Prokaryotic Cell Structure and Function: A Comprehensive Guide, Study notes of Microbiology

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MICROBIOLOGY EXAM 1 STUDY GUIDE (CHAPTER 1-7)

CHAPTER 1: Microorganisms and Microbiology ● What are the differences between prokaryotic and eukaryotic cells? ○ Prokaryotic cells- include the domain archaea and bacteria. They differ from eukaryotic cells because they lack membrane bound organelles and contain a nucleoid rather than a nucleus. They also can have plasmids ○ Eukaryotic cells do indeed have membrane bound organelles and have a nucleus ● Differentiate between properties of all cells and properties of some cells ○ ALL CELLS: ■ Metabolism: cells take up nutrients, transform them and expel wastes ● Genetic: replication, transcription, translation ● Catalytic: energy and biosynthesis ■ Growth: Nutrients from the environment are converted into new cell materials to form new cells ■ Evolution: cells evolve to display new properties. Phylogenetic trees capture evolutionary relationships ○ SOME CELLS: ■ Differentiation: some cells can form new cell structures such as a spore ■ Communication: cells interact with each other by chemical messengers ■ Genetic exchange: cells can exchange genes by several mechanisms ■ Motility: some cells are capable of self propulation ● Life on Earth through the Ages ○ The first cells ■ The first self replicating entities may not have been cells ■ How long ago did the first cellular life appear? About 4 billion years ago ■ What were the first type of bacteria? Anoxygenic phototrophic bacteria ■ Which bacteria started to arise at about 3bya? Cyanobacteria ■ When did modern eukaryotes arise? 2 bya ○ What is a Last universal common ancestor? Common ancestral cell from which all cells descended ● Phylogeny ○ Is? Evolutionary relationships between organisms ○ How can relationships be deduced? By comparing genetic information in the different specimens ○ Who were multicellular organisms ancestors? Eukaryotic microorganisms ● How do microorganisms exist? In microbial communities ○ What is that!? Population of interacting assemblages of microorganisms ● The Extent of Microbial life ○ Where are microbes found? In almost every environment imaginable ○ What is the global estimate of cells 5x10^30 cells ● Microorganisms as disease agents ○ They can be both beneficial and harmful to humans

○ What is resolution? The ability to distinguish two adjacent objects as separate and distinct ○ How is it determined ■ By the wavelength of light used and numerical aperture of the lens ■ Limit of resolution for the light microscope is? 2 micrometers ● Improving Contrast in Light Microscopy ○ What do Differential stains do? ■ separate bacteria into groups ○ What is one of the most widely used stains in microbiology? ■ Gram stain ○ What two major groups can bacteria be divided into ■ Gram positive and gram negative ○ Describe the gram stain procedure and describe results at each step ■ 1. Flood the heat fixed smear with crystal violet for 1 minute. Result is all cells will be purple ■ 2. Add iodine solution for 1 minute. Result is all cells remain purple ■ 3. Decolorize with alcohol breifly for about 20 seconds. Result is that gram positive are purple and gram negative are clear ■ 4. Counterstain with safranin for 1-2 minutes. GP are purple GN are pink to red ○ Fluorescence Microscopy is used to visualize specimens that? Fluoresce ■ The emit light of one color when illuminate with another color of light ○ Cells fluoresce naturally called? ■ Autofluorescence ○ Some must be stained with? ■ A fluorescent die like DAPI ○ Cyanobacteria fluoresce what color? ■ Red ○ What did Osamu Shimomura do? ■ Won the nobel prize for isolating GFP from the jellyfish (fluorescent protein) ● Electron Microscopy ○ How do electron microscopes image cells and structures ■ They use electrons ○ What are the two types and describe them? ■ Transmission electron microscopes: shoots the electrons through the specim ■ Scanning electron microscopes: a layer of metal is melted with high heat on the surface of the specimen and the microscope scans that surface ● Cell Morphology ○ What is morphology ■ Cell shape ○ What are the three major ones?

■ Coccus= spherical or avoid ■ rod= cylindrical shape ■ Spirillum= spiral shape ○ What are some unusual shapes ■ Spirochetes, appendaged bacteria and filamentous bacteria ○ There are many variations on basic morphological types ● Membrane Structure ○ Composition of membranes? ■ Fatty acids point inward to form hydrophobic environment and hydrophilic heads remain exposed to external environment or the cytoplasm ○ How thick is the membrane ■ 8-10 nm wide ○ It’s got embedded proteins ○ How is it stabilized ■ By hydrogen bonds and hydrophobic interactions ○ It’s somewhat fluid ● Peptidoglycan ○ Species of Bacteria are separated into two groups based on Gram Stain ○ Gram positive cells wall describe? ■ Can contain up to 90% peptidoglycan ■ Common to have teichoic acids embedded in the cell walls ● What are lipoteichoic acids? Teichoic acids covalently bound to membrane lipids ○ Gram Negative Cell walls ■ Total cell wall contains about 10% peptidoglycan ■ Most of the cell wall is composed of outer membrane aka the liposaccharide layer ● The LPS consists of core polysaccharides and O-polysaccharide ■ Endotoxin: the toxic component of LPS ● Vocab from lecture 2 ○ Inoculum- cell material used to initiate a microbial culture ○ Specimen ○ Aperture- ○ Magnification- see above ○ Resolution- see above ○ Putrefy ○ Aseptic technique- the manipulation of sterile instruments or culture media in a such a way as to maintain sterility ○ Sterile- free of all living organisms and viruses ○ Ocular- ○ Infection- ○ Heat-fixed ○ Differential stain

○ An energy requiring metabolic reaction ● Where does most of our knowledge concerning microbial metabolism come from? ○ It is mostly based on the study of laboratory cultures, and we need to have pure cultures to see what the microbes do. Energy Classes of Microorganisms ● Microorganisms can be grouped into energy classes ● What type of trophs are microorganisms that use chemicals as an energy soure and organic chemicals (glucose, acetate etc) as the electron source? ○ Chemoorganotrophs ● What about microbes that use chemicals as an energy source and INorganic chemicals (H2, fe, H2S , water etc.) as a source of electrons. ○ Chemolithotrophs ● What about organisms who you light as their source of electrons? ○ Phototrophs ● How to name a metabolism you use ○ EEC ■ E- energy (photo/chemo) ■ E- electron source (organo/litho) ■ C- carbon source (auto/hetero) ● What are plants? ○ Photolithoautotrophs ● What are humans? ○ Chemoorganotrophs Electron Donors and ELectron Acceptors ● Energy from oxidation-reduction reactions is used in the synthesis of energy rich compounds. ● What is an electron donor ○ the substance oxidized in the redox reaction ● What is the electron acceptor ○ the substance reduced in the redox reaction ● What does the redox tower represent ○ The range of possible reduction potentials ● What is located at the top of the tower? ○ The reduced substance that will donate the electrons ● What is located at the bottom of the tower ○ The oxidized substances that accepts electrons ● The farther down the electrons drop, the greater the amount of energy released ● What facilitates redox reactions? Are these substances consumed?

○ NAD+ and NADH facilitate redox reactions without being consumed: they are recycled

● Chemical energy that is released in redox reactions is often stored in certain phosphorylated compounds… what is an example of two compouns where chemical energy is stored ○ ATP and coenzyme A ● Fermentation occurs when…. ○ There is an absence of oxygen ● What happens to the membrane in respiration? ○ The cell membrane becomes positive on the outside and negative on the inside Glycolysis ● Glycolysis is a common pathway for the catabolism of glucose and its anaerobic ● You end up with two pyruvates and 2 ATPs Respiration ● During electron transfer, several protons are released on outside of the membrane. Where do these protons originate from? ○ Protons originate from NADH and the dissociation of water ● What does the movement of protons out and the movement of electrons into the cell cause? ○ A pH gradient because the inside of the cell becomes negative and alkaline and the outside becomes electrically positive and acidic ● What is the function of ATP synthase ○ It is the complex that converts proton motive force into ATP, there are two components to ATP ● What is the citric acid cycle? ○ The pathway through which pyruvate is completely oxidized to CO ● What is released/ manufactured during the citric acid cycle per glucose molecule? ○ 6 CO2 and NADH and FADH as well as ATP ● The citric acid cycle plays a key role in catabolism and biosynthesis ● What types of electron acceptors are used in anaerobic respiration? How about aerobic respiration? ○ Anaerobic respiration= S, SO4, NO ○ Aerobic respiration= oxygen CHAPTER 4: MOLECULAR MICROBIOLOGY

● What is the functional unit of genetic information? ○ The gene ● What are genes composed of? ○ DNA ● Name the three main informational macromolecules in the cell ○ DNA, RNA, Protein ● What is the central dogma? ○ DNA---> transcription----> mRNA-----> translation---> Protein ● What is an operon and what kind of cells is it found in?

○ They are genetic elements that replicate independently of the host chromosome ● How long are plasmids and what do they carry on them? ○ Usually 1kbp to greater than 1mbp and they carry nonessential genes but often very helpful genes ● What is an R plasmid? ○ An R plasmid is a resistance plasmid and they confer resistance to antibiotics and other growth inhibitors ● What is meant by the fact that DNA replication is semiconservative? ○ It means that each of the two progeny double helices will have one parent strand and one new strand ● What is the precursor to each new nucleotide? ○ Deoxynucleoside 5’- triphosphate which is dNTP ● In what direction does replication always proceed in? ○ 5’ to 3’ ● What is the function of DNA polymerase? And which is the primary DNA polymerase used in replicating chromosomal DNA in E. Coli. ○ DNA polymerase catalyzes the addition of dNTPs and there are five different kinds in E. coli but DNA polymerase 3 is the one primarily used. ● DNA polymerases require primers. How are primers made? ○ They are made from RNA by Primase ● Where does DNA synthesis begin in prokaryotes ○ At the origin of replication ● What is the replication fork? ○ The zone of unwound DNA where replication occurs ● What is the function of DNA helicase? ○ It unwinds the DNA ● In what strand does continuous extension of DNA happen? ○ In the leading strand ● Where are the okazaki fragments found ○ On the lagging strand ● Is DNA synthesis unidirectional or bidirectional. What does this mean? ○ Bidirectional; it means that there are two replication forks moving in opposite directions. ● How many base pairs does does DNAP add per second? ○ 1,000 base pairs ● Is DNA replication accurate? How many errors occur per base inserted? ○ Yes very accurate. The mutation rate is 10e- 8 to 10e- 11 ● What is the promoter and how it is recognized? ○ The promoter is the site of initiation of transcription and it is recognized by sigma factor of RNA ● Where does transcription stop? ○ At sites called transcription terminators ● Why is it good that transcription involves only smaller units, like a single gene, of DNA?

○ Because it allows the cell to transcribe different genes at different rates ● Prokaryotes often have genes clustered together and they are all transcribed at once as a single mRNA. What is an mrna that encodes a group of cotranscribed genes called? ○ A polycistronic mRNA ● What is an operon? ○ A group of related genes cotranscribed on a polycistronic mRNA ● What is the advantage of an operon ○ It allows for the expression of multiple genes to be coordinated

CHAPTER 5: Microbial Growth and Growth Control ● Binary Fissions ○ What is growth? ■ Increase in the number of cells ○ Binary Fission? ■ Cell division following enlargement of a cell to twice its minimum size ○ Generation time? ■ time required for microbial cells to double in number ○ Cell division: each daughter cells recieves a chromosome and copies of all other cell constituents to exist as an independent cell ○ Cells must elongate before they can divide ○ What happens if growth is not accurate? ■ The cells could get 1% loner every time or 1% shorter every time ● Fts Protiens and Cell division ○ What are Fts proteins ■ They are filamentous temperature sensitive proteins that are essential for cell division in prokaryotes they help to form the septum ○ When does the FtZs ring form? ■ After the DNA replicates ○ What do min proteins do and where are they found? ■ They are found coiled around the outside of the cell and they collapse into the cell to for the FtsZ ring. They facilitate the location ■ There are four types MinC, MinD and MinE ● MreB and Cell Morphology ○ Describe a prokaryotic cytoskeleton ■ It is dynamic and multifaceted ○ What is MreB? ■ Major shape determining factor in prokaryotes ○ What is crescentin? ■ Shape determining protein produced by vibrio-shaped cells of Caulobacter crescentus ● Peptidoglycan Synthesis ○ How does the cell wall grow in cocci?

separated too and they come in clumps that could be hard to count as well. ○ What is a second method for enumerating cells in liquid sample? ■ Use of a flow cytometer ● Viable Counts ○ Viable cell counts (plate counts)? ■ Measurement of living, reproducing population ○ What is a method to get a viable count ■ Spread plate method ○ Explain the spread plate method ■ A sample is pipetted onto surface of agar plate (0.1 ml or less) → sample is spread evenly over surface of agar using sterile glass spreader → it is incubated and you get a bunch of surface colonies ○ What should you do to obtain the appropriate colony number? ■ The sample counted should always be diluted ○ What number of colonies gives you the maximum statistical validity? ■ A plate containing 30-300 colonies ● Spectrophotometry ○ Turbidity measurements are indirect, rapid and useful methods of measuring microbial growth ○ What does a spectrophotometer do? ■ Measures turbidity ■ What is the measurement referred to as? ● Optical density ○ What do you have to establish to relate a direct cell count to a turbidity value? ● A standard curve ○ What could be some problems with this method? ● Microbes that form clumps or biofilms in liquid medium ● Temperature Classes of Microorganisms ○ Temperature: major environmental factor controlling microbial growth ○ What are cardinal temperatures? ■ The minimum, optimum, and maximum temperatures at which an organism grows ○ What happens when a cell gets too hot or too cold? ■ Cold: membrane gelling transport processes so slow that growth cannot occur ■ Hot: protein denaturation collapse if the cytoplasmic membrane thermal lysis ○ Microorganisms can be classified into groups by what? ■ Growth temperature optima ○ Psychrophile? ■ Low temperature optima ○ Psychrotolerant?

■ Can tolerate low temp but not ideal ○ Mesophile? ■ Midrange temperature ○ Thermophile? ■ High temperature ○ Hyperthermophile? ■ Very high temperature, uses Taq polymerase ● Oxygen and Microbial Growth ○ Aerobes? ■ Require oxygen to live ○ Anaerobes? ■ Do not require oxygen and may be even be killed by exposure ○ Facultative anaerobes ■ Can live with or without oxygen ○ Aerotolerant anaerobes ■ Can tolerate oxygen and grow in its presence even though they cannot use it ○ Microaerophiles ■ Can use oxygen only when it is present at levels reduced from that in air ○ What are the toxic forms of oxygen that can be formed in the cell? ■ Superoxide, hydrogen peroxide, hydroxyl radical ■ These can all be damaging to a cell

CHAPTER 6: MICROBIAL GENOMICS ● What is genomics? ○ Discipline of mapping, sequencing, analyzing, and comparing genomes ● How many prokaryotic genomes are sequenced and available? ○ Thousands ● What was the first genome to be sequenced and how many base pairs long was it? ○ RNA virus MS2; 3569bp ● What was the first CELLULAR genome to be sequenced and how many base pairs did it have? ○ Haemophilus influenzae ; 1,830,137 bp ● When did the miniaturization of reaction size and increased computer power happen? ○ Second generation sequencing ● The ability to sequence single molecules of DNA occured in when? ○ Third generation sequencing ● What generation of sequencing measure release of protons when a deoxyribonucleotide is added? ○ Fourth generation sequencing ● What is annotation? It is known as the what in genomics? ○ Converting raw sequence data into a list of genes present is the genome. It is known as the bottleneck in genomics

● What are paralogs? ○ Genes within an organism whose similarity to one or more genes in the same organism is the result of gene duplication ● What are orthologs ○ Genes found in one organism that are similar to those in another organism but differ because of speciation ● What are transposons? ○ Pieces of DNA that can move between chromosome, plasmids, and viruses ● What are insertion sequences? ○ Pieces of transposable DNA whose genes only encode transposition ● The genomes of bacterial species consist of two components, the pan and core. What are these two components? ○ The core genome is basically shared by all strains of the species. The pan genome on the other hand includes all the optional extras present in some but not all strains of the species. ● What are pathogenicity islands? ○ Chromosomal islands containing genes for virulence