Fastidious organisms Difficult to isolate on ordinary culture due to special nutritional factors. E.g.

. helicobacter pylori needs blood agar plates. E.g. mycobacterium leprae hasnt been cultivated (only isolated from humans +armadillos) Viable non-culturable (VNCs) Environmental bacteria + fungi that havent been cultured as their growth conditions arent understood, yet: We see them by live dead staining We amplify their genes from dna in soil and seawater and they match those of no other We know there are nutrient cycle enzyme activities, but not whats doing them! Thinking simply -Stock cubes are like agar- meat and vegetable extracts enable them to grow. - in reality though, we use mixtures of 20 amino acids in a solution with some glucose and buffer containing salts (THE SALTS are important for electron transport proteins of bacteria like cytochromes and other enzymes) photo autotrophs just need simple salts and sulphur medium and light! (remember media must be sterilised before) Blood Agar Pathogenic bacteria like streptococci lyse red blood cells via beta haemolysis (IMPETIGO). These are cultured by blood agar (5-10% mammalian blood- sheep or horse) Lab or no lab? Careful media design helps study in lab, but not all studied ones have been cultured- Mycobacterium leprae (leprousy causer) has 2 hosts, humans and armadillos- has only been cultured in lab animals. -Bdellovibrio are heterotrophs but need bacterial prey like E.coli in buffer for culture! Vital conditions 1)GAS- some microbes can grow in little or no oxygen (early earth anaerobic), and some are killed by it (the ones under showcase cinema, our guts, ocean floor vents). obligate aeorobes/anaerobes- grow well under both, but dont need O2. Can often guess conditions (e.g colon-anaerobic, skin-aerobic). 2) temperature-

Psychophiles- low temp optima (4) mesophiles- mid-range (39) thermophiles (60) hyperthermophiles(88)

3) pH- acidophioles like lactic acid bacteria, alkalophiles prefer higher. Mid-range for e.coli type bacteria is 6.7-7.2.

Aseptic technique -Simply using sterile tools and avoiding atmospheric contamination, but also preventing escape and harm.

open in clean air work near a Bunsen, driving the microbes up flame neck of flask when opening to remove surface microbes presterilised tools for inoculation dispose of contaminated into disinfectants or by incineration/autoclavation Isolating Pure Cultures solid medium- solidified by 1.5% agar method a, b or c (to follow) Check for isolated colonies where a single microbial cell divided again and again to form millions disinfect surfaces or solutions contaminated with microorganisms. a) streak plating- loop resterilised between streaks so no carry over, ABCDE, E will have single colonies despite the 10,000,000 bacteria applied at point A. b) spread plate- <0.1ml pipetted onto AGAR platespread evenlycount surface colonies c)pour plate- sample pipetted onto STERILE plate mix well with inoculum count colonies liquid cultures Defining Bacteria -Usually single celled, haploid microorganisms with a projartotic structure and double stranded dna as genetic material. Each cell lives as self-supporting creature - Bacteria have shapes due to cell wall structures and arrangements of proteins that control their synthesis. Some are: a) Streptococcicoccus round shape. Form long chains b)Bacillus stearothermophilus c) Rhodospirillumd) Treponema/ Borrelia e)Caulobacter f) Streptomycesg) Borrelia burgdorferi wobbly shape spirillum Caulobacter crescentus: Gram ve, lakes and streams, flagellated or stalked, produce many antibiotics, in soil -LYMES DISEASE, a spirochete- highly coiled spirochetes are distinct because of their lengthwise flagella which run in periplasmic space causing twisting motions for movement

Surface structures- not all are compulsory: -Capsule (S layer)- part of cell envelope. Monomolecular layer of proteins and glycoproteins. Have pores -Fimbriae- mostly gram ve, some +ve- thinner + shorter than flagellum. 3-10nm diameter, to several microns long. Used to adhere to animal cells or objects. Sex pili. -Flagella- type 3 secretion machinery -Cytoplasmic membrane- internal to the cell wall. Gram ve inner membrane. Inner structures -polyhydroxybutyrate granules- bacteria store and degrade it- its in plastic bottles too.Its a fuel. -Sulfur storage granules -polyphosphate granules

Simple microscopic staining - and then light microscope examination reveals morphology. You need electron MS and ve staining with electron dense materials like uranium. -GRAM STAIN- purple then pink Gram +ve if they are purple and retain the first stain Gram ve if pink and lose the first dark purple stain (going colourless) and then getting stained by the pink. Capsule Is Loose slimy layer outside SOME bacteria (called glycocalyx) Hydrophilic polymer made up of sugars like fructose and glucose prevents pathogenic bacterium being enveloped by phagocytes in hosts (e.g. Haemophilus influenzameningitis) may allow attachment (e.g. streptococcus mutans causing plaque on teeth- holes are anaerobic, acids produced when they have sugar anaerobically, making holes) produced by several soil bacteria revealed by indian ink staining!- clear halo around bacterium as it cant penetrate S-Layer -Present in several species (may be called RS layer) -Usually external to everything- an alternative to capsule. -Rigid crystalline lattice made of proteins/glycoprotein subunits arranged regularly. -Protein subunits self-assemble- form sheets several molecules thick. -Resistance to proteolytic enzymes, resist phage infection. -Function is protective (aeromonas salmonicida- fish pathogen- prevents phagocytosis). Serritia protected from predatory attack by Bdellovibrio . Like a suit of protein armour. Fimbriae (AKA type 1/common pili) -Straight, usually still, filament protrusions from surface. -0.2-1micron long, 7nm wide, 100s per cell -Protein arranged in a helix to form cylinder- new molecules added at base as it grows -Function allows attachment to eachother/surfaces. Some bacteria have them to adhere to specific tissues- Neisseria gonnorrhoeae attaches to genital epithelium, urogenic E.Coli attach to intestinal epithelium. Special sugar-protein tips encoded by genes to attach to correct tissue. -Special type IV ones (pseudomonas aeruginosa) ratched from side to side allowing crawling- called twitching Conjugative pili (sex/F pili- as some encoded by F plasmid) -straight, still, but retractable. Carry CERTAIN plasmids. -7nm wide like fimbriae, but 1-5microns long and only 1-2 per cell. -Made of cylindrical helix of protein (pilin) molecules. -Allows transfer of genetic material. -Leads to antibiotic resistance in hospitals by gene transfer from soil bacteria to pathogens. Flagella Description: Helical filaments

15nm wide, 2-10microns long, variable in number (rodobacter sphaeroides has 1, Eschericia coli has 5-12, Proteus mirabilis may have 200). Arrangement of flagella- peritrichous- branching all directions, polar- at poles Components: filament=hollow cylinder of a helical protein array of flagellin sub units. There are other proteins Rotate at speed from basal motor complexes in cytoplasmic membrane like propellers=swimming. ion-motive gradient (usually H+) across cytoplasmic membrane. Ions interact electrostatically with the motor proteins causing rotation. NO ATP HYDROLYSIS REQUIRED flagellar filaments grow from distal end- self assembly- monomers secrete from cell, travel up hollow centre. no eukaryotic homologue- the genes arent retained in our cells. present in majority of bacteria. swimming allows optimum environment arrival for growth, reaching infection site, e.g. vibrio fischeri- chemoattracted to amino acids released by fishstay in the fishes eye increased density of cells=increased release of pheromone= increased growth rate= bioluminescence via switching on of lux genes help fish find mate. POSSIBLE EVOLUTION? - Type III export systems in salmonella, pathogenic E.Coli and Shigella and Yersinia pestis (for injecting toxins into human cells) are homologous to the basal proteins in flagella, which also have export roles in exporting flagellar components. i)in salmonellatip of exporter protein integrates into human cell and delivers effector proteins, modifying host cell cytoskeleton causing actin polymerisation and pseudopod formation making salmonella get engulfed and internalised ii)in E. Coli the type III secretion system injects proteins that polymerise human cell actin causing pedestals to rise up and firmly attach bacteria. The normal human microvilli are effaced (removed). Similar for salmonella and shigella- cause membrane ruffles + engulfment. The outer membrane -Outside of gram ve (some +ves have a special one eg mycobacteria (SEE TB LATER ON), but thats special) -Lipid bilayer, inner is phospholipid, outer is lipopolysaccharide. LPS- hydrophobic Lipid A tails, bound to sugar core made of heptose and KDO (ketodeoxyoctonoic acids), which is bound to a long hydrophilic carb chain, the O antigen which sticks out into the medium. O antigen Sugar core A porin. Lipid A tails x 3 The O antigen being a hydrophile stops hydrophobes from getting close. So E.COLI with O antigens are more resistant to antibiotics and bile salts than a mutant without- important in the gut where bile salts are emulsifying fats- the e.choli arent getting destroyed. LPS found only in nature in bacteria and toxic to man, its an endotoxin as its only released when the bacteria lyse. Salmonella and Shigella cause cell death when they lyse= bleeding and diarrhoea. outer membrane has porins (proteins arranged in circles), allowing molecules of certain size into periplasm. They open and close according to osmotic conditions (too small to let bile salts in) Some are also binding sites for phages (e.g. lambda binds to maltose pore protein).

Periplasm = NOT a space. 20-40% of whole cell volume. Contains thin peptidoglycan wall + many proteins. Lysosyme can destroy the peptidoglycan- found in tears-line of defence. Penicillin targets peptidoglycan. Its proteins Binding proteins for aminos, sugars, ions, co-factors. E.g. leucine, galactose, thiamine, phosphate. Degradative enzymes- proteases, nucleases, etc. Detoxifying enzymes e.g. beta lactamases which break penicillin down. Cytochromes required to keep electron-transport continuing and therefore energy production. Cell walls -Lots of similarity between the 2 types, OM is the main difference. -Resist large osmotic pressure on membrane due to high solute conc of cytoplasm. -Consist of peptidoglycan= N-acetylglucosamine (G) + N-acetylmuramic acid (M) along with Lalanine, D-alanine, D-glutamate and L-lysine or diaminopimelate (DAP). the peptidoglycan sheet is made of alternating M and G molecule chains cross linked with amino acids. M-G-M-G-M-G-M-G-M-G-M-G <- alternating m and g I I I <- amino acid links M-G-M-G-M-G-M-G-M-G-M-G

The differences between the 3 membranes though... 1.Gram + POSITIVE -Inc. Bacillus, Streptococcus (sore throat- strepsils?), bifidobacterium (Yakult). -Cell walls are THICK and and are 90% peptidoglycan and 10% teichoic acid (which is -vely charged and gives the outside of the bacterial cell a net negative charge)

-Retain the first stain due to thickness -Special note: mycobacterium TB is unusual in that it forms thick outer layers including a 'mycomembrane'. The outer layer makes a kind of makeshift outer membrane of mycolic acid and adds a capsule to the outside, forming a lot of protection agains white blood cells when it's causing TB. 2. Gram NEGATIVE -Escherichia, Salmonella, Yersinia (BLACK DEATH), etc. -THIN cell walls with no teichoic acids. Connected to outer membrane by lipo-protein. And finally... 3. The Cytoplasmic membrane -The cell membrane/plasma membrane. Or in gram -ve, the inner membrane. -8nm thick- a phospholipid bilayer with proteins- polar heads outwards -ESSENTIAL for life- forming a selective barrier against environment. -Sometimes, instead of a smooth layer, it's invaginated to form an intracytoplasmic membrane (ICM) containing photosynthesis stuff (like Rhodobacter use, as they are phototrophs) or containing magnetosomes for Aquaspirillum magnetotacticum). -Function? Specific proteins on membrane controlling septum formation, xsome segreg. And bacterial cell division. It's like a light grade oil, viscous and means proteins and other molecules free to move within it. Site of ATP synthesis and anchor for flagella in many. -AN IMPORTANT NOTE- at least 200 different proteins in the membrane- 10-20% of the total cellular protein of the bacterium is here. A KEY DIFFERENCE TO US- their membrane makes ATP so needs the proteins, whereas our cells have mitochondria (which are basically bacteria using THEIR membranes to make ATP ;D). -The proteins it contains: Permeases- transport toxins, metabolic products, extracellular enzymes and structural components out, but transport IN growth substrates and cofactors. Type 3 system flagella... Biosynthetic enzymes- produce lipids, cell wall and surface structures. Energy generating complexes- most energy runs on ATP- produced by e- trans chains and ATPases- a series of proteins in the membrane. 3.5. The Cytoskeleton -Made of MreB, a distant relative of actin. Role? directing pepridoglycan biosynthesis Chromosome segregation Positioning of septa for cell division Note that when they divide the septum (cross wall at the centre) must have the circular xsome copied and divided either side of the septum. The MreB cystoskeleton and Fts proteins help this happen!- the FtsZ ring contracts and pinches the cell into 2. Secretion and import Gram -ve bacteria have lots of different secretion methods for different compounds, but remember foods are imported too.

Cytoplasm (cytosol) -Has both nuclear DNA and plasmid. -Polysomes -Storage materials -Proteins -Vitamins -Cofactors -ions -ATP and other high energy compounds.

The vast majority of cytoplasmic reactions occur here- some 2000. There's free exchange between all areas which= a rapid rate.

This is where the polysomes are. They are the site of protein synthesis in bacteria. There's no ER, so they're in the cytoplasm with the RNA (which has no nucleus to be contained in). = 20 ribosomes attached to an mRNA molecule as well as cofactors and tRNA molecules. 1000 per GROWING cell Ribosomes process along mRNA, decode it, use tRNA molecules to make the ppeptide chain an amino at a time :D= fast access to mRNA and fast conversion of mRNA to protein. This is why E.Coli divide every 18 mins- why bacteria can express genes and adapt so quickly. -The polysomes wait around the DNA and don't diffuse away Storage -They don't use fat as energy store, instead they store gycogen or polyhyydroxyaklanoates in granules (e.g. polyhydroxybutyrate PHB stored by many inc. rhodobacter. - Sulfur or polyphosphate sometimes stored. Other inclusions No organelles (remember, they're thought to be organelles in eukaryotes). BUT -Magnetosomes- 300-700nm long and 50-100nm wide, 100 per cell. -Gas vesicles- membrane made entirely of protein and not phospholipid. Permeable to gas but not water so good for buoyancy. Ribbed to form hard shell -Endospores- resting structures formed by many like Clostridium and Bacillus anthracis. Made by sporulation. Only grow when lacking an essential nutrient. Extremely resistant cells. 1. Vegetative cell turned into nongrower, heat-resistant. Divides then consumes spore itself 2. Spore produced and can remain dormant for years 3. Mother cell dies and spore is released- right conditions allow it to grow. allows terrorists to use the spores. Using the example of anthrax: Type 1: cutaneous anthrax- the OK. -Common in people handling animal products in developing countries. Bacillus anthracis spores get into a cut and a black leathery scar develops. CAN BE TREATED (20% mortality).

Type 2: gastrointestinal anthrax -Very rare, caused by eating spores in undercooked putrid meat from anthrax infected animal. Blood poisoning, swelling of abdomen and lymph nodes. RARE BUT FATAL. Type 3: pulmonary anthrax -Rare in normal life, but bio-warfare... Inhale the spores in dust or air, they release live bacteria upon germination. Flu difficulty breathing + death after 2-3 days. Extra note on anthrax: -Virulence is coded for on plasmids. B. anthracis has 2 of them, and they contain 3 toxin genes (tag, lef and cya) and capsule genes (this capsule is more like an S layer but made of poly-D-glutamate and not sugars). toxin gene products produce toxic proteins like... PA protective antigen, which binds to immune system phagocyte receptors and blocks them, allowing LEF lethal factor to enter immune system cells and other host cells= death. The cya mentioned above is involved in oedema and swelling. NEW LECTURE Primary Metabolism- looking at how bacteria convert the foods they consume into usable energy via ATP for synthesis of cell components, for division and movement. Flagella are key here. Taxis -Remember this from vibrio fischeri and the pinecone fish. Different types of it photo taxis- bacteria swimming out of shadows in a petri dish chemotaxis- swimming towards chemicals. Flagellation: -Uses PMF (proton motive force) like ATP synthase does- remember type III. H+ made by e- transport enters via motor protein change conformation of loop of MotA's. These push on the FliG's which are attached to the rotar make flagellum rotate. The protein CheY phosphate is made by bacteriums tactic sensors. under -ve tactic stimulus, it binds to the motors and causes them to disengage from Mots, stopping them and starting them causing the 'stumble' and randomness of choosing a new direction.

CheY-P Now onto primary metabolism The most important, geared towards growth (e.g. energy metabolism) and cell component synthesis.

(NB. Secondary metabolism isn't essential for growth- may occur later as stationary phase when toxic metabolites and antibiotics and pigments may be secreted by non growers). -In most bacteria its done by binary fission (exceptions are filamentous bacteria dividing differently). -Different foods depending on species (REMEMBER API TESTS) though ALL require synthesis of lipids, carbs and proteins (all CHO) for cell bodies. Some take up ready made (amino acids, peptides, fatty acids, sugars) Others (autotrophs) take up 1 type- e.g. sugars and convert. Some even just fix O2. -Feeding a bacteria glucose or peptone containing amino acids gives C,H,N to build molecules. C,H, O from glucose convert to lipids with added phosphate transported in by bacteria. = lipid membranes simple carbon molecules from sugars/CO2, N from nitrates/NH3 + phosphate = DNA + RNA or ATP -Bacterial enzymes degrade sugars they take up/synthesise by CO2 fix. This pathway is called glycolysis. At the end of glycolysis, pyruvate is made. -Aaerobically growing?= citric acid cycle (TCA/Krebs cycle) -Anaerobically growing?= fermentation products made (inc. ethanol) instead.- more labourious way of making cellular molecules. - The enzymes are in the cytoplasm- some attached to the membrane, some bound to a 'production line'. -Importantly NADH and some FADH are made during these processes, e.g. in glycolysis and the krebs cycle. Some NADH is USED in glycolysis, but then LOADS is made in krebs. -Electron transport chain is fed ions and electrons by NADH, and this electron transport feets ATP production chemical electrical potential is produced- proton motive force specialised proteins let the H+'s back into the cell- these are ATPase and the flagellar motor, which have parts rotates by the passage of the H+'s. HOW DOES THE ATP SYNTHASE WORK?- see separate sheet drawing. Protons being pumped -Dehydrogenases, cytochromes and quinones passing electrons to eachother. Dehydrogenases and cytochromes contain metals that carry charges. Quinones have aromatic rings and can move quickly in the fats of the membrane. -1st electrons from food (e.g. pyruvate, succinate) or chemicals made during breakdown of these. -Dehydrogenase proteins take these electrons and are passed along according to energy states. -Bacteria use a siderophore to suck in this Iron molecule- important. -As the electrons pass onto the cytochromes, the negativity makes them change shape and open a proton pore, pumping those H+'s out- electrons pass on and the pore shuts till the next electron. -Electrons reach the terminal cytochrome in the chain, binding to oxygen molecules making O-. Oimmediately binds to 2H+ to give H2O -Anaerobic bacteria don't use O2... they bind sulfur and make H2S instead BACTERIAL GENETICS How do we go from DNA mRNA peptide protein (an enzyme)? How do we use DNA polymerase in the PCR?

Small Genomes Bacteria have small genomes to encode cell function (E.Coli has 4000). Bacterial genes were the first to be studied as they're easily purified, only 1 of each gene, and mutations easily revealed phenotypes -Gene products are translated only when needed- transcriptional regulators. E.g. spore formers only express the genes when starved! Chromosomes -Carry the genes needed for normal growth. About 4Mb in E.Coli (4,000,000 bases/4000Kb) to 0.58Mb in Mycoplasma genitalum. Average gene= 1-2Kb long. Average genome= 4Mb (inc plasmid) - Haemophillus influenze- 1st bacterium with sequenced genome- now there are hundreds all the time - Usually single unique copy of the xsome per cell (Rhodobacter an exception: 0.9Mb and 3Mb) -Circular loop of double stranded base paired DNA- not like eukaryotes. BUT some have linear xsomes (Borrelia burgdorferi or streptomyces lividans Chromosome packing -Highly packaged, tightly wound up and supercoiled to fit inside. -Histone-like (the spools DNA winds round in euk.) proteins bind and package- they are HU and HNS- they require NO specific sequences for DNA binding This means DNA must be unwound and rewound for replication or transcription to mRNAs BUT if all unwound at once, it wouldn't fit in the cell. SO... genes are arranged on xsomes in clusters coding for related functions. Sometimes in 'operons' (group of genes whose expression is co-ordinated from an operator or promotor region). In some cases these show they were transferred in a block of DNA from another bacterium evolutionarily. Regulatory proteins compete with H-NS for DNA binding at some points on the xsome for binding, allowing genes clustered at points to be expressed at once- useful in a host. Organisation within the chromosome -Bacterial genes are densely packed with little space (a few hundred base pairs) between genes- NO INTRONS AT ALL. -20Kb can contain 13+ genes in bacteria, but in humans 1 gene could be longer than this (introns). -Genes in bacteria can be on either strand but always run 5'-3' -Sometimes overlap in bacteria. -Commonest form of regulation is having different promoter regions recognised by different proteins, which then activate or repress transcription by changing RNA polymerase activity. Some xsome facts -They're haploid -Located in cytoplasm with no nucleus- PROkaryotes. We call it a nucleoid. -Replication is bidirectional from 'OriC' to 'terminus' E.Coli -Divides every 18 minutes, yet 40 minutes to do a round of xsome replication.

-So... 'well fed' E.Coli initiate replication before the first round reaches the terminus. Testing for this?... Bacterial Genomics Robotic technology runs the sequencing. Pathogens concentrated on, as drug companies can make back the initial cost with the therapeutic information they gain. D-aspartate (-ve) E-glutamate (-ve) K- lysine (+ve) R- arginine (+ve) P- Proline (form bends in proteins). C & H (cystine and histidine)- binding the metal in e- transport. W,F,Y (tryptophan, phenylanine, tyrosine)- hydrophobic aminos in membrane transporters. Polymerase Chain reaction -Use heat stable DNA polymerase (Taq or Pfu) from a thermophile (this enzyme usually copies xsomes of bacterium before cell division). -DNA polymerase will start synthesis from any template single strand DNA that has a short double strand at the beginning (primer). (done from the OriC in original bacterium) -If you wanna get a certain gene, you need to know start and end sequences Say it's a mammoth... ...You'd need primers from the start and end of an elephant gene and just hope they're the same in the mammoth. Say it's just a bacterium without a genome sequence... Purify the enzyme and get some protein sequence from the beginning and end of the protein by nibbling off amino acids with trypsin and analysing what they are. E.g. MADTEACHER, you'd get MA (met, ala) and ER (glu, arg). Go on the internet and order single strands that code for this sequence. 1) Purify xsomal DNA from bacterium you want to clone 2) Melt DNA (95C) breaking base pairs 3) Cool (55C) and add primers to bind on to these (forward and reverse) 4) heat a little more (72C) and Taq or Pfu enzymes read SS extension making a new copy strand = 2 AMPLIFIED COPIES- repeat 30 times heating and cooling.' A PCR machine does all this stuff- heating and cooling and whatever else. ALSO you get overshoots at first, but gradually more and more are right within the range you want.

Cloning bacterial genes -Bacteria naturally take up and express genes.

-The gene we've just cloned- the bacteria may be a wild type for it (already has a copy of the gene intact) or a mutant (with a defect in the gene). -If it's a mutant we can attempt to 'complement' the defective gene in the bacterium by adding another gene. Genes can be carried on plasmids or on defective phages. Gene cloning in bacteria -Insertion into bacteria using transformation or transduction- these are natural. genes clones into a bacteriophage genome or onto a plasmid 'vector'- then replicated in the cell. bacteriophages transfer DNA or RNA into bacteria in nature to replicate their OWN genome and can mistakenly transfer bits of bacterial DNA from one to the next plasmids usually transferred by conjugation. ----> We modify these natural processes in the lab TRANSFORMATION -'naked' DNA or plasmids (ie. From dead bacteria) is naturally taken up by 'competent' (competence=ability to take up naked dna from environment) bacteria (e.g. heliobacter) OR bacteria treated to be made competent. Phages -Natural viruses of bacteria. RNA or DNA genome encoding their coat and other proteins allowing them to infect. -Don't produce own ATP- rely on host for replication also. - In bacterial lawns, plaquess form if susceptible to phage lysis. 1 plaque= 1 infected bacterium replicating phage and lusing -Tails are for docking, injection devices for actively inserting DNA -The phage DNA (OR RNA) codes for nucleases to degrade bacterial SNA, sigma factors and sometimes RNA polymerases to make more phage genomes. -Bacterial ribosomes translate phage mRNAs to give phage enzymes (like genome stuffase) and phage structural proteins (head, coat, tail, neck). So new ones are made with the bacterial shell and its resources. TRANSDUCTION -sometimes phages can accidentally package a piece of broken bacterial genome in place of a phage genome in their phage heads. When they're released and infect a new bacterium they 'transduce' it by adding these genes to it. If bacterial lysis genes are removed from a genome by a scientist, then phages can be used as molecular syringes to inject DNA into cells. Genes can be cut and inserted into the phage genome instead of the lysis genes to test their function in bacterial cells- this is being used in genetic engineering. Plasmids Like mini bacterial xsomes -Occur naturally, carrying, e.g. antibiotic resistance genes. -double stranded loops with their own origin of replication -c3000bp 100kb size range. -Replicated inside the bacterium by its own DNA polymerase.

-Recombination leads to plasma being integrated onto the bacterial xsome so it replicates when the xsome replicates in cell division. Why plasmids are good... -They code for antibiotic resistance genes (e.g. ones that have protein pumps to export certain antibiotic type/enzymes to degrade an antibiotic... They can be selected for on the basis of their ability to encode resistance to antibiotics- bacteria must replicate the plasmid to stay resistant. Some plasmids can be conjugated into bacteria and carry parts of the bacterial chromosome with them from one bacteria to another sometimes... though a few bacteria are naturally competent... :) so alternatively, plasmids can be taken up by bacteria made to be competent by chemical treatment or by treating them to electric shocks. -Restriction endonucleases- natural enzymes- can cut at specific DNA sequences. after this cutting, DNA from different species can be joined together! Cut sites may be chosen so that genes fit together, then DNA LIGASE joins these bits together. It's then replicated and expressed in the bacteria :) the cut DNA fragments can be visualised by gel electrophoresis and then cut out and purified for cloning into plasmids . There's a gene called lacz, and when this is interfered with, it stops working and using an indicator leaves it colourless. When present the indicator turns it blue. A cut is inserted into this fragment of the plasmid- if successful, the enzyme is inactive and no blue. If unsuccessful, the enzyme turns the sugar blue, meaning we can discard these. DNA Libraries Can be made if all pieces of a genome are cut with restriction enzymes and joined into individual copies of plasmids (1 fragment per plasmid). Plasmids cut up in different places, and the bits are ligated in. Then you've made a library. Uses Easy to study as bacteria are haploid, so removing a gene= phenotype removed. For some properties, plate assays can test this. If you have a mutant for a gene you can learn about what the gene does and you can clone the gene from a wild type... Use mutagenesis methods to find mutants. Feed chemical nucleotide base analogues to misincorporate into DNA: e.g. 5BU can wrongly base pair with G substitutions. UV light gives TT dimers side by side on DNA whhich often are repaired to a single T giving deletion of bases.

Reminder of unique bacteria -Caulobacter crescentous- differentiates into stalked and swarmer cells.

-Bacillus subtilis- differentiates into spores under conditions of nutrient limitation. -Myxococcus- form raised fruiting bodies when starved- organ like multicellular behaviour- a glimpse of how ancient unicellulars evolved into multicellulars like us. Fruiting body is formed, ones at top differentiate to fruits, each fruit contains a spore (v. resistant). These spores are at the top and so are more likely to disperse further. Biofilms -Soy sauce uses it for soy beans, yeast fermentation to beers uses it, tooth plaque, slime on rocks/algae...

-Begin as 'planktonic' (mobile) bacterial cells, settle on surfaces/the body, form structures that protect the cells deep inside! Depending on where the bacteria are in the biofilm, they form different structures. attachment: adhesion of a few cells to suitable solid colonization: intercellular communication, growth and polysaccharide formation. Development: More growth + polysaccharide. -Includes pink biofilms that reduce Fe2+--> Fe3+ in rivers. Pseudomonas biofilms -mucous in lungs of cystic fibrosis patients is a place pseudomonas biofilms grow, affecting breathing. The ones at the center of the mushroom shaped biofilms are protected from antibiotics :/ Biofilms in the world: -ships-barnacle slime

-teeth-plaque, decay, acids, streptococcuus mutants

Porins- proteins arranged in circular groups spanning the membrane (see outer membrane in gram ve) Phages- viruses that infect bacteria- they inject genetic material in from its protein capsid Binary fission-

Defining prokaryotes -No nuclear membrane thus xsomes lie in cytoplasm. -Most v. small- 1x2 microns. 350 bacteria across the full stop. -SOME LARGE- 500 MICRONS- diffusion limits size.