Microbiology - Lecture notes - 1.17.12 PDF

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Microbiology: What is it and why we study it? 1-6. Syllabus 7. Overview Slide 8-16. What is microbiology? Fascinating facts about Microbiology The study of cellular (living) and acellular (non-living) biological particles that are too small to see with the un-aided eye We need instruments, lens, magnifying glasses, and microscopes in order to see this type of life Non-Living particles can also impact life. IT IS NOT, the study of any ONE group of organisms. Microbiology has no boundaries 9-16. Fascinating Facts Although small, microorganisms play central roles in human activities and the web of life on the planet Thus we may think of microbiology as the foundation of the biological sciences 1. Bio Mass – Microbes comprise about 50% of the earth’s biomass. There is more microbial biomass on the planet than plant or animal (estimated 5 x 10 30 cells) There are amazing discoveries EVERY single day 2. Diversity –Most branches on the tree of life are microbial a. Eukaryotes b. Archaea c. Bacteria 3 domains of life, Microorganisms are found in all 3 domains of life. Viruses do NOT appear on the tree of life, because they are considered NON-LIVING Focus is on Micro Bacterial and Archaea biology 3. Time—The oldest microbial fossils are about 3.8 billion years old. The oldest multicellular fossils are about 600 million years old. Microbes have shaped the world we live in. Cyanobacteria oxygenated our planet Every plant and every animal has evolved in the presence of microbe organism In the absence of microbes other life would never have arisen and would not survive today.

4. Habitat Diversity – Boiling Hot Spring Antarctic Sea Ice Salt Evaporation Ponds Antarctic Dry Valley Soil 5. You are just another habitat as far as a microbe is concerned There are 10 times more bacteria in your body than human cells! Microorganisms play central roles in human activities 6. Deep Sea Microbial Mat Most microbial cells do NOT reside on the planet’s surface, but instead live within it’s subsurface— an unexplored frontier They live within our planet, at least 3 km, has life in it. 7. Microbiology requires us to rethink some central biological concepts a. A species – Asexual reproduction b. Methods of gene acquisition Microorganisms can share genes laterally; conjugate with whomever you want Horizontal Gene Transfer Bacteria can accustom to antibiotics faster, why? Because of how they transfer genes 17-22. Cells. The microbial cell, microbial populations and communities, cell shapes The cellular microorganisms Microbes– Unicellular, hence microbial cells are capable of doing everything an organisms does Must be able to adapt, must have all the necessary equipment in order to withstand any type of environment Will not find a single cell, but instead a population Plants & animals –Multi-cellular, cells differentiate / become specialists. Different cells have different activities

Microbes in nature A bacterial population is a group of cells derived from a single parent by successive divisions A microbial community is a mixture of microbial species in nature A habitat is the environment where a particular species normally lives. The study of microorganisms in their natural environment is call MICROBIAL ECOLOGY The 3 domains of Life Examples of microorganisms can be found in each domain In this course we will focus on the PROKARYOTES: The Bacteria and Archaea “Before the nucleus came” These are NOT primitive life forms What is a cell—Living Cells Cell—A unit of life, and is compartmentalized, the compartmentalization is achieved by membranes Diverse compartment that is in flux with its environment Membranes keep good stuff in and bad stuff out. It constrains the nutrients within the cell, metabolizes the cell, they have to be somewhat permeable Membranes and Wall are different things Membrane  compartmentalization Wall strength and integrity All cells have membranes, but plant cells have a wall also In eukaryotic cells you see membrane bound organelles and a nucleus which is also membrane bound. In prokaryotic cells, no nucleus is present, the genetic material is free floating in the cells, and there is a NUCLEOID All cells are made up of the same materials (macromolecules) Four Types of macromolecules 1. 2. 3. 4.

Lipids Nucleic Acids –DNA (genes) & RNA Proteins Polysaccharides

Shapes (Morphology) of some bacteria and Archaea Coccus / Cocci Rod / Bacillus/ Bacilli Spirochete Spirillum/ Spirilla Filaments

Review in DNA Replication – Chapter 6 http://www.youtube.com/watch?v=49fmm2WoWBs&feature=youtu.be 3. Fidelity of DNA synthesis –Proofreading activity Mutation rate during DNA synthesis is incredibly low; 1 in every 108 – 1011 bases Why?

Question—DNA Polymerase 1 removes 5’-3’ direction and proofreading occurs from 3’-5’ 4. Termination of Replication Opposite the Origin (O) is the terminus of replication (T) where the two replication forks collide Origin of replication –Where replication and DNA synthesis begins Replication forks collide at the terminus Ter Sites within the DNA are recognized and bound by the Tus proteins. This blocks the progression of the replication forks The newly generated daughter chromosomes remain linked, like links of a chain. This structure is called a concatemer and is resolved by Topoisomerase IV

5. Supercoiled DNA in a Cell Bacteria maintain an ordered chromosome

1. The origin (O) and terminus (T) are located toward poles 2. DNA forms staked loops orthogonal to cell axis

6. DNA Polymerases catalyze the addition of dNTPs In E. Coli there are 5 DNA polymerases (DNA polymerase 1-V) DNA polymerases synthesize DNA in a 5’-3’ direction All DNA polymerases require a free 3’OH group to allow synthesis to start For DNA replication 3’OH groups are provided by a primer In most cases, the primer is a short RNA molecule 7. Transcription in Prokaryotes –Chapter 6

8. Overview of Information Flow in ALL living systems Replication  Transcription  Translation Template –Used to synthesize the mRNA, (5’-3’ direction), then has to be the bottom strand (3’ -5’) Noncoding Anti-sense Runs 3’-5’ direction Minus Strand Coding—5’3’ (coding strand) Positive Sense Non-template strand

9. Genes Functional units of genetic information Are transcribed into RNA All genes run in a 5’-3’ direction, but can be found on either strand of DNA Can encode proteins, mRNA, rRNA, tRNA, or even regulatory RNA RNA has a functional and genetic role RNA has the potential to encode for protein 10. Types of RNA Messenger RNA (mRNA) codes for protein sequences and is therefore translated Ribosomal RNA (rRNA) is not translated. It is part of the ribosome (23s, 16s, 5s) Molecular weight of ribosomal RNA differ in prokaryotic cells Transfer RNA (tRNA) is not translated. It “translates” mRNA sequences into amino acid sequences at the ribosome Regulatory RNA, such as Ribozymes have catalytic activity 11. RNA vs. DNA a. Backbone contains ribose instead of deoxyribose b. RNA is typically single stranded c. Uracil is substituted for thymine d. Also synthesized 5’ to 3’ e. Uses DNA as a template f.

No primer required –de novo synthesis (RNA synthesis can start by itself)

12. Review of DNA/ RNA Structure and Function DNA – Double Stranded DNA, Nucleotides, Antiparallel strands, Hydrogen bonding, Complementarity, Denaturation and secondary structures within DNA molecules RNA—Single Stranded molecule, polarity, nucleotides, secondary structures

Inter vs. Intra 13. Transcription in Prokaryotes

Promoter –Molecular switch that turns a gene on and off Messenger RNA gets synthesized by the Transcription start site (+1) when promoter is on Always downstream AUG—Translation start site NOT transcription 14. RNA Synthesis: Transcription Transcription is the faithful copying of the template strand of DNA into RNA 5’ -3’ Transcription is carried out by a multi-subunit enzyme called RNA polymerase Unlike DNA polymerase, RNA polymerase does not need a primer to start synthesizing the RNA 5’ -3’ direction Faithful –No errors made 15. RNA Polymerase: The Transcription Factory

Eukaryotes have 3 RNA polymerases, which transcribe different types of RNAs. Prokaryotes have one RNA polymerase whose activity is modulated by sigma factors Core is sufficient for RNA synthesis, assuming RNA is already anchored with its template Sigma assists in binding to appropriate promoters 16. The Function of Sigma Sigma Factors

act as a prokaryotic transcription initiation factors

Sigma factors associate with the core RNA polymerase enzyme to create holoenzyme

Holoenzyme recognizes and binds specifically to promoter sites on DNA The sigma subunit confers promoter binding specificity by directly interacting with the -10 and -35 regions of the promoter DNA Sigma will never bind to DNA alone, always will bind as Holoenzyme 17. Different Sigma Factors Recognize Different Promoter Consensus Sequences

Different sigma factors have different biochemical affinities for core enzymes For a given sigma factor, promoters close to the consensus are strong whereas those diverging from the consensus are weak. Anti sigma factors that stop a sigma factor from binding to the core 18. RNA Polymerases of the 3 Domains

19. 3 Steps of Transcription Initiation, Elongation, Termination 20. Step 1: Transcription Initiation Requires a promoter sequence Promoters typically extend approximately 250 base pairs upstream of the transcription start site Promoters are regions that specifically allow the RNA polymerase enzymes to bind

Promoters typically contain 2 sequence motifs; -10 (similar to TATA box) and -35 sequence Promoters also bind DNA-binding proteins which control gene expression Promoters are found “upstream” of the gene they control. The gene is “downstream” of its promoter

-10 and -35 interact with sigma factor of a holoenzyme 21. Transcription Initiation Sigma factor of the holoenzyme specifies the site of transcription initiation by recognizing a promoter Promoters are recognized by -10 and -35 consensus sequences and a transcriptional start site, usually a purine Sigma is lost during elongation

22. Different sigma factors recognize different promoter sequences

23. Transcription Initiation RNA polymerase holoenzyme binds DNA Sigma interacts with the -10 and -35 sequences Once RNA polymerase is positioned correctly at the transcription start site the DNA helix is unwound Nucleotides enter the RNA polymerase and the RNA polymerase starts to move along the DNA, away from the promoter 24. Step 2: Elongation Once RNA polymerase escapes from the promoter region, ELONGATION begins RNA synthesis starts at the transcription start site As RNA polymerase moves into the gene an RNA molecule is made that is complementary to the template strand of DNA and an exact copy of the coding strand Is sigma lost during elongation? Yes. 25. Overview of Transcription

26. Overview of Transcription

28. Step 3: Transcription Termination Transcription terminates at sequences in the nascent RNA strand called “Terminators” Types of Terminators Intrinsic Terminators –Stem loops formed by inverted repeats GC Rich  AT rich Rho Dependent Terminators –Rho protein knocks off Polymerase that has paused at Rho dependent termination site

29. Overview of Transcription

Essentials of Virology

4/4/2012 2:35:00 PM

Viruses in Nature 

Viruses are the most abundant biological particles on earth, typically found in concentrations of 10x higher than cells



Probably all organisms have viruses which infect them



Viruses don’t always harm the host and some even carry genes that benefit the host

General Properties of Viruses 

Viruses are non-cellular biological particles



They do NOT carry out Respiration or Biosynthesis



They rely on host cells for energy and material needed for viral replication



Viruses usually exists in one of two forms – intracellular or extracellular

Extracellular form- The Viron 

Metabolically inert, transmission particle

Intracellular Form –Essential for Viral Replication 

1. Attachment



2. Penetration



3. Synthesis of nucleic acid and protein



4. Assembly and packaging



5. Release (lysis)

Overview of Information flow in all cells 

Replication of DNA



Transcription of RNA



Translation of Protein

Viral Genomes Page. 237

Viral Classification 

Based on their host o

Bacterial & Archaeal – Bacteriophages

o

Animal- Medicinal importance



o

Plant-Agricultural importance

o

Other kind of eukaryotic viruses

Viral Taxa-Formal Classification o

Class, Orders, families, genus and species

o

Members of a Viral family all have similar virion structure and genome structure and replication strategy

o

Viral Families have the suffix – viridae eg. Poxviridae

Roads to Transcription in Viruses with Different Genome Types 

Page 246



Baltimore Classification scheme

Virus Shapes and Sizes

Virus Symmetry: Helical Nucleocaspids 

The nucleic acid genome assumes a helical configuration, surrounded by the protein capsid



Example: Tobacco Mosaic Virus

Virus Symmetry: Icosahedral Nucleocapsids



An icosahedron is a 20-sided object and is the most efficient arrangement of subunits to form a closed shell. o

Example: Human Pailloma Virus

Enveloped Viruses 

Nucleocapsid = Nucleic Acid + Capsid (composed of capsomeres) o



NAKED VIRUS

Nucleocapsid + Envelope o

ENVELOPED VIRUS

Complex Viral Particles 

Comprised of Several Parts o

Example: Phage T4 of E.coli

Enzymes in Virions 

Virions are metabolically inert



Why then, do some virions contain enzymes?



Examples: o

Bacteriophages contain lysozyme (enzyme) that makes a small hole in the peptidoglycan layer allowing nucleic acid to enter the host (Penetration)

o

Retroviruses replicate with DNA intermediate need reverse transcriptase (enzyme). Other RNA viruses require their own RNA dependent RNA polymerases (enzyme)

o

These enzymes are required because the host cell does not have these enzymes

Plaque Assay 

Molten Top Agar – Pour Mixture onto nutrient agar plate & incubate



Outcome? Phage Plaques

Viral Growth / Propagation – Plaque Forming Units 

Plaques = zones of lysis



It is assume that each plaque has originated from one single virion

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PFU’s – Plaque forming units

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PFU’s are typically lower than number of virions seen with EM = efficiency of plating

4/4/2012 2:35:00 PM

4/4/2012 2:35:00 PM

Principles of Bacterial Genetics

4/13/2012 10:49:00 PM

Chapter 1: Hallmarks of Life 

Evolution- Cells evolve to display new biological properties. Phylogenetic trees show the evolutionary relationship between cells

 

Mutations are inherited changes in the nucleotide base sequence of genomes



Genetic alteration can also be brought about through Recombination – PHYSICAL Exchange of genetic information



3 major pathways lead to genetic material transfer in prokaryotes

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Prokaryotes can also alter their genetic material through RECOMBINATION



Definitions o

Genotype-Nucleotide sequence of the genome

o

Phenotype-Observable characteristics of the organism

o

Wild Type-

o

Mutant-Genetic changes; genes are never mutants, always MUTATED. Mutants = proteins with mutations

o

Auxotroph-Specifically mutant forms of life that have a nutritional requirement

o

Prototroph-A mutant that doesn’t have a nutritional requirement 

o



Auxotroph is derived from phototroph

Selection vs. Screening 

Screening- Evaluating organisms dependent on their phenotypes



Selection- Living vs. Dying

Point Mutations o

Errors are repaired, not mutations

o

Point mutation- Single nucleotide that is being changed for another nucleotide

o

Transitions vs. Transversion 

Purine for Purine ; Pyrimidine for Pyrimidine



Purine   Pyrimidine



Purine = A, G



Pyrimidine = C, T, U

 o

Silent Mutations

o

Replacement Mutations 

o

Insertions vs. Deletions (INDEL) 

o

Missense vs. Nonsense Causes frame shifts

 Which of these mutations occurs most frequently? Missense (faulty protein) 

Missense Mutations do not necessarily affect the protein in a positive or negative way

o



Most mutations have no affect at all

Spontaneous Mutations o

Mutation Rates—Errors in DNA replication occur at frequency 10-6 – 10-7 per kilobase pair during a single round of replication



Mutation in RNA genomes o

Mutation rate s in RNA genomes 1000 fold higher than in DNA genomes. Why? 

Viruses have RNA genomes



Have proof reading activity but NO REPAIR enzymes to replace the error

o

RNA dependent RNA polymerases have proof reading activity, but lack enzymes necessary to repair RNA that damaged. Hence, errors in replication are corrected but error introduced at other times are not.



Induced Mutations o

Mutagens – Nucleotide Base Analogs 

Induced mutations require mutagens



5-Bromouracil looks similar to Thymine ; 2-Aminopurine looks similar to Adenine

 o

Mutagens—Intercalating Agents lead to single base pair insertions and deletions (INDELS); not analogous to Nucleotides

o 

Can be planar molecules that insert themselves between the stacked base pairs

 o

Mutagens—Radiation 

Assists to study different wavelengths



Ionizing radiation damages DNA



UV light damages DNA 

Pyrimidine Dimers

 

Causes Thymines and Cytosine are adjacently next to each other in DNA





DNA Repair Systems o

Direct Reversal – Bases that have been chemically modified (By alkylating agents, pyrimidine dimers). Identity of the base is still intact so no template strand required

o

Repair of single strand damage-Damaged DNA removed from one strand. Template strand provides information for DNA repair 

o

Example: Base excision repair and nucleotide excision repair

Repair of double strand damage- Especially dangerous. Repair by...