Lecture 12 Deaths from dangerous gut bacteria hit historic high o While other health care related infections have been decreasing in recent years cases of clostridium difficile or C diff continue rising o It is a bacterium that also happens to form spores that produce toxins that affect the colon the large intestine o About 14 000 Americans die each year from the infection according to the CDC o C diff tends to hit people who are taking antibiotics for some other illness The antibiotics wipe out other beneficial microbes increasing the chances that C diff will make them sick o In addition health care facilities should also sterilize everything an infected patient has come into contact with to eliminate any spores which can contaminate areas for months o Side note some people carry C difficile in their intestines naturally and it does not constantly cause disease in them Antibiotic and stationary phase cultures o Stationary phase Death rate growth rate o Dead organism no biological activity o If organisms are growing they need to be to be synthesizing new cell walls o New cell walls is where penicillin acts o Culture dies How do organisms survive antibiotic treatment Basic mechanisms of bacterial resistance to antibiotics o Activity spectrum o Mechanism of resistance o Inactivation of antibiotic o Change the target Nalidixic acid streptomycin Substitutions in target change binding Nalidixic acid one change in gyrase Streptomycin o Change the ribosome protein o Change the ribosome RNA o Reduced permeability o Efflux Change the transport protein stop transport P aeruginosa and other gram negatives Pump it out as it comes in Costs energy up the concentration gradient o Change the pathway Folic acid sulfonamide Use a different path Use a different enzyme Macromolecules and genetic information o Functional unit of genetic information is the gene o Genes in cells composed of DNA o Offspring acquire genes from parents by inheriting chromosomes Inheritance of genes o Genes Are the units of heredity Affect an organism s traits phenotype Are segments of DNA o Each gene Has a specific locus position on a DNA molecules Loci plural of locus Chromosomes can contain thousands of genes Cow of the future PmoC o Mc Methane monooxygenase has 3 distinct protein components pmoA PmoB o Rumen contains huge populations of microbes including cellulolytic bacteria and methanogens generate CH4 o Want to reduce methane production by cow propose that M capsulatus might be useful and want to explore whether genes that enable Mc to process methane can be introduced into a microbe that can be maintained in the cow rumen o The structural genes for this enzyme lie in a three gene operon named pmoCAB o The operon codes for three integral membrane polypeptides that are the 3 subunits of methane monooxygenase PmoC 23 kDa m PmoA 27 kDa PmoB 45 kDa o E coli gram negative straight rod may contain a capsule motile by peritrichous flagella Faculative anaerobe chemoorganotroph having both a respiratory and a fermentative type of metabolism Optimal growth temp is 37 degrees Celsius Oxidase negative catalase positive D glucose and other carbohydrates Gene name Protein name lacZ LacZ Protein name based on function Beta galactosidase pmoA PmoA Particulate Methane Monooxygenase o Prokaryotic chromosomes are supercoiled and highly compacted o Double stranded circular chromosome Number of chromosomes Archaea One haploid Plasmids present In some cells Type of nucleic acid In cytosol in nuceoid and plasmids Histones present Yes Eukarya With one exception two or more typically diploid In some fungi and protozoa In nucleus and in mitochondria chloroplasts and plasmids in cytosol Yes Bacteria Single haploid copies of one or rarely two In some cells frequently more than one per cell In cytosol in nucleoid and plasmids No though chromosome is associated with a small amount of nonhistone protein DNA structure o 4 nucleotides found in DNA Adenine A Guanine G Cytosine C thymine T o Backbone of DNA chain is alternating phosphates and the pentose sugar o Phosphates connect 3 carbon of one sugar to the 5 of the adjacent sugar deoxyribose DNA vs RNA DNA double helix o Bases adenine thymine Uracil guanine cytosine o Structure see pic o All cells and some viruses have DNA in double stranded molecule o Two strands are antiparallel o Two strands have complementary base sequences Adenine always pairs with Thymine Guanine always pairs with cytosine o Two strands form a double helix DNA structure o Chain of nucleotides A T G C o Complementary nucleotides o Hydrogen bonding o Double strands antiparallel o Helix Elongating a new DNA strand 5 to 3 only o DNA synthesis is catalyzed by an enzyme called DNA polymerase which can add nucleoside triphosphates to the 3 end only Dehydration synthesis o There is an existing DNA strand then a triphosphates nucleotide is added When it is added the OH group is taken off the existing DNA strand and diphosphate is released the energy is used for the synthesis of the longer DNA strand What is left is the longer DNA strand with the nucleotide and one phosphate ex guanine nucleotide dGMP The double helix pairs 1 kbp o Size of DNA molecule is expressed in base pairs 1 000 base pairs 1 kilobase o 1 000 000 base pairs 1 megabase pairs 1 Mbp o E coli genome 4 64 Mbp o Each base pair takes up 34 nm of length along the helix o 10 base pairs make up one turn of the helix DNA structure o Major groove o Minor groove Chromosome and other genetic elements o Genome entire complement of genes in cell or virus o Chromosome main genetic element in prokaryotes o Other genetic elements include virus genomes plasmids organellar genomes and transposable elements Plasmids o Replicate separately from chromosome o Great majority are double stranded o Most are circular o Generally beneficial for the cell i e antibiotic resistance DNA replication o Templates and enzymes o The replication fork o Bidirectional replication and the replisome o Proofreading and termination Templates and enzymes o DNA replication is semiconservative each of the two progeny double helices have one parental and one new strand o Precursor of each nucleotide is a deoxynucleoside 5 triphosphates dNTP o Replication ALWAYS proceeds from the 5 end to the 3 end Priming DNA synthesis o DNA polymerase cannot initiate the synthesis of polynucleotide They can only add nucleotides to the 3 end of an existing strand o An initial nucleotide strand is required A primer made of RNA