Lecture 11 Methylococcus capsulatus is a Gram negative bacterium first discovered in a Roman bath that is a methanotroph it can grow using methane as a carbon source The complete genome of the strain from the Roman baths M capsulatus Bath has been sequenced Methane Utilization by M capsulatus This 1st key step is catalyzed by an enzyme called methane monooxygenase Mc Methane Monooxygenase Has 3 Distinct Protein Components PmoA PmoB PmoC Oxidation of Methane by M capsulatus Methane methanol formaldehyde a Rumen in the gastrointestinal tract of cow b A fistulated cow Rumen contains huge populations of microbes 1010 1011 per ml including Cellulolytic bacteria Methanogens generate CH4 Cow doesn t produce methane bacteria in rumen do Cows eat food with a lot of cellulose and cows have microbes in rumen that are capable of degrading cellulose how they can live on such a cellulose rich diet they don t digest the cellulose themselves though Cows eat feed etc broken down to cellulose and sugars Sugars enter various fermentative pathways Get catabolism of cellulose anabolism of products for biologically relevant molecules in cell Sugars get converted to pyruvate lactic acid acetate formate formate can be converted to methane and CO2 Cow gets volatile fatty acids out of this symbiosis cows microbes o Acetate propionate butyrate these fatty acids can pass through rumen wall and into cow s bloodstream cows can use these for energy Symbiosis o o Cows get energy molecules they can use from fermentative processes of the microbes The microbes get a food source they can use protective environment Dr Kenmore decides to apply for funding from Cow of the Future Foundation M capsulatus might be useful we should explore whether genes that enable Mc to process methane can be introduced into a microbe that can be maintained in the cow rumen New strain E coli pmoCAB How to know that E coli can survive in cow s rumen Competitions between microbes in same niche competition between orgs trying to live in rumen You review the literature and find the structural genes for this enzyme lie in a 3 gene operon named pmoCAB operon codes for the 3 integral membrane polypeptides that are the 3 subunits of methane monooxygenase PmoC 23 kDa PmoA 27 kDa PmoB 45 kDa pmoCAB when we refer to the 3 subunits together PHENOTYPE Escherichia coli Gram negative Straight Rod May contain a capsule Motile by peritrichous flagella Facultative anaerobe chemoorganotroph having both a respiratory and a fermentative type of metabolism Optimal growth temperature is 37oC Oxidase negative catalase positive D glucose and other carbohydrates are catabolized Everything we ve studied so far are phenotypes things you can observe about a cell colony on the cellular or enzymatic level Do they grow with fermentation phenotype GENOTYPE Phenotypes all come from the genotype the entire collection of genes in a microbe o Genes encoded either on chromosomes or on plasmids in bacteria made of DNA double helix Genes encode peptides Phenotypes talk about actions of Colonies are pink in MacConkey s agar because cells are producing an enzyme beta lactamase which breaks down the lactose available in MacConkey s agar making it into glucose and other things fermentation of glucose leads to pH change indicated by pink colony because colony is picking up the dye indicator of the media as well o There s a piece of DNA on chromosome that s been expressed protein s action the phenotype Gene Protein Names in Prokaryotes Rules for gene and protein names in prokaryotes 3 lowercase letters capital letter e g lacZ gene LacZ protein lacZ makes protein LacZ which is called beta galactosidase indicative of its function Clicker Bacteria have 1 Linear DNA molecules 2 Circular DNA molecules with some exceptions some have a number of linear pieces of DNA in their genotype 3 Both 4 Neither Prokaryotic Chromosomes Are Supercoiled and Highly Compacted Double stranded circular chromosome most common Without Supercoiling Compacting the DNA of a Bacterial Cell Wouldn t Fit Into the Cell Gently lysed cell and took away supercoiling of chromosome in terms of sheer length this is all the length of the circular chromosome falling out of the cell DNA is too long to pack into cell bacteria twist chromosome over and over onto itself compacting its physical size until it fits inside the cell These are much more highly organized than we thought they were genes that need to be expressed are generally on top of supercoiliing genes that don t need to be expressed are often buried underneath Independent piece of DNA plasmid 1 chromosome per cell in proks Plasmid extrachromosomal pieces of DNA bacteria some archaea and within Eukarya typically only either fungi or protozoa Circular or linear dsDNA in bacteria archaea usually have circular DNA usually in the cytosol no nucleus in proks Archaea do have histones Backbone of DNA chain is alternating phosphates and the pentose sugar deoxyribose Phosphates connect 3 C of one sugar to 5 of the adjacent sugar DNA Structure Four nucleotides found in DNA Adenine A Guanine G Cytosine C Thymine T DNA v RNA Bases Structure Adenine Thymine Uracil Guanine Cytosine Ribose sugar know this because there s an O attached to every carbon o In deoxyribose OH H at position 2 5 attached to O attached to triphosphate The Double Helix All cells and some viruses have dsDNA Two strands are antiparallel Two strands have complementary base sequences Adenine always pairs with Thymine Guanine always pairs with Cytosine A T G C Two strands form a double helix Clicker Question Chargaff s Rules of A s and T s are equal to each other of C s and G s are equal to each other DNA Structure Chain of nucleotides A T G C Complementary nucleotides Hydrogen bonding Double strands antiparallel Helix H bonding between bases 2 H bonds between A and T 3 between C and G Antiparallel 3 4 5 phosphate because the 2 strands are running antiparallel to each other 5 should be across from 3 on other strand Each strand has a 5 end and a 3 end o o o 3 end pairs with 5 end 3 has hydroxyl 5 has a phosphate Prime strand goes on in either direction beyond the part of the strand you re looking at so you just call the ends of what you re looking at 3 and 5 DNA Structure DNA grows via reaction between 3 OH of base already on DNA strand and 5 phosphate of base coming in Structure of the DNA chain and Mechanism of Growth Chain that s growing 3 hydroxyl on chain 5 phosphate nucleotide to be added to chain 2