SCH2235 Microbiology Exam
Give an example of a eukaryotic microbe involved in food spoilage.
Ergot
Describe a fungus
Can be single celled like yeast or multicellular • Eukaryotic, spore-bearing • Chemoorganoheterophs (eat everything) with absorptive metabolism • Saprophytes - absorb nutrients from dead organic material by releasing degrative enzymes and osmotrophic (absorb soluble products) • Lack chlorophyll • Reproduce sexually and asexually
What diseases are caused by prions?
Cause degenerative diseases in humans and animals • Scrapie in sheep • Bovine spongiform encephalopathy (mad cow) • Creutzfeldt-Jakob disease and variant (vCJD) in humans • Kuru in humans • Fatal familial insomnia
Give an example of a eukaryotic microbe involved in human disease
Caused by fungi that are not usually pathogenic to a healthy person but under certain conditions they can produce severe infections Includes: Aspergillus, Candida, Cryptococcus, Mucor and Rhizopus
What is the bacterial cell cycle?
Cell cycle is a sequence of events that begins at the formation of new cells and continues through the next cell division Peptidoglycan in cell growth • Spherical/coccoid o Build new peptidoglycan only at the midcell, where the septum will form during division. o New cells have one new and one old cell hemisphere • Rod cells o Side wall elongation - produce new cell wall at the side of the cell and not at the poles - placement thought to be due to position of MreB homologues o Preseptal elongation - As division begins FtsZ polymerisation forms a Z ring and new cell all growth is confined to the mid cell o Division - Rod shaped daughter cells are formed with one new pole and one old pole
What are the 4 major mechanisms of antimicrobial drugs?
Cell wall synthesis inhibitors Protein synthesis inhibitors Metabolic antagonists Nucleic acid synthesis inhibition
Explain the process of conjugation
Conjugative plasmids (F plasmid- fertility) can transfer copies of themselves to other bacteria during conjugation. F factors contain the information for formation of sex pilus. F+ and F- cell bind for DNA transfer, transfer is unidirectional (type IV secretion from cell to cell).
How is antimicrobial resistance transmitted?
Immunity genes • Resistance genes that exist in nature to protect antibiotic producing microbes from their own antibiotics Horizontal gene transfer • Transferred immunity genes from antibiotic producers to non-producing microbes
What is a satellite (acellular organism)?
Infectious nucleic acids (DNA or RNA) • Satellite viruses - encode their own capsid proteins when helped by a helper virus (required for replication) • Satellite RNA/DNA do NOT encode their own capsid proteins • Encode one or more gene products • Require helper viruses for replication • E.g. human hepatitis D virus is satellite and requires human hepatitis B virus
Parasitism
symbiosis in which one organism benefits from the other and the host is usually harmed • One is a parasite but there is some co-existance between the host and parasite - most viruses
Mutualism
type of symbiosis where both partners gain from the association and are metabolically dependent on each other. • Zooxanthella - Dinoflagellates are harboured by marine invertebrates - they provide organic carbon to the host • Coral has pigments that protect algae from UV • Ruminants - microbial community have a mutualistic relationship • Cows - acetate, CO2 and H2 are used by methanogenic archaea to generate methane
How do viruses replicate?
Requirements: 1. A living cell 2. A receptor to allow the virus to enter the cell 3. A way to leave the cell Mechanism 1. Attachment - viruses attach to the cell membrane 2. Penetration - by endocytosis or fusion - into a vesicle or fused with the envelope 3. Uncoating - by viral or host enzymes 4. Biosynthesis - production of nucleic acid and proteins - through ribosomes as do not have their own 5. Maturation - nucleic acid and capsid proteins assemble 6. Release - by budding (enveloped viruses) or rupture
How do retroviruses reproduce?
Retroviruses (such as HIV) are different to other viruses • Produce a provirus that integrates into the host DNA and is replicated along with the host DNA • Allows virus to exist indefinitely in the host genome • When coming out of latency the provirus directs replication of new RNA strands as well as capsids and envelope proteins • Convert ssRNA to dsDNA using reverse transcriptase • dsDNA integrates into host cell genome and serves as a template for mRNA synthesis and genome synthesis
Describe the difference between Sanger sequencing and Next generation sequencing
Sanger - chain-termination DNA sequencing - the chippy choppy one • Synthesis of a new strand using the DNA to be sequenced as a template • Mix single strands of DNA with a primer that is specific for the DNA being sequenced • + DNA polymerase I, deoxynucleotides (x4), labelled deoxynucleotide in each tube AT, T, C & G tubes • DNA synthesis occurs; random insertion of the labelled dideoxy-NTP generated DNA fragments of different lengths • Auto - uses 4 different coloured dyes instead of radiolabelled ddNTP • Fragments are separated by electrophoresis and laser beam determines the order Next Generation sequencing • DNA cut into short sequences • Small oligonucleotides called adaptors are added to the ends of the fragments • One end adheres to a solid substrate, the other anneals to a primer that initiates PCR • PCR produces many copies of the fragment and these are sequenced sequentially • Reversible chain termination sequencing is the most frequently used technique
What is epidemiology and why it is used?
Science that evaluates occurrence, determinants, distribution and control of health and disease in a defined human population. Determines • Causative agent • Source/reservoir of disease agent • Mechanism of transmission • Host and environmental factors that facilitate development of disease within a defined population • Best control measures Terminology • Sporadic disease - occurs occasionally and at irregular intervals • Endemic disease - maintains a relatively steady low-level frequency at a moderately regular interval • Hyperendemic diseases - gradually increase in occurrence frequency above endemic level but to epidemic level • Outbreak - Sudden, unexpected occurrence of disease usually focal or in a limited segment of population • Epidemic - sudden increase in frequency above expected number - index case is the first case in an epidemic • Pandemic - increase in disease occurrence within large population over wide region Public health surveillance - protects populations and improves health of communities via education etc
Describe the basic structure of viruses
• All contain a nucleocapsid comprised of nucleic acids (DNA or RNA) and a protein coat (Capsid) • Some consist of only a nucleocapsid, others have additional components. • Envelopes come from the cell the virus has infected
What is a slime mould?
• Also in Supergroup Amoebozoa • At various times have been classified as plants, animals and fungi • Protoplasm creeps along over logs etc • Acellular slime mould - streaming masses of colourful protoplasm • Cellular slime mould - strictly amoeboid (stays in amoeboid shape)
What is a lichen?
• An association between specific fungi (mycobiont) or green algae or cyanobacteria (phycobiont) • Fungus takes nutrients from the phycobiont but protects it form environmental conditions
How do metabolic antagonist antimicrobial drugs function?
• Antimetabolites - antagonise or block metabolic pathways by competitively inhibiting key enzymes • E.g. sulphonamides (analog of PABA - folic acid synthesis), Trimethoprim (combines with sulfa drugs to increase treatment effectiveness) • Sulfa drugs competitively inhibit PABA folic acid synthesis by pathogens • Trimethoprim - interferes with folic acid production - side effects include GI pain and photosensitivity
What makes up a gene?
• Basic unit of genetic information • Most in bacteria encode proteins • Most have a promoter at the start • Promotor binds and correctly orients the RNA polymerase that synthesises the mRNA • Always before the start codon • Leader sequence is the first part of the gene that is transcribed but it is not translated
Explain viral multiplication
Viral Entry and Uncoating Either the viral genome or the entire nucleocapsid can enter the cell after attachment - this varies between viruses and naked/enveloped viruses • Fusion of the viral envelope with host membrane; nucleocapsid enters. • Endocytosis in vesicle; endosome aids in viral uncoating • Injection of nucleic acid Synthesis Stage Some viruses reorganise host cell membranes to form membranous structures that enclose the genetic replication machinery (protection against hosts immune system) • Gene expression and protein synthesis is tightly controlled • Genes are often referred to as early (1 hour), middle (2.5 hours) or late genes (10+ hours) depending on when they are expressed and can activate temporally • Early proteins are often involved with cell control • Middle involved with replication of genome • Late involved with self-assembly or the capsid Assembly • Late proteins are important in assembly • Complicated but varies • Bacteriophages - assembled in stages • Some are assembled in the nucleus • Some in the cytoplasm • May be seen as paracrystalline structures in the bacterial cell Virion Release • Nonenveloped viruses and bacterial viruses often lyse the host cell (lysozyme) • Viral proteins may attack peptidoglycan or the plasma membrane (holin creates holes in E.coli) • Enveloped viruses use budding o Viral proteins are placed onto host membrane o Nucleocapsid may bind to viral proteins o Envelope derived from host cell membrane, but may be Golgi, ER or other membrane
What are bacteriophages?
Viruses that infect bacteria
Predation
relationship in which one organism captures and feeds on another • Attack and kill their prey - Vampirococcus and Myxococcus
Amensalism
relationship in which the product of one organism has a negative effect on another organism. • Antibiotics produced by fungi and their effect on bacteria • Bacteriocin produced by bacteria • Antibacterial peptides produced by insects and mammals • Organic acid production during fermentation
What are Koch's postulates and what do they tell us?
- 4 criterium used to identify causative effect of an illness - The microorganism or other pathogen must be present in all cases of the disease - The pathogen can be isolated from the diseased host and grown in culture - The pathogen from the pure culture must cause the disease when inoculated into a healthy susceptible individual - The pathogen must be re-isolated from the new host and shown to be the same as the original pathogen
How did Carl Woese and Geofre Fox use rRNA to determine that Archaea were their own domain (and not genetically similar to bacteria)?
- Carl Woese sequenced the 16S rRNA gene in prokaryotes and 18S rRNA in eukaryotes. - Identified Archaea to be very different from bacteria and eukaryotes
Why are archaea no longer considered "prokaryotes"?
- Carl Woese sequenced the 16S rRNA gene in prokaryotes and 18S rRNA in eukaryotes. - Identified Archaea to be very different from bacteria and eukaryotes
What archaea features are similar to bacteria
- Contains circular double stranded DNA like bacteria - Contains gas vacuoles - May possess a flagella, fimbriae and pilus like structure
Describe light microscopy
- Different types - bright field, dark-field, phase-contact, fluorescence and confocal. Compound use two lenses. - Light shines through the specimen - Objective lends produces the first image - magnified by the ocular lens to obtain a magnified virtual image.
Broadly speaking, how do bacteria evolve?
- Mutation of genetic material lead to selected traits - Bacteria and Archaea increase genetic pool by horizontal gene transfer within the same generation. - HGT - the acquisition of genetic info by transfer from an organism that is not its parent
Why are archaea able to survive at extremes of temperature/pH?
- Organisms that can survive at extreme temperatures (hyperthermophile) and pH (acidophiles and alkophiles) - Contain DNA, membrane and enzyme modifications that help withstand extremes of temperature and pH - Stabilized membrane proteins - different membrane lipid compositions - ether linkages
Why are some bacteria better adapted than others to survive at higher temps?
- Protein structure is stabilised by - more H bonds, more proline, more chaperones etc - Histone-like proteins stabilise DNA - Membrane stabilised by - more saturated, branched and higher MW lipids, or more ether linkages (archaeal membranes)
How can you best treat (or prevent) viral infections?
1) Route of infection and break the chain • Sexually transmitted - barrier protection • Faecal/oral - wash hands after bathroom/nappies etc • Fomites - PPE • Food borne - cook properly, avoid, recall contaminated food • Airborne - isolation of infected people, face masks are limited 2) Vaccination • Not always the best option • Can be inactivated, attenuated, antigenic fragments, live or killed virus 3) Antiviral medications • Stop nucleic acid replication • Stop virus from entering cells Other: Interferon to stop virus spreading to new cells and Gamma globulin (pre-formed IgG)
Describe the features used to classify viruses.
1. Genome structure 2. Life cycle 3. Morphology 4. Genetic relatedness
Describe what a bacterial operon is.
A bacterial operon is a cluster of genes whose expression is controlled by the presence of a repressor protein that attaches to the operator that is upstream of the genes.
What is a strain?
A genetic variant or sub-type of a microorganism E.g. Bacteria - Escherichia Coli strain EHEC or Clostridium difficile strain NAP1 Viruses - Hepatitis A genotype I, II and III
What is a species?
A group of interbreeding or potentially interbreeding natural populations that is reproductively isolated form other groups. E.g. Clostridium difficile
What is a Shine-Dalgarno sequence?
A leader sequence that precedes the start codon and is used to orient the mRNA correctly on the ribosome.
What is an exotoxin?
A toxin that is released by a living bacterial cell into its surroundings. Can cause damage to a host by destroying cells or disrupting cellular metabolism. Stimulate the production of neutralising AB antitoxins - A for toxic effect ad B for binding to cells Can be secreted or released during lysis of the cell E.g. Clostridium difficile toxins A and B, tetanus
What do aerobes produce to protect themselves from ROS?
Aerobes produce protective enzymes • Superoxide dismutase (SOD) • Catalase • Peroxidase Note: Aerotolerant anaerobes and microaerophiles produce SOD but not catalase
What components make up the chain of infection?
Agent • Pathogens can be acquired form animate sources - humans and animals • Infection passed from animal to human are zoonoses • Pathogens can be acquired from inanimate sources • Reservoir = natural environmental location in which the pathogen normally resides Pathogenicity • A pathogen must contact a host and survive within it to cause a disease • 1) suitable environment • 2) source of nutrients - in competition with eukaryotic host cells and commensal organisms • 3) protection from harmful elements in the environment - virulence factors allow a pathogen to outcompete host cells and existing microbiota and resit their defences Host Susceptibility • Two main factors - defence mechanisms of host and pathogenicity of pathogen • Nutrition, genetic predisposition, and stress also play a role in host susceptibility
Explain the mechanisms behind antimicrobial resistance
An increasing problem as once resistance originates in a population it can be transmitted to other bacterial species • In an abscess or biofilm maybe growing slowly and not susceptible to antibiotics • Resistance mutants can arise spontaneously and are then selected for • Variety of mutations can lead to resistance • Mechanisms • Enzymatic destruction of drug • Prevention of penetration of drug • Alteration of drugs target site • Rapid ejection of the drug • Resistance genes are often on plasmids or transposons that can be transferred between bacteria
Competition
An interaction between two organisms attempting to use the sae resource (nutrient, space etc). One may outcompete the other for the resources or they may coexist at lower levels because they're sharing the limiting resource
What is an opportunistic pathogen?
An organism that is not usually pathogenic and part of the normal body flora but can cause disease when the host is immunocompromised
What are Archaea?
Archaea are diverse cells that do not contain peptidoglycan in their cell wall. They have similar features to both bacteria and eukaryotes but all have circular double stranded DNA. Some are able to live in extreme environments such as above 80 degrees (these are called extremophiles).
How do archaea reproduce?
Asexually by binary or multiple fission, fragmentation, or budding. Mitosis and meiosis do not occur!
How do bacteria reproduce?
Binary Fission where a parent cell divides into two genetically identical daughter cells. 1. Young cell at early stage 2. Parent cell prepares for division by enlarging its cell wall, cell membrane, and overall volume. DNA replication starts 3. Septum begins to grow inwards as chromosomes move towards opposite ends of the cell - other cytoplasmic components are distributed to the two developing cells 4. Septum is synthesized completely through the cell centre, creating two separate cell chambers 5. Daughter cells are divided - some separate completely and others remain attached forming chains, doublets, or other cellular arrangements.
Which eukaryote is responsible for red tides?
Dinoflagellates
What is a plasmid?
Double stranded DNA molecule that can exist and replicate independently of the chromosome. A plasmid is stably inherited but is not required for the host cell's growth and reproduction • Plasmids can be used to clone different genes into bacterial chromosomes - 'cloning vectors'
Describe electron microscopy
Electrons replace light is the illuminating beam . Their wavelength of electron beam is much shorter than light, resulting in much higher resolution - allowing for the detailed study of microbial morphology.
What factors influence the effectiveness of antimicrobial drugs?
Factors influencing antimicrobial drugs • Ability to reach the site of infection • Depends on mode of administration o Oral - may be destroyed by stomach acid o Topical - hard to get appropriate concs o Parenteral - intravenous - maybe excluded by blood clots or necrotic tissues • Susceptibility of pathogen to the drug • Ability of drug to reach concs in the body that exceed minimum inhibitory concentration (MIC) of pathogen o Amount administered o Route of administration o Speed of uptake o Rate of clearance by the body
What may support the growth of obligate anaerobes?
Facultative anaerobes! These can utilise the available oxygen.
Describe the process of translation.
Final step in expression of protein encoding genes where mRNA translated into AA sequence of polypeptide chain. • Start codon is AUG or GUG. • Stop codon: UAA, UAG or UGA • In bacteria this is preceded by a leader sequence (named the Shine-Dalgarno sequence). This helps to orient the mRNA correctly on the ribosome. • mRNA strand is oriented on the ribosome and anticodons on tRNA molecules complement the mRNA strand. • Each codon codes for a single protein= polypeptide chain formed. • Polypeptide chain is translocated from cytoplasm to plasma membrane or periplasmic space with aid of molecular chaperones (proteins such as DnaK) (includes transport proteins, ETC proteins ect) OR secreted from cytoplasm to external environment (includes hydrolytic enzymes for nutrient breakdown)
Explain the process of transformation
Genes are transferred from one bacterium to another in solution- horizontal gene transfer. Uptake of "naked DNA" by a competent cell followed by incorporation of the DNA into the recipient cell's genome.
Understand how sequence analysis is used to determine how "related" different bacterial isolates are.
Genome annotation • Process that located genes in the genome map • Identifies each open reading frame in genome (>100 codons that is not interrupted by a stop) • Binding site at the 5' end and terminator at the 3' end • To see how familiar they are
What is an endotoxin?
Lipopolysaccharide complex associated with the outer membrane of gram negative bacteria. Lipid and polysaccharide molecure composed of an O-antigen, outer core and inner core joined by a covalent bond.
What is a halophile?
Microorganisms that will grow in the presence of NaCl Halophiles • Optimal growth in the presence of NaCl or other salts at a conc > 0.2M Halotolerant • Highest growth rate at low NaCl • Growth at higher rate but rate is slow Moderate halophile • Growth occurs over a range of NaCl concs • Fastest rate is at moderate NaCl conc Extreme halophiles • Require salt conc of 2M and 6.2M • Some cell growth at moderate NaCl but fastest at high concs • Extremely high conc of potassium • Cell wall, proteins, and plasma membranes require high salt to maintain stability and activity Facultative halophiles • Tolerate high osmotic pressure
What is a transposon?
Mobile genetic elements that jump between chromosomes. • Can spread between species • Major way that bacteria are able to rapidly transmit antimicrobial resistance Composite transposons • Contain genes for antibiotic resistance - some have multiple resistance genes • Can move rapidly between plasmids and through bacterial population Gene cassettes • Sets of resistance genes • Can exist as separate genetic elements • Can be part of transposon, integron or chromosome.
What is a virulence factor?
Molecules produced by microorganisms that add to their effectiveness and enable them to become established in a host or maintain a disease state. E.g. the S layer of Clostridium difficile or the capsule of some strains of Klebsiella pneumoniae
How can viruses cause cancer?
Multistep process - mutations in multiple genes May involve oncogenes - cancer causing genes- proto-oncogenes and tumour suppressor genes • Proto-oncogenes and tumour suppressor genes are normal cellular genes • Proto-oncogenes allow cell division to occur - they are expressed if the cell receives an appropriate signal • Tumour suppressor genes provide a checkpoint to cell growth • Viruses can alter the normal regulation of cell growth/differentiation by mutating these genes directly or changing their expression. Mechanisms • Viral proteins bind host cell tumour suppressor proteins (no longer a growth checkpoint) • Virus can carry an oncogene into host cell and insert into genome - stimulates activity of cellular proto-oncogenes and alters cell regulation • Insertion of strong viral genetic elements next to cellular oncogene causing overexpression.
What might have the earliest cells consisted of?
RNA surrounded by liposomes
Give an example of a pathogenicity factor (e.g. haemolysis, coagulase)
Pathogenicity is the ability to cause disease E.g. coagulase enzyme produced by Staphylococcus aureus that enables the conversion of fibrinogen to fibrin.
Cooperation
Positive but not obligatory interaction between to different organisms • Benefits both in the relationship • Differs from mutualism as not obligatory
Describe the basic structures of prions.
PrPSc -abnormal prion proteins - misfolded PrPc prion proteins
Explain how genetic mutations can sometimes be repaired.
Proofreading • Correction of errors in base pairing made during replication • Errors corrected by DNA polymerase Recombination • Repairs DNA with damage in both strands • Involves recombination with an undamaged molecule - in rapidly dividing cells, another copy of chromosome is often available • RecA protein catalyses recombination events
Explain how a bacterial operon functions in controlling the metabolic processes in bacteria.
Some operons are inducible, meaning that they can be turned on by the presence of a particular small molecule. Others are repressible, meaning that they are on by default but can be turned off by a small molecule.
What was the theory of spontaneous generation and how did Louis Pasteur "debunk" it?
Spontaneous generation states that living organisms can originate without descent from similar organisms. He debunked this though his goose neck flask experiment. Flasks with a broken neck or tilted grew bacteria where the ones that were let to sit did not.
How do fungi reproduce? - through hyphae and spores
Spores are important for fungal dissemination and survival. They are used in identification of fungal species. Reproduce from hyphae, spores may break off an reproduce, all parts can reproduce!, not all spores germinate and spores are weaker than bacteria • Gametes are formed • Can be homothallic (self-fertilising) or heterothallic (different but compatible mycelium is required) • Sexual and asexual reproduction for some fungi - remember
What causes genetic mutations?
Stable, heritable changes • Point mutations are the most common • Occur due to alteration of single pairs of nucleotides • May result in addition or deletion of pairs • Larger are less common • Insertions, deletion, inversions, duplication and translocation of nucleotide sequences • Can be spontaneous or induced Spontaneous Mutations • Arise without exposure to external agents • May result from errors in DNA replication - base tautomerisation AC and GT base instead of AT and GC • May also be from mobile genetic elements such as transposons • Can be carried forward Induced Mutations • Caused by agents that directly damage DNA - physical or chemical • Base analogs - structurally similar to normal bases but mistakes occur when they are incorporated into a growing polynucleotide chain o Chemical - 5-bromouracil, 2-Aminopurine • DNA modifying agents - alter a base causing it to mis pair o Physical - UV or x-rays • Intercalating agents - distort DNA to induce single nucleotide pair insertions and deletions o Chemical - proflavine, acridine orange Point Mutations • Silent mutation - change nucleoside sequence of codon - but not the encoded AA • Missense mutation - a single base substitution that changes codon for one AA into codon for another AA • Nonsense mutation - converts a sense codon to a top codon • Frameshift mutation - results from insertion or deletion of one or two base pairs in the coding region of the gene
Commensalism
Symbiosis in which one individual gains from the association (the commensal) and the other is neither harmed nor benefited • Fermenting bacteria promote rapid growth of acid tolerant species • Biofilm formation - initial coloniser helps others attach
What is a Genus
Taxonomic category that ranks above species and below family. E.g. Clostridium
Describe the differences between lysogenic and lytic growth cycles of bacteriophages.
Temperate phages have two phases to their life cycles. The lysogenic cycle allows the genome of the virus to be replicated passively as the host cell's genome is replicated. Certain environmental factors such as UV light can cause a switch from the lysogenic cycle to the lytic cycle. In the lytic cycle, new virus particles are made and released when the host cell lyses. Virulent phages are limited to just the lytic cycle! • Lysogenic cycle- Virus (bacteriophage) attaches itself to the host cell and injects DNA and proteins. Phage DNA circularises inside the cell. The phage DNA then integrates into the host's chromosome, becoming a prophage. Host cell then replicates normally, replicating the viral DNA along with it. Occasionally the prophage may excise itself from the host chromosome- initiating a lytic cycle. • Lytic Cycle- Virus (bacteriophage) attaches itself to the host cell (bacteria) and injects DNA and proteins into the cell. Phage DNA circularises. New phage DNA and proteins are made using the hosts organelles and ATP and are assembled into virions. The virions lyse the cell and are released.
What was Leeuwenhoek's important contribution to microbiology?
The first to identify microorganisms using a microscope - identified protozoa and improved the microscope
Define mortality rate
The rate of deaths due to a disease in a population
Define morbidity rate
The rate of disease in a population
Define prevalence rate
The rate of new cases of the disease in a population
Explain why the term prokaryote is somewhat controversial
The root of the 'tree of life' is present on the bacterial branch, suggesting that bacteria and archaea are not linked phylogenetically • Thought that the Archaea and Eukarya evolved independently to Bacteria • Suggested by Carl Woese in the 1970's on the basis of rRNA and confirmed by analyses of SSU rRNA
Describe why it is important to know what normal flora (bacterial species) are expected to be present at a particular site.
To determine whether the bacteria present may be causing disease. • Normal microbiota or microflora are microbes regularly found at an anatomical site • Relationship begins at birth • Normal flora affected by immunity, genetics, general health and environment • Usually mutually beneficial o Normal microbiota often prevent colonisation by pathogens (C. Diff) o Bacterial products can be beneficial (K produced by E. Coli) • Opportunistic pathogens o Members of normal microbiota that produce disease under certain circumstances • Compromised host o Debilitated host with lowered resistance to infection
How do cell wall synthesis inhibiting antimicrobial drugs function?
• Blocks enzyme catalysing peptidoglycan cross-link formation • Prevents synthesis of complete cell walls leading to cell lysis • Acts only on growing bacteria that are synthesizing new peptidoglycan • E.g. Penicillin V & G, semisynthetic penicillins (broader spectrum than natural), Vancomycin, cephalosporins and bacitracin • Penicillin resistant organisms produce beta-lactamase which hydrolyses beta lactam ring of penicillin • Vancomycin important for treatment of antibiotic-resistant staph and enterococcal infections
Cytoskeleton - what is its role? Why are they different to bacteria and archaea?
• Composed of actin filaments, intermediate filaments and microtubules • Provides cell structure and movements
Describe the functional differences between DNA and RNA.
• DNA is double stranded polymerised nucleotides - A, T, G and C • RNA is single stranded polymerised nucleotides - A, U , G and C • DNA replicates and stores genetic information - blueprint for all genetic information within an organism • RNA converts DNA genetic information to a format used to build proteins and moves to ribosomes • DNA is found in the nucleus • RNA is found in the nucleolus and moves to specialised regions of the cytoplasm
How do bacteria replicate their genome?
• DNA is replicated and segregated prior to division • Most chromosomes are circular • Single origin of replication - site at which replication begins • Terminus - site at which replication is terminated, located opposite to the origin • Replisome - group of proteins needed for DNA synthesis • Bidirectional replication - DNA replication proceeds in both directions from the origin • Origins move to opposite ends of the cell
Sterilization - How does it work? Provide an example, the advantages and disadvantages and when it would be used.
• Destruction or removal of all viable organisms including living cells, spores and acellular (viruses) entities. • Example: autoclaving, UV radiation, dry heat sterilization • Advantages: Inactivate bacteria and most bacterial spores • When used: To sterilize laboratory equipment
Explain the differences in replication between bacteria, eukaryotes and archaea.
• Different chromosome structure and replication machinery • Multiple chromosomes (eukaryotes) are much larger and linear (rather than a closed circle) • Eukaryotic DNA is wound around histones • Archaea also have histone-like proteins Eukaryotic transcription • Occurs in nucleus • RNA products must move to cytoplasm • Most eukaryotes have genes with their own promotor and the transcript produces one protein • Genes are split or interrupted - exons (coding regions) and introns (RNA that isn't translated). Differences in promotors • Region of DNA at the start of a gene that RNA polymerase binds to before beginning transcription. • Eukaryotic and archaea promoters share sequences • Eukaryotes and archaea also both use transcription factors that bind to the DNA and line up the polymerase correctly.
Mitochondria - what is its role? Why are they different to bacteria and archaea?
• Energy production through use of CAC • Electron transport • Oxidative phosphorylation • Membrane bound - bacteria and archaea do not have membrane bound organelles
Flagella and cilia - what is its role? Why are they different to bacteria and archaea?
• External structures present in some eukaryotic cells • Cilia are shorter (5-20µM) than flagella (100-200µM) • Move in a wavelike fashion, not rotating like in bacteria • Membrane bound cylinder structure - basal body in the cytoplasm at the base of each structure
What is a water mould?
• Grow on the surface of fresh water and moist soils • Feed on decaying vegetation • Phytophthera infestans - caused the great potato famine • Phytophthera cinnamomi - causes jarrah dieback
How does osmotic pressure impact bacteria?
• High osmotic pressure causes plasmolysis of cell membrane • Cell growth is inhibited as plasma membrane pulls away from the cell wall • Adding salt or other solutes to solution increases osmotic pressure (preserved foods) • Salting foods and sweetening condensed milk draws water out of microbes stopping growth.
What happens when a bacterium is put into hypotonic and hypertonic solutions?
• Hypotonic solution - lower osmotic conc • Water enters the cell and cell swells and may burst • Hypertonic solution - higher osmotic conc • Water leaves the cell, membrane shrinks from cell wall - plasmolysis may occur • The water will go to where the concentration is the highest
Describe the basic structures of viroids.
• Infectious agents composed of closed, circular ssRNAs RNA does not encode gene products • Replication requires host cell NA-dependent RNA polymerase - viroid used as the template for synthesis of new viroid RNA. • Pospivirodae - intrastrand base pairining with single stranded loops and replicates in the nucleus • Avsunviroids - stem loop structure of intramolecular base pairing. Replicate in the plastids • Can produce latent infection or cause severe disease • Can be caused by using RNA silencing - a process that usually protects the eukaryote from infection with RNA viruses
How do nucleic acid synthesis inhibiting antimicrobial drugs function?
• Inhibits nucleic acid replication and transcription • Not as selectively toxic (bacteria and eukaryotes don't differ in nucleic acid synthesis) • E.g. Quinolones and rifampin
Lysozomes - what is its role? Why are they different to bacteria and archaea?
• Intracellular digestion - hydrolases to digest molecules and pathogens • Only found in animal cells • Combine with a phagosome to make a phagolysosome
What are protists?
• Lack a common evolutionary heritage and do not have the same level of tissue organisation as plants, fungi or animals - are an artificial grouping. • Includes algae (photosynthetic protists) and protozoa (chemoorganotrophic protists) • Asexual replication - usually by binary fission BUT can also reproduce sexually (leads to a diverse nuclei) • Protist undergoes mitosis then cytoplasm divides by cytokinesis
What is the typical growth curve for bacteria?
• Lag phase • Log phase • Stationary phase • Death phase • Long term stationary phase - the up and down bit
What are the three main capsid shapes?
• Large macromolecular structures that serve as a protein coat for the virus • Protects viral genetic material and aids in its transfer between host cells • Made of protein subunits - protomers that aggregate to form capsomeres • Capsid shapes are described as helical, icosahedral or complex Helical Capsids • Shaped like hollow tubes with protein walls that enclose the nucleic acid • Promoters self-assemble - often forms a rigid tube • Size of capsid depends on the length of the nucleic acid Icosahedral Capsids • An icosahedron is a regular polyhedron with 20 equilateral faces and 12 vertices • Efficient way to enclose a space • Capsomers - ring or knob shaped units of 5/6 protomers. Pentamers - 5 subunit capsomers hexamers - 6 subunit capsomers Complex capsid • Some viruses do not fit into the category of having helical or icosahedral capsids e.g. T4 bacteriophages
Smooth endoplasmic reticulum - what is its role? Why are they different to bacteria and archaea?
• Lipid synthesis
How do protein synthesis inhibiting antimicrobial drugs function?
• Many antibiotics bind to bacterial ribosome (either subunit), others inhibit other steps in protein synthesis. • E.g. Tetracyclines and Macrolide (erythromycin), chloramphenicol and streptomycin • Tetracyclines - Broad spectrum bacteriostatic sometimes used to treat acne- combines with 30S ribosomal subunit inhibiting binding of aminoacyl-tRNA molecules to the A site of the ribosome • Erythromycin - broad spectrum bacteriostatic - binds to 50S ribosomal subunit inhibiting peptide chain elongation - used for patients who are allergic to penicillin
Describe viral envelopes
• Many viruses are bound by an outer flexible membranous envelope layer • Animal virus envelopes (lipids and carbohydrates) usually arise from hot cell plasma or nuclear membranes • Make the virus susceptible to certain disinfectants Peplomers • Envelope proteins (encoded by the virus) that project from the envelope surface as spikes • Involved in viral attachment to host cell (hemagglutinin of influenza virus) • Used for identification of the virus • May have enzymatic or other activity (neuraminidase of influenza virus) • May play a role in nucleic acid replication
What is a dinoflagellate?
• Most commonly found in marine plankton - some responsible for phosphorescence and toxic red tides • Two flagellae - whirling propulsion • Some are endosymbionts - photosynthetic reef-building coral • Form motile cells called zooxanthellae - 3 distinct habitats. • 1. Mucous layer at the surface • 2. The gastrodermal cavity • 3. Coral skeleton
Understand how you can use BLAST (basic local alignment search tool) to assign unknown gene sequences.
• ORFS are presumed to encode protein • BLAST (basic local alignment search tool) - base by base comparison of 2+ gene sequences and assign tentative function of gene or protein structure • Proteins that don't align with known AA sequences fall into two categories o Conserved hypothetical proteins that match known sequences in databases but don't yet have an assigned function o Proteins of unknown function are products of genes unique to that organism (but may match other organisms genes as more genomes are sequenced in full) • Full gene annotation allows for construction of a physical map of the entire genome - may be colour coded
Chloroplasts - what is its role? Why are they different to bacteria and archaea?
• Organelle called a plastid - present in photosynthetic protists and plants • Photosynthesis - trapping light energy and forming CHO from CO2 and H2O • Membrane bound - bacteria and archaea do not have membrane bound organelles
Golgi apparatus - what is its role? Why are they different to bacteria and archaea?
• Packing and secretion of materials for various purposes • Lysosome formation
Describe the process of transcription.
• Polycistronic mRNA is often found in bacteria and archaea • Contains directions for >1 polypeptide catalysed by a single RNA polymerase • After binding, RNA polymerase unwinds the DNA • Transcription bubble is produced - moves with polymerase as it transcribes mRNA from template strand • Within the bubble a temporary RNA:DNA hybrid is formed • Termination occurs when core RNA polymerase dissociates from template DNA • DNA sequences mark the end of gene in the trailer and the terminator - lots of AAAAA • Some terminators require the aid of the rho factor for termination • Transcription pauses when the RNA polymerase reaches a rho-dependent pause site. Trailing rho protein catches up and dissociates the RNA polymerase from the DNA
What factors influence the effectiveness of sterilization and disinfection?
• Population size - larger populations take longer to kill • Population composition - different sensitivities • Agent concentration - not a linear relationship, increase in conc. kills rapidly • Exposure duration - longer direction, more killings • Temperature - higher temperatures, more killings • Local environment - pH, viscosity, conc. of matter (biofilms less susceptible to agents)
How do prions reproduce?
• PrPc (prion protein) is present in normal form (abnormal is PrPSc) • PrPSc causes PrPc to change confirmation to abnormal form • Newly produced PrPSc molecules convert more normal molecules to the abnormal form through unknown mechanism Neural Loss • PrPc and PrPSc interaction causes PrPc to crosslink and trigger apoptosis • PrPc conversion causes neuron loss - PrPSc is the infectious agent • All prion caused diseases have no effective treatment and result in progressive degeneration of the brain and eventual death.
Rough endoplasmic reticulum - what is its role? Why are they different to bacteria and archaea?
• Protein synthesis
Ribosome - what is its role? Why are they different to bacteria and archaea?
• Protein synthesis • Sedimentation co-efficient of 80S (70S is bacteria) 60S and 40S subunits • Molecular chaperones aid the proper folding of proteins after synthesis and assist with the transportation of proteins into eukaryotic organelles
Sanitization - How does it work? Provide an example, the advantages and disadvantages and when it would be used
• Reduction of microbial population to levels deemed safe (based on PH standard) • Example: Heat or chemical sanititizing • When used: In a laboratory, bleach to kill bacteria on equipment.
Nucleus - what is its role? Why are they different to bacteria and archaea?
• Repository for genetic information • Stores cells genetic info and chromosomes • Chromosomes are made from chromatin - complex of DNA and protein (inc histones). Also change and move all of the time (dynamic) • The histone bead is called a nucleosome that allows DNA compaction to occur - some archaea have histones • Surrounded by nuclear envelope that has pores - continuous with ER at some points • Nucleolus is prominent - not membrane enclosed and plays a major role in ribosome synthesis • Have paired chromosomes in a nuclear membrane - bacteria and archaea do not have membrane bound stuff
Describe the difference between rooted and unrooted phylogenetic trees.
• Show inferred evolutionary relationships in the form of multiple branching lineages connected by nodes • Identified sequences at tip of branches - the operational taxonomic unit • Nodes represent a divergence event • Length of brand represents number of molecular changes between two nodes • Rooted - has node that serves as common ancestor • Unrooted - represents a phylogenetic relationship but does not provide an evolutionary path Rooted Phylogenetic Trees • Unique node known as the 'root' that is the reference to the most recent common ancestor • Sometimes the edge lengths can be interpreted as time estimates • Each node is a taxonomic unit • Gives evolutionary information Unrooted Phylogenetic Trees • Show how the different leaf nodes are related • Do not give any evolutionary information
What are amoeba?
• Single cell eukaryote that moves through the use of pseudopodia
How are 16SrRNA sequences used to characterize microbial populations?
• Small subunit rRNA (SSU rRNAs) o Sequences of 16S and 18S rRNA the most powerful and direct method of inferring microbial phylogenies and making taxonomic assignments at the genus level • Comparative Analysis of 16S rRNA sequences o Oligonucleotide signature sequences found o Short conserved sequences specific for a phylogenetically defined group of organisms o Either complete or specific rRNA fragments can be compared o When comparing their relatedness is represented by a % sequence homology - 70% is the cut off for species definition
Antisepsis - How does it work? Provide an example, the advantages and disadvantages and when it would be used
• The prevention of infection of living tissue by microorganisms • Example: Alcohol or chlorohexidine, antiseptics and chemical agents that inhibit the growth of micro's when it is applied to tissues • Advantages: Kills and inhibits growth by microorganisms • Disadvantages: Alcohols may be slow acting, may leave a residue, may cause irritation or staining • When used: In medical procedures
Explain the process of transduction
• The transfer of bacterial genes by viruses. • Viruses (bacteriophages) can carry out the lytic cycle (host cell is destroyed) or viral DNA integrates into the host genome (becoming a latent prophage) • Generalised transduction- any part of bacterial genome can be transferred, occurs during lytic cycle of virulent phage. During viral assembly, fragments of host DNA mistakenly packaged into phage head. • Specialised transduction- Carried out only by temperate phages that have established lysogeny. Only a specific portion of bacterial genome is transferred. Occurs when prophage is incorrectly excised.
Endoplasmic reticulum - what is its role? Why are they different to bacteria and archaea?
• Transport of minerals
How do bacteria respond to changes in pH
• Try to maintain neutral cytoplasmic pH • Some exchange potassium for protons using an antiport system. • Some exchange sodium for protons • Some synthesize new proteins (acid shock and heat shock proteins and help refold denatured proteins)
Describe a yeast
• Unicellular fungus • Non-filamentous (spherical or oval shaped) • Widely distributed in nature - white powdery coating on leaves and in the air • Fission yeasts divide symmetrically • Budding yeasts divide asymmetrically as the bud on the end does not divide • Reproduce by budding o Parent cell forms a protuberance (bud) on the outer surface o Cell nucleus divides and 1 migrates into bud - cell wall forms and bud moves away o 1 parent yeast can produce 2-4 daughter cells • Pseudohyphae are formed when they fail to break from parent call (forms long chains) - pathogenicity
What influences the ability of viruses to infect different types of cells?
• Virus must interact with a host cell receptor • If receptor is present on the cells of numerous animals, the virus will be able to infect multiple species (rabies) • Some viruses have very specific hosts - particular bacterial strain within a species • Some viruses only infect specific cells within a complex eukaryote - e.g. respiratory cells infected by cold virus • No receptors have been identified for plant viruses - usually damage of the host cells allow virions to be transferred between plants.
Why does UV kill bacteria?
• Wavelength most effectively absorbed by DNA is 260nm • Must be directly exposed to microbial surface • Mutations cause death - DNA damage can be repaired • Causes thymine dimers to form in DNA
How does ionising radiation impact microbes?
• X-rays and gamma rays • Mutations = death (sterilisation) • Disrupts chemical structures including DNA - if small dose, damage may be repaired • Deinococcus radiodurans - extremely resistant to DNA damage
Disinfection - How does it work? Provide an example, the advantages and disadvantages and when it would be used.
• the killing, inhibition or removal of disease causing (pathogenic) organisms. • Example: 70 % ethanol • Disadvantages: Some spores and microorganisms may remain • When used: To disinfect the benchtop in a laboratory