms of transcription and regulation protein translation today: tool for plasmid production protein production experiments in evolution, ageing host-pathogen relations mobility new genes: Crispr-Cas gene network and regulation of the bacterial genome

Jacques Monod, 1910 -1976. : “All that is true for E. coli, is true

Jacques Monod, 1910 -1976. : “All that is true for E. coli, is true for the elephant. ” What is true of one E. coli strain is not even true of the next. . . Prof. Mark Pallen, University of Birmingham

http: //ecolihub. org/ http: //ecoli. bham. ac. uk/ http: //education-portal. com/academy/lesson/escherichia-coli-e-colias-a-model-organism-or-host-cell. html#lesson books. google.

http: //ecolihub. org/ http: //ecoli. bham. ac. uk/ http: //education-portal. com/academy/lesson/escherichia-coli-e-colias-a-model-organism-or-host-cell. html#lesson books. google. hr/books? isbn=0387008888