Microbiology Final Exam

Lysis favored when

-Many hosts -Threatened host

Group VI: RNA reverse-transcribining; retroviruses

(+) strand RNA Package reverse transcriptase instead of RNAP RNA → DNA

Name two types of virulence factors/strategies that are used by pathogens for colonization of the host cells?

Flagella/chemotaxis; Pili/fimbriae; Adhesins

Carbs

Food composition Mold predominates Degrades food by hydrolysis, tomatoes in particular Little odor Ergotism - ergot alkaloids create vasoconstriction

In the specific case of vancomycin resistance in Enterococcus faecalis, whole genome sequencing evidence suggested that resistance in this pathogen arose from which mechanism?

HGT

The increase in emerging infectious diseases of all kinds observed in the 1980s correlates with the _______ epidemic

HIV/AIDS

Fertilized crop soil

High nutrients → lysis

Type IV Secretion System

Homologous to conjugation Similar to conjugation pilus Modified to secrete proteins only or proteins + DNA Can secrete protein from cytoplasm or periplasm

Phage Lambda - Model Lysogeny System

ds-DNA phage Infects E. coli K12 Genome: -Early promoters & operators: OL, PL, PRM, OR, PR, PRE -Late promoters (for lytic cycle): PAQ, PR

Influenza

ss-RNA (-) sense Segmented genome (each protein present separately)

Antiviral targets

1) Antibodies (vaccines) - block cell entry 2) Amantidines - block uncoating 3) HAART therapy (HIV) - blocks mRNA synthesis 4) Reverse transcriptase inhibitors - blocks mRNA synthesis 5) Proteases - degrade viral proteins 6) Neuraminidase inhibitors i.e. Tamiflu - blocks deparature check this

Describe the difference between a viral capsid and a viral envelope. How are these two things related to virions?

A capsid refers to the protein shell that surrounds a virion's nucleic acid. The viral envelope is membrane-like structure derived from the host cell's membrane. A virion is a non-cellular particle that infects a host cell, which can have a RNA or a DNA genome encased in a proteinaceous capsid. Virions can be enveloped (in the case of influenza) or not enveloped (in the case of phage lambda).

Type III Secretion System

Homologous to flagellar synthesis Triggered by cell-to-cell contact b/w host & bacteria Creation of pore complex in host cell membrane Effector proteins (13 different) transported to base of needle by chaperones ATPase unfolds effector proteins to transport through needle Ex: Salmonella pathogenesis -Actin rearrangement -Affect activity of host proteins that trigger apoptosis

Nosocomial Infections

Hospital-acquired infections, 10% hospital patients get Often caused by bacteria that are members of normal microbiota Many resistant hospital strains

Group II: ss-DNA virus

Host DNAP → complementary DNA strand ds-DNA transcribed by host RNAP

Sialic acid

Host cell receptor, attached to galactose Hemagglutinin binds to this

Lysogeny Logic for Bacteria

Immunity from other viruses Lysogenic conversion - can alter surface character of host (change phenotype) - Ex: Salmonella phage epsilon changes enzymes making lipopolysaccharide - Ex: Diptheria: B-phage → viral toxin kills RBC when [Fe] ↓

Why would the U.S. and Europe have high incidence of Multidrug-resistant tuberculosis? Why would Europe have MERS and the U.S. have West Nile disease outbreaks?

In the case of multi-drug resistant tuberculosis, the development of these drug-resistant strains has occurred in the U.S. and Europe because of 1) the widespread use of antibiotics and 2) noncompliance of patients in taking full courses of antibiotic treatment. MERS-CoV (Middle East Respiratory Syndrome Coronavirus) emerged in 2012. It was first reported in Saudi Arabia in 2012 and has since spread to several other countries, including European countries, largely due to international travel. So far, all cases of MERS have been linked through travel to, or residence in, countries in and near the Arabian Peninsula. The largest known outbreak of MERS outside the Arabian Peninsula occurred in the Republic of Korea in 2015. The outbreak was associated with a traveler returning from the Arabian Peninsula. By contrast, West Nile virus has appeared in the U.S. in part, we think, because of climate change. West Nile virus is transmitted between humans and animals by mosquitoes. It is endemic to warmer environments, such as the Mediterranean and North Africa-hence the name "West Nile virus." Why the sudden appearance of West Nile virus in the United States? The answer is not known for certain, but some climatologists argue that West Nile and other emerging pathogens arise from unusual weather patterns related to global climate change. In 1999, the unusually warm winter enabled a few mosquitoes carrying the virus to survive until the spring. The spring that followed was drier than usual, forcing birds to spend more time at dwindling water pools, where mosquitoes were concentrated. The July heat wave then increased the rate of viral replication in the mosquitoes. As the cycle continued, mosquitoes re-infected birds, which then infected more mosquitoes. Eventually, the cycle included mosquitoes infecting humans.

Lysogeny to Lysis Induction

Lysis = response to environmental factors that threaten the host (ex: UV) DNA damage changes RecA → CI cleavage → CI levels decrease → cI gene transcription decreases → Cro protein increases (OR available) → Lysis

Group III: ds-RNA

Need RNA-dependent RNAP to make RNAP by transcribing directly from RNA genome Requires RNAdRNAP to make mRNA & polymerase has to be packed as part of genome for process to be successful Ex: Rotavirus

The antiviral drug Tamiflu inhibits the protein Neuraminidase. Which step in viral replication would Tamiflu inihibit?

Viral departure from host cell

Hemagglutinin

Viral protein, targets virus to host cell to bind to sialic acid Trimer Projects away from cell, makes protein spikes that come out of virion which recognize sialic acid Interaction of sialic acid & HA triggers host uptake of cel

General ecologic role of viruses

Virus can promote species diversity Selective killing limits dominant species which allows competitive species to persist

Phage

Virus that infects bacteria (bacteriophage)

Describe the molecular biological detail behind why the lytic phase is triggered by UV light.

DNA damage alters RecA protein levels, which results in CI protein cleavage. As CI protein levels drops, c1 gene transcription also decreases. As the CI protein levels drop, Cro proteins levels rise (OR available), leading to lysis.

Target of quinolones

DNA synthesis

Metronidazole

DNA synthesis Prodrug - harmless until activated Metabolized by cofactors ferredioxin & flavodoxin

West Nile

+ ssRNA Flavivirus family (dengue/yellow fever) Endemic where it is warm 1999 NY Outbreak

Tetracycline

Affects 30S 4 fused cyclic rings Block binding tRNAs⁺ to A site -Distorts A site, prevents incoming tRNA from binding, stops elongation Doxycycline - can interefere with bone development

Aminoglycosides

Affects 30S Cyclohexane ring & amino sugars Cause translational misreading of mRNA Unusual in that it's bactericidial Includes streptomycin, gentamycin

Rifampin

RNA synthesis Binds B subunit of RNAP Prevents elongation step transcription

Target of macrolides

RNA synthesis (50S subunit) --check this

Capsid

protein shell that surrounds virion's nucleic acid

Influenza Characteristics

3 main: A, B, C (different aa sequence in envelope proteins) Sialic acid receptor → attacks multiple types of human cells Fast evolving RNA genome → constant ∆s in envelope proteins

Positive (+) stranded RNA from viruses such as Polio can be directly translated into mRNA once the virus enters the host cell. However, an additional enzyme is required in order to generate more genetic material for viral replication by this group of viruses. This enzyme is:

RNA replicase

Actinomycin D

RNA synthesis Prevents initiation step Binds to DNA from any source (not selectively toxic)

Chloramphenicol

RNA synthesis (50S synthesis?) -- check this

Protein synthesis antibiotics

i.e. Tetracycline - bacteriostatic Difference b/w prokaryotic & eukaryotic ribosomes = selective toxicity Most antibacterial antibiotics work by binding & interfering w/ bacterial rRNA function Generally bacteriostatic Can affect 30S or 50S

Lysogeny Evolutionary Advantages for Phage

Low host abundance - virus can keep reproducing when there are many more phages in environment than host - Multiple of same phage can infect 1 host Nutrient deprivation - virus stays within dormant host during

Newborn gut

Low host cell density → Lysogeny

Temperate Bacteriophages

Lytic infection - replication immediately, kills host (T4) Lysogenic infection - phage remains within host cell as prophage - Inserts genome into host chromosome

Drugs that affect 50S

Macrolides Chloramphenicol

Cro

Lysis

CIII protects...

CII

2 types of epidemics

Common source Propagated

CI

Lysogeny

Microevolutionary change

Patient mutation may occur in basepair

"Brewer's yeast" refers to this industrial strain used in beer and wine production:

A. Saccharomyces cerevisiae

Parts of Pathogenicity Systems

ATSE 1. Attachment 2. Toxins 3. Secretion Systems 4. Extracellular immune avoidance

Ciprofloxacin (fluoroquinolone)

DNA synthesis Binds DNA gyrase, causes complex to block DNA replication

Lipid rich biofuels can be produced in photobioreactors from this type of microbe:

Microalgae

Microbial food generation

Microalgae: photobioreactor Chorella pyrenoidosa

DNA synthesis antibiotics

Quinolones (bactericidal)

Lysogeny favored when

-Few hosts -Strong hosts

2 purposes of viral replication for genetic material

1) Template to make proteins for virions 2) Template to make new genetic material for virions

Attachment: Adhesin

Any microbial factor that promotes attachment For viruses - capsid/envelope proteins For bacteria - varied strategies bind specific host cell receptors

antibiotic that stops cell growth and reproduction falls into this category.

Bacteriostatic

Ethanolic fermentation

Beer/wine

Chloramphenicol

Blocks peptidyl transferase

Saccharomyces cerevisiae

Brewer's yeast - production of alcoholic beverages

Homolactic acid fermentation

Buttermilk

Baltimore scheme

Classifies viruses based on: DNA/RNA ss or ds Linear or circular genomic material Whole or segmented

Diphtheria

Corynebaacterium diphtheriae - Lives in pharynx, makes toxin → diphtheria - blocks eukaryotic protein synthesis Only the toxin causes symptoms Gene encoding toxin: in genome of bacteriophage that lysogenizes C. dip (B phage)

Outcome: Lysis

Cro binds OR → blocks PRM PRM → CI (Cro blocks CI production) ↓ Lysis

Pseudomonas putida

Efficiently degrades nicotine Example of biodegradation/remediation Bacterial catabolism of nicotine

Index case

First case of epidemic

Activation by CAP

Helix-turn-helix-domain

: Bacterial capsules are used by pathogens during this phase of infection:

Immune evasion

Macrolides

Inhibit translocation Erythromycin Azithromycin

John Snow is the considered the father of epidemiology. Describe Snow's contribution to the field of epidemiology, in relation to the cholera epidemic of the 1850s in London.

John Snow developed the first known statistically rigorous, geographical analysis of disease outbreak. This happened during the serious outbreak of Cholera in London in 1854. Snow visited addresses or diarrheal cases and conducted first geographical analysis of disease cases, allowing him to find the source of the disease. Water in London was pumped from separate wells located in different neighborhoods. There was a close association between the density of cholera cases and a single well located on Broad Street. This was John Snow's great contribution to epidemiology - one of the first formal and extensive biogeography studies of pathogenesis. Vibrio cholerae was not recognized until 1905, 50 years later.

Heterolactic acid fermentation

Keffir

Low host cell density

Many lambda phage infects same cell ↓ Favors production of CII & CIII ↓ CII activates CI which leads to lysogeny

Envelope

Membrane-like structure derived from host cell's membrane

Food spoilage

Microbial changes that render a product obviously unfit or unpalatable for consumption

MERS-CoV

Middle Eastern Respiratory Syndrome - Coronavirus Emerged 2012; 1st reported in Saudi Arabia

Acidic fermentation of dairy products

Milk fermentation starts by lactic acid fermentation w/ Lactobacilus & Streptococcus Followed by rennet proteolysis (by chymosin & pepsin) - renders casein insoluble Cleaved peptides coagulate into semisolid curd -Separated from liquid protein whey

Mycoplasma mycoides genomics

Mycoplasma mycoides genome → yeast → genomic modification → M. capricolum Example of synthetic microbe

A brand new antibiotic is discovered from an unculturable marine bacterium. It is found to target Gram positive aerobic bacteria exclusively. Which best describes the where this drug falls on the antibiotic activity spectrum?

Narrow

Alkaline fermentation

Natto: whole soybeans

Virion

Non-cellular particle that infects host cell

Anticipation

Phenotype gets worse/progresses through generations Ex: trinucleotide repeat disorders i.e. Huntington's CAGCAGCAG More copies, if you have certain # repeats, you have disease, # repeats can grow → worse Replication error - more likely to occur when go through male germline

List three general factors that contribute to the emergence of infectious diseases in the world today.

Population density Agricultural practices (clearing of land, changing wildlife habitats) Land usage (also changing wildlife habitats) Hunting practices (eg bushmeat) Other practices (domestication of animals as pets)

Macroevolutionary change

Rearrangement of large segments of DNA in single event

Extracellular Immune Avoidance

Secrete thick capsule: Streptococcus pneumoniae, Neisseria meningitidis Make proteins to bind Abs -S. aureus cell wall protein A --Binds Fc fragment --Abs attach "upside down" --Prevents opsonization (macrophage response) -Cause apoptosis of phagocytes -Alter suface antigens

Once inside phagosome, pathogen might use hemolysin to break out

Shigella dysenteriae, Listeria monocytogenes Break out of phagosome, move throughout cytoplasm into adjacent cells by forming actin tails

Primary pathogen example

Shigella flexneri

Antigenic drift

Small ∆s envelope proteins

Selective toxicity

Specific for targets w/ few side effects Ex: Penicillin

How does seafood spoil

Spoils fast because unsaturated FA rapidly oxidize Psychotrophic bacteria - reduce trimethylamine oxidase to fishy-smelling trimethylamine

Roundup-ready plants that are resistant to glyphospate were engineered using what system from A. tumefaciens?

The Ti plant gene transfer vector

Clostridium botulinum

Toxin inhibits fusion of synpatic vesicles → prevents activation of muscle cells → flaccid paralysis

PR

Transcribes Cro

Zoonotic diseases

Transmitted from animals to humans Many zoonoses due to practices like international travel

Type IV Pili

Twitching & gliding motility Continually assemble & disassemble Grow from inner membrane SecA independent In broad-spectrum of Gram negative bacteria Pilin protein: PilA PilC1 & YI form attachment tip Assembly/disassembly requires hydrolysis of NTP

Taq polymerase

Thermus aquaticus For bioprospecting

Diphtheria Toxin Iron Response

Toxin genes (dtx) controlled by repressor (DtxR) → binds Fe Fe ↑ (as in body) → not enough Fe to activate DtxR → dtx gene expressed → Toxin made → target cells killed → iron released for bacterial use `

Types of Secretion Systems

Type II Type III Type IV Type VI

Question 2: Which type of secretion system bears the most evolutionary similarity to the bacterial conjugation apparatus?

Type IV

Malolactic fermentation

Uerococcus oeni bacteria

What is the role of neuraminidase in virus replication within a host?

When the new virus particles (virions) are ready to exit their current host cell and infect new host cells, there is one final problem. The hemaglutinin protein spikes that stick the virus to the cell in the first place still want to bind to the sialic acid resides on the mammalian cell surface. If they do, the virus would get tethered to the cell it is trying to leave. This is where the other envelope protein comes in. Neuraminidase cleaves the bond attaching the sialic acid residues to the rest of the protein embedded in the host cell. This helps to release the virus from the cell. So the virus is now free to infect a fresh cell and repeat its replication cycle

How did air travel contribute to the emergence of Zika virus?

Zika virus has spread all over the world due in large part to increased air travel. From late 2012, we find an increase in the number of travelers arriving in Brazil from countries with reported Zika infections, including French Polynesia. This increase in visitors to Brazil from ZIKV-affected countries coincides with the period during which Zika is estimated to have entered the Americas. The time that Zika is estimated to have entered into the Americas is determined using a phylogenetic analysis, where the American-type Zika (clade B) is observed to have evolved from the French Polynesian-type Zika (clade A) around 2013 (based on estimated rates of viral evolution). If the Zika epidemic in Brazil did indeed arise from a single introduction, then the virus must have circulated in the country for at least 12 months prior to the first case being reported in May 2015. There are two published hypotheses for how ZIKV came to be introduced into Brazil, during (i) the 2014 World Cup soccer tournament or (ii) the Va'a sprint event held in Rio de Janeiro during 2014. Alternatively, introduction could have occurred during (iii) the 2013 Confederations Cup soccer tournament. The data we have at hand suggest that the introduction of Zika to the Americas predated events (i) and (ii). Although the phylogeny dates are more consistent with the Confederations cup, that event ended before ZIKV cases were first reported in French Polynesia. It seems that the best predictor of clade B Zika emergence in Brazil is total international travel from all Zika-infected countries to Brazil across 2013-2014.

Tularemia (Francisella tularensis), Lyme Disease (Borrelia burgdorferi), Bird flu (avian influenza), and Ebola are all examples of _______, diseases that can be transferred from animal to human hosts.

Zoonotic diseases

Chemical means of preserving food include all of the following EXCEPT: a. addition of copper b. use of sulfates c. use of organic acids, such as sorbic acid, as preservatives d. use of nitrites

a. addition of copper

What are the key takeaways from the story of gene regulation of the Francisella Pathogenicity Island?

1) The virulence of pathogens is highly regulated at the level of gene expression (and in the case of the FPI, at the transcriptional level). Common regulatory themes, such as basic transcriptional repression and activation, are used to control the expression of genes on these pathogenicity islands. While we talked about a specific regulator (PigR) in a specific system (Francisella), the same types of transcription factors (homologues) can be found in a wide range of bacterial pathogens. 2) Pathogenicity islands commonly contain genes that encode for many of the important virulence factors we have talked about: exotoxins/endotoxins; secretion systems; adhesins. 3) To study virulence factors and their mechanisms of action requires a combination of scientific approaches including biochemical approaches, molecular genetic approaches, in vivo gene expression studies, and genomics methods.

5 Steps of Influenza Viral Replication Cycle

1. Attachment 2. Entry & uncoating 3. Replication 4. Assembly of new virions 5. Exit from host & transmission

Mechanisms of Antibiotic Resistance

1. Modify target so no longer antibiotic (vancomycin) 2. Pump antibiotic out of cell using transport proteins (tetracycline) 3. Add modifying groups that inactivate the antibiotic (chloramphenicol) 4. Destroy antibiotic before it gets to target (penicillin)

List three kinds of microbes that are consumed as food:

1. Mushrooms, red algae 2. Baker's/Brewer's yeast (S. cerevisiae) 3. Lactobacillus, Streptococcus (in yogurts)

9 classes of Exotoxins

1. Plasma membrane (alpha toxin) 2. Cytoskeleton - can stimulate actin polymerization or depolymerization 3. Ribosome activity (Shiga) 4. Cell division 5. Signaling pathways (E. coli ST) 6. Cell-cell adhesion - proteases cleave proteins binding host cells to each other 7. Vesicles 8. Exocytosis - alter movement of nerve cells in cytoplasmic vesicle membrane where they release NT 9. Immune system regulation

4 steps in peptidoglycan synthesis

1. Precursors made in cytoplasm: UDP-NAG & UDP-NAM peptide 2. Bactoprenol (lipid carrier) - carries precuroses across membrane then is recycled 3. Transglycosylases - polymerize precursors to existing cell wall structure 4. Transpeptidases - cross-link side chains

Purpose of fermentation

1. Preserve food (limit microbial growth - NH3, carboxylic acid, ethanol) 2. Improve digestibility: break down fibers 3. Add nutrients (i.e. vitamins) & flavor molecular (ex: esters & sulfur compounds

EIDs caused by

1. Reemergence of disease through antibiotic resistance & recombination w/ alternate hosts 2. Intense interactions w/ wildlife

Stage II: Entry & uncoating

2 ways 1. Envelope or capsid fusion with host cell membrane & nucleoprotein core releases into cytoplasm - Virion fuses directly with membrane 2. Endocytosis - Endosome & viral membrane fusion releases nucleoprotein core into cell (can be in cytoplasm or @ nuclear membrane - Endosome that formed around virus particle fuses with lysosomes (low pH) -- When fuses → increases pH & acidification → Fusion of virus envelope with lysosome membrane → Virus uncoating & release nucleic acids into cells * Acidification caused by lysosome fusion Influenza has an ion channel in the virion's outer membrane so when it's endocytosyzed, this lets H⁺ enter the virus particle → changes pH → fusion of virus membrane with endosomal membrane → release material into host

What are the different traits of viruses that are used in classifying viruses? How are they described taxonomically?

A virus is a non-cellular particle that infects a host cell, where it reproduces. The virus particle, or virion, consists of an infective nucleic acid (either DNA or RNA) contained within a protective shell made of protein, called the capsid. Viruses are classified based on the composition of their genomes (i.e. DNA-based vs. RNA-based). Viruses don't have their own rRNA genes (i.e. no common gene region like 16S rRNA among living organisms). Their small and very diverse genomes don't have enough conserved sites to classify viruses based on genetic similarity. Instead, they are split into groups based on whether their genome is DNA-based vs. RNA-based, single-stranded vs. double-stranded (i.e. the Baltimore scheme). Viruses can also be classified based on a number of other characteristics. For example, they can be classified by the shape and size of the virus - closely related viruses are similar in these characteristics. In some cases, the host range of the virus and its mode of spread (by insect for example) play a role in classification. Closely related viruses usually infect the same or related hosts. However, viruses with extremely different hosts can show surprising similarities in genetics and structure. For example, both rabies virus and potato yellow dwarf virus are enveloped, bullet-shaped viruses of the rhabdovirus family. Viruses are organized into families, genera and species. The family name ends in "viridae". Both the genus and species names end in "virus". For influenza, the family is the Orthomyxoviridae

Which of the following microbes is associated with contaminated canned goods? A. Clostridium botulinum B. Escherichia coli C. Listeria monocytogenes D. Aspergillus niger

A. Clostridium botulinum

Swiss cheese is the product of which of the following types of fermentation?

A. Propionic acid fermentation

Food spoilage includes

Acids = sour taste Oxidation of fats = rancidity Decomposition of proteins = putrefaction Alkalinity = bitter taste

Chemical means of food preservation

Acids added as salts -Sodium benzoate, potassium sorbate, sodium propionate -Act by crossing cell membrane in protonated form & releasing protons at higher intracellular pH Nitrites -Inhibit aerobic respiration of bacteria & effectiveness enhanced at low pH -Inhibit iron-sulfur clusters of aerobic bacteria -Not effective for controlling gram negative (Salmonella & E. coli) Sulfites -Inhibit aerobic respiration of bacteria -Effectiveness enhanced at low pH -Commonly introduced to arrest fermentation @ desired time & may be added to wine preservatives to prevent spoilage & oxidation at several stages of winemaking Esters -FA esters & benzoic acid esters Other organic compounds -Cinnamon contains benzene derivative - eugenol (potent antimicrobial agent)

Chloramphenicol Resistance

Addition of acetyl groups prevents it from binding to 50S

Type I Pili

Adhere to mannose residues Produce static attachment Grow from outer membrane Assembly: E. coli pap system

Narrow spectrum

Affect small range antibiotics Zones in on pathogenic microbial culprit Drawback: v hard to diagnose illness based on symptoms alone

Broad spectrum

Affect wide range of organisms Often first prescribed to give more tests Drawback: also target resident flora -C. diff -Yeast infection

Roundup: glycohosphate produced by

Agrobacterium Strain CP4 Roundup ready plants: resistance to glycophosphate Transferred w/ A. tumefaciens

Ti plasmid

Agrobacterium tumefaciens - ubiquitous in soil Encodes multiple functions DNA transfer into cell induces plant cells to make foor for bacterium Complexes with plant, plant releases phenolic compounds that bacteria may recognize In cell environment: VirA - detects chemical signal from wounded plant -Binding activates VirA which activates VirG Vir genes on plasmid -Expression requires activation of VirG Genes are VirD1, VirD2 An endonuclease cleaves @ 24 bp sequence repeats @ end of T-DNA → cell produces ss T-DNA molecule attached to VirD2 VirD2 directs transfer of T-DNA into plant cell (similar to conjugation) T-DNA integrated into plant genome in nucleus T-DNA genes expressed in plant cells Genes cause synthesis of cytokinin & auxin which activate rapid plant cell division which leads to undifferentiated mass (tumor growth in plant - Crown gall disease) Opine synthesis & N -Source of C for bacterium Used to genetically engineer plants Engineers replace Ti w/ yfg

Describe at least 3 different roles of Agrobacterium sp. in generation of GMO products and services for agriculture.

Agrobacterium tumefaciens is a ubiquitous soil borne pathogen responsible for Crown Gall disease in plants. It is called a disease mainly because the galls produce ugly growths on ornamental plants, but the plants are not killed by the presence of the bacteria. The plasmid-transfer system in A. tumefaciens is responsible for much of the genetic engineering work that has been done in plants. When the molecular nature of crown gall disease was worked out, it became clear that the Ti plasmid and its T-DNA had great potential to be a natural vector for the insertion of recombinant DNA into plant chromosomes. Basically the way this works is that a single gene of interest is inserted into the plasmid. That plasmid then gets inserted into an Agrobacterium host cell. The bacteria interact with a single plant cell and inserts the T-DNA into plant cell chromosome. The Ti plasmid has been modified and the tumor-producing and opine-producing genes have been knocked out, so no tumor is formed and the bacteria don't grow. That single plant cell is then cultured to produce a full plant, with each cell of that plant expressing the new gene. Because the gene will be part of the plant's genome, it will be transmitted to offspring in seeds. There are many plants now grown in the US that have been genetically modified via A. tumefaciens. Examples include plants with herbicide resistance genes, which lowers the amount of chemical spraying that needs to be done. The nutrient content of certain crops has also been manipulated. Golden rice produces lots of beta-carotene from a gene isolated from daffodils and soy has also been manipulated lower saturated fat content. In addition to the plasmid system, Agrobacterium is resistant to the Roundup herbicide glyophosate. The herbicide glyphosate kills plants by inhibiting an enzyme necessary for making aromatic amino acids. The broad-spectrum herbicide glyphosate, the active ingredient of Roundup, inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, the enzyme catalyzing the penultimate step of the shikimate pathway toward the biosynthesis of aromatic amino acids. In 1996, Monsanto introduced the Roundup Ready soybean, a genetically engineered crop resistant to glyphosate. In the few years after, Roundup Ready cotton, maize, and various other crops also made their debut. While almost all plants are susceptible to glyphosate's grip, the beautiful thing about genetic engineering is that the genes need not be from similar organisms. A gene encoding the resistant enzyme from Agrobacterium was cloned, modified for expression in plants, and transferred into important crop plants like soybeans. When sprayed, the plants containing the bacterial gene are not killed.

Question 3: Exotoxins that disrupt the cell membrane are often known as this type of toxin:

Alpha

Drugs that affect 30S

Aminoglycosides Tetrayclines

How do we classify antibiotics? When is it appropriate to use a broad-spectrum (vs. a narrow-spectrum) antibiotic? What are the down-sides of using a broad-spectrum antibiotic?

Antibiotics are classified based on the types of organisms they infect. Those that affect a wide range of organisms are called broad spectrum. Those that affect a small range of organisms are called narrow spectrum. Antibiotics that fall into the "broad spectrum" category, like chloramphenicol and tetracycline, are effective of both aerobic and anaerobic, Gram-positive and Gram-negative bacteria. It is very difficult to diagnose the specific bacteria causing an illness based on symptoms alone. As a result, when a person with an infection comes into a hospital, they are often given a broad-spectrum antibiotic because the culprit is unidentified. Lab work can then be done and within a few hours or days, the person can be given a more narrow spectrum antibiotic that is targeted towards killing the pathogen that is responsible for making that person sick. However, because they are broad spectrum, these antibiotics don't just go after pathogens. They also target our resident flora, many of which we want there. If we lose our native flora, that can allow other pathogens normally kept at bay by our native flora to take over and cause secondary infections. An example of this is a disease called pseudomembranous colitis. It is caused by a bacterial species that normally lives in our colons in very low abundance called Clostridium difficile. When some people take a course of antibiotics, this organism gets knocked down like all the rest of the bacteria in the colon, but it grows back faster. Clostridium produces two protein toxins (TcdA and TcdB proteins) that damage the walls of the colon. They do this by glucosylating small GTP-binding proteins in the cell, causing actin condensation, cellrounding, and cell death. If untreated, it can lead to death, with symptoms similar to septic shock. Yeast infections in women are another example of secondary infections, where the normal bacteria in the vagina are disrupted, which allows yeast to take over.

Compare and contrast the ways in which antiseptics, disinfectants, and antibiotics work.

Antiseptics are things we apply to our skin like the hand sanitizers that don't require washing. Disinfectants are things that get applied to inanimate objects, such as phenols, which are too harsh for the skin. The main difference between antiseptics/disinfectants and antibiotics is that the mode of action for the first is really broad. The alcohol sanitizers work by disrupting cell membrane and denaturing proteins in the cell. This mode of action has the same effects on our cells, so antiseptic and disinfectants don't show selective toxicity. As a result, we can use them to kill microbes, but we can't ingest them, because they would have equally bad consequences for us. Antibiotics work by binding to some molecule important to bacteria, which impairs its function.

Penicillins

B-lactam ring resembles D-Ala-D-Ala piece of peptidoglycan Bind transpeptidase/transglycosylase (penicillin-binding proteins PBP)

Penicillin Resistance

B-lactamases break lactam ring, so cross-linking proteins no longer bind to penicillin

Anthrax Toxin

Bacillus anthracis: Gram +, spore-forming Lethal because secretion of plasmid-encoded tripartite toxin Core subunit = "protective antigen" (PA) -Binds host surface where human protease cleaves off a fragment (B subunit) Can self-assemble in membrane to form pores Edema Factor - bind to PA rings; carried into cell (A subunit) - Activates CAMP production - Adenylate cyclase Lethal factor - protease, cleaves kinases, shuts down signals that recruit immune cells to fight infection Complex endocytosed PMF unfolds toxins, translocates then across endocytic membrane

Describe the role of Bacillus thuringiensis in generation of GMO products for agriculture.

Bacillus thuringiensis is a live pathogen on plants that kills insects. Bacillus thuringiensis (Bt) sporulates on the surfaces of plants. However, in addition to the spore, the sporulating cell makes a separate parasporal body that cradles the spore. The parasporal body contains crystallized proteins that are toxic to moth and butterfly larvae feeding on the plant. After the insect or insect larva ingests the insecticidal protein crystals, the alkaline environment in the insect midgut dissolves the crystals, and insect proteases inadvertently activate the proteins. Activated insecticidal proteins (Bt toxins) insert into the membrane of the midgut cells and form pores that lead to a loss of membrane potential, cell lysis, and death of the insect through starvation. Bt toxin has been genetically introduced into plants. It was cloned into a plasmid vector under control of a chloroplast rRNA promoter, then transferred into tobacco plant chloroplasts by microprojectile bombardment. This yielded transgenic plants that expressed Bt toxin at levels that were extremely toxic to larvae from a number of insect species.

What are some economic and social factors that promote the persistence of antibiotic-resistant bacteria? How have bacteria evolved or acquired resistance, given the selective pressure of antibiotics they've experienced over time?

Bacteria can evolve quickly in response to strong selective pressure. Antibiotics are exactly that - an incredibly strong selective pressure that favors the evolution of resistance. The more an antibiotic is used, the more chance to bacteria have to become resistant. This is a problem because the prescription of antibiotics has been something that has been loosely regulated. A second issue is the widespread use of antibiotics in agriculture. It has been found that animals given antibiotics grow bigger faster, which has economic benefits to producers. It is hypothesized that the constant exposure of the animals to antibiotics may be serving as incubators for the development of resistance that could move via horizontal gene transfer into pathogens (however, there are not any document cases of this just yet, thankfully!). Also, the widespread use of antimicrobials in soaps and other solutions that really do not need them to be effective results in the exposure of microbes to drugs on a large-scale basis, which allows them many opportunities to evolve resistance. Finally, two final social factors are important. One is the increased global travel that allows microbes that have gained resistance to travel very quickly into other parts of the human population. The last one is incompletion of treatment regimes, often due to poverty. Drugs can be expensive and or require prohibitive travel to acquire (like at a clinic in rural areas). When the choice is between food/time versus proper medical treatment, it is not an easy decision to make and sometimes completing treatment loses.

Proteins (putrefaction) or fats (rancidity)

Bacteria predominate Breakdown of peptides & amino acids produces odorants Cadaverine & putrescine

RNA synthesis antibiotics

Bactericidal Rifampin & Actinomycin D

Type VI Secretion System

Bacteriophage-like-injection of effector proteins "Poison darts" Repurposed phage tail parts normally used to deliver phage DNA into bacterial host cells VipA/B homologous to T4 tail sheath VgrG homologous to T4 spike complex Vibrio cholerae, E. coli, Pseudomonas

Penicillin resistance of bacterial strains can be overcome by addition of this type of small molecule to the drug treatment regime of the patient.

Beta-lactamase inhibitors

Stage I: Attachment

Binding of HA & sialic acid triggers host uptake of virus

Bacillus thuringiensis

Bio-pesticides example Produces parasporal body -Made during sporulation as intracellular protein toxin crystal -Acts as insecticide for specific insects Bt = insecticide -Does not accumulate in environment

Describe gene regulation by the Catabolite Activator Protein. How does CAP achieve specific regulation of its gene targets?

CAP is a DNA binding protein that activates transcription at target promoters by stabilizing the binding of RNA polymerase at the promoter and thus promoting transcription initiation. CAP responds to the secondary messenger molecule cyclic AMP (cAMP). In the absence of cAMP, CAP does not bind promoter DNA, it is in an inactive conformation. However, when conditions occur that result in an increase in cAMP concentrations, cAMP binds to CAP, and induces a conformational change in the protein that allows it to bind to specific DNA operator sites upstream of CAP regulated promoters. CAP protein has a special sequence of amino acids - a helix-turn-helix motif - that allows it to interact with the phosphate backbone of the DNA, forming a specific DNA-protein binding interaction. Once cAMP::CAP complex has bound to the promoter region, it interacts with the alpha subunits on the core RNAP polymerase to promoter RNAP binding and transcription at the promoter. In this way CAP upregulates (increases) the transcription of genes that have a CAP binding site upstream of the -35 and -10 regions of the promoter sequence.

Key to Lysis-Lysogeny Switch

CII regulates CI

Streptococcus pyogenes

CRISPR-Cas9 Cas9 - endonuclease that can KO genes or replace w/ modified forms LacI represses Cas9 IPTG derepresses Cas9 dCas9 protein + gRNA inhibit transcription of luciferase gene

Structure of T4 phase

Capsid (head) - Linear ds-DNA Tail -sheath & internal tube Fibers Collar & neck Base plate "Early genes" - proteins favoring viral replication (w/in 2 minutes) Phage DNA begins replication after about 5 minutes "Later genes" - proteins for new virions Timing regulated by manipulating host RNAP & clustering genes in genome Life cycle is 22 minutes from injection to host lysis Phage entry injects nucleic acids into the host

Alternate Induction Plan

Cell density ↓ in nutrient ↓ → Lysogeny Nutrients ↑ → cells ↑ activity & #s → Lysis Proteases made more when nutrients ↑ → ↑ FtsH to cleave CII → Less CII → Less CI → More Cro → Lysis

Gramicidin

Cell membrane integrity Cyclic peptide made by Bacillus brevis Inserts into membrane as dimer, forms cation channel through which ions leak

Daptomycin

Cell membrane integrity Lipopeptide produced by Streptomyces roseosporus Forms ion channel that leaks K⁺ Vs. MRSA

Polymyxin

Cell membrane integrity Polypeptide made by Bacillus polyxin Destroys cell membrane like a detergent Topical

Target of Cephalosporins

Cell wall synthesis

Vancomycin

Cell wall synthesis Binds ends of peptides Prevents action of transglycosylase/transpeptidases Same step as penicillin, different activity Glycopeptides bind to D-Ala-D-Ala part of peptide & block cross-linkage

Cycloserine

Cell wall synthesis Inhibits formation of D-Ala-D-Ala dipeptide precursor

Targets of Antimicrobial Agents

Cell wall synthesis - penicillin, vancomycin (bacteriocidal) Cell membrane integrity DNA synthesis RNA synthesis Protein synthesis Metabolism

Cephalosporin

Cell wall synthesis inhibitor

Propionic acid fermentation

Cheese

Describe two examples of food preservation: one physical and one chemical example.

Chemical methods of food preservation involve antimicrobial agents, which are chemical substances that either kill microbes or slow their growth. (1) Acids are generally added as salts (e.g. sodium benzoate, potassium sorbate, sodium propionate). These acids act by crossing the cell membrane in the protonated form and then releasing their protons at the higher intracellular pH. (2) Nitrites inhibit aerobic respiration of bacteria, and their effectiveness is enhanced at low pH. Nitrites inhibit iron-sulfur clusters of aerobic bacteria. They are not as effective for controlling gram-negative enteric bacteria (e.g. Salmonella and E. coli). These substances, however, may have harmful effects on humans; nitrites can be converted to toxic nitrosamines. (3) Nitrites and sulfites inhibit aerobic respiration of bacteria, and their effectiveness is enhanced at low pH. Sulfites are commonly introduced to arrest fermentation at a desired time and may also be added to wine as preservatives to prevent spoilage and oxidation at several stages of the winemaking. Note - these substances, sulfites cause allergic reactions in some people. Physical methods of food preservation include canning. In canning, the most widespread and effective means of long-term food storage, food is cooked under pressure to attain a temperature high enough to destroy endospores (typically 121°C). Food slated for canning could be under-processed or could spoil prior to canning, but the main danger of canning is contamination of food during transfer into the final container. This occurs via leakage of contaminated water into cans during cooling process.

Applications for Ti Plasmid

Cloning vector for introducing foreign genes into plants Ex: GM plants: cotton with herbicide, detox genes, soybeans with lower saturated fat content, w/ more iron & B-carotene roundup

Opportunistic pathogen example

Clostridium difficile

What does it take to be a pathogen

Colonization: Adhesion, immune evasion Ex: Pili, flagella, adhesions, capsule Persistence: Nutrient acquirement, continue immune evasion Ex: Iron acquired via siderophores, catalase & superoxide dismutase, using specific nutrients, toxins that kill cells & release contents Spread & disseminate: Degradation of connecting proteins & molecules Ex: Toxins that modify host cells, cause actin rearrangements, collagenases, proteases All of these encoded by genes that make VFs

Penicillin inhibits transglycosylase and transpeptidase enzymes. It achieves this inhibition by mimicking the structure of which molecule in the cell wall synthesis pathway:

D-alanine dipeptide

Current hotspots of emerging infectious diseases include the Northeastern USA, Western Europe and Japan. In 2-3 sentences explain what these areas might have in common that predicts EIDs.

Commonalities between these regions that contribute to the emergence of infectious disease include dense populations, overuse of antibiotics (as is common in developed nations), and urban development and displacing of wildlife/populations.

Discuss the pros and cons of GMOs as we understand them based on data from the early 2010's.

Cons: ¥ Reduced biodiversity ("ecological vacuum") - insects and disease can exploit ¥ Intellectual and property rights disagreements ¥ Unknown consequences if genes from GM crops find their way into other species. Farmers who have crops contaminated with GM plants can be sued by Monsanto for illegally obtaining the seed without paying Monsanto ¥ Unknown risks to birds, insects, other animals that consume GM plants Pros: ¥ Potential reduced herbicide/pesticide use ¥ Potential reduced GHG emissions ¥ Potential for reduction in hunger and poverty ¥ Another reason Roundup Ready crops theoretically result in a new environmental benefit is that farmers no longer have to till their cropland. In 2010 alone, CO2 emissions have been reduced by 28 billion kgs - the equivalent of taking ~12.4 million cars off the road. Poverty in developing countries was at 46% of the population in 1990, while in 2005 it decreased to 27%. Furthermore, since 1995, GM technology has reduced chemical pesticide use 37 percent, increased crop yields 22 percent, increased farmer profits 68 percent since the early 1990s. However, Roundup ready plants have increased herbicide use 382.6 million pounds from 1996-2008. Crop years 2007 and 2008 accounted for 46% of the increase in herbicide use over 13 years across herbicide-resistant crops. Herbicide use on herbicide-resistant crops has risen a remarkable 31.4% from 2007 to 2008. Genetically engineered crops reduced overall pesticide use in the first several years of commercial introduction, but have increased pesticide use by 20% in 2007 and by 27% in 2008.

Once inside phagosome, pathogen might mature in acidic environment

Coxiella burnetti → Q fever Lysosome fusion & intracellular replicaton Phagosome - lysosome fusion, different into form able to replicate in phagolysosome → "inclusion bodies"

What is the microbiological reason behind the separation of milk into curds and whey during the cheese production process?

Curd formation results from two kinds of processes: acidification, usually as a result of the microbial production of lactic acid; and treatment by proteolytic enzymes such as rennet. The curd may then be separated and processed to varying degrees, depending on the type of cheese. Cheese production begins by lactic acid fermentation of milk by Lactobacillus and Streptococcus species. As the pH of milk declines below pH 5 (due to this fermentation - production of lactic acid), the acidic amino acid residues of caseins become protonated, eventually destabilizing the tertiary structure. As the casein molecules unfold (or "denature"), they expose hydrophobic residues that regain stability by interacting with other hydrophobic molecules. The inter-molecular interaction of caseins generates a gel-like network throughout the milk, trapping other substances, such as droplets of milk fat. This protein network generates the semisolid texture of yogurt, a simple product of milk acidified by lactic acid bacteria. In most kinds of cheese formation, an additional step of casein coagulation is accomplished by proteases such as rennet. Rennet is a complex of enzymes produced in the stomachs of ruminant mammals. During cheese production, milk protein coagulates to form a semisolid curd. As in all fermented foods, microbial metabolism generates by-products that confer characteristic aroma and flavor. Casein catabolism generates flavor molecules. Extracellular enzymes break down casein into peptides and amino acids, which are taken into the bacterial cell by membrane transporters. The amino acids are fermented to volatile alcohols and esters. In some cases, they combine with sulfur to form methanethiol and other sulfur-containing odorants characteristic of cheese.

Agrobacterium tumefaciens can be used to transform the DNA of plants. Describe this process, including how A. tumefaciens manages to "farm" these plants for carbon resources.

DNA transfer from Agrobacterium tumefaciens to eukaryotic cells is the only known example of interkingdom DNA transfer. This interaction starts with the bacteria attaching themselves to the plant in an area where the plant has been damaged, because the plant produces phenolic compounds as part of the wound response. These plant phenolics trigger a two component regulatory system (VirA sits in the cell membrane and phosphorylates VirG). VirA and VirG are encoded on a plasmid called a Ti plasmid (tumor inducing). They are always expressed in the bacterium. VirG-P then activates transcription of VirD genes, which mediate the excision of a segment of the Ti plasmid called the T-DNA and transfer of T-DNA by conjugation to plant cells. In the plant cell, the T-DNA enters the nucleus and integrates into the plant genome. The T-DNA contains genes encoding plant hormones, and these genes have promoters recognized by plant cells. In the plant, the expression of these genes and production of hormones causes the plant cell to divide, leading to the formation of the tumor. The T-DNA also induces the plant cells in the tumor to produce opines. These are unusual derivatives of amino acids and sugars that can be used by A. tumefaciens as a source of carbon and energy but not by the plant. Meanwhile, back in the bacterium, the Ti plasmid encodes enzymes that allow the bacteria to catabolize those opines. Opines are unusual compounds and few bacteria besides A. tumefaciens carrying a Ti plasmid can use them. Thus, any other bacteria that might be in the area are not serious competitors for these compounds. In effect, the Ti plasmid allows the bacterium to use the bacterial sex (conjugation) to "farm" the plant cells, creating a tumor that provides nutrients for the bacteria.

Coccolithovirus (CLV)

DNA virus, infects coccolithophores Part of nucleo-cytoplasmic large DNA virus group (includes mimvirus) Parts of CLV genome transcribed in cytoplasm Affects diploid form of alga (haploid stage is resistant to virus; EHUX uses this strategy to evade infection)

ELISA Assays

Detect antigens or Abs in nanogram & picogram quantities Antigen from virus attached to wells, patient serum added, Ab binds to viral antigen Often use secondary antibody linked to colorimetric enzyme reaction for detection

Pathogenicity

Determines how easily pathogen can cause disease (infectivity) & virulence (severity) Measured with infectious dose & lethal dose

PANTHER (Pathogen Analyzer for Threatening Environmental Release)

Developed in 2008 to detect pathogen in air in as little as 3 minutes Can detect dozen particles per liter of air Detects up to 24 pathogens i.e. bioterror agents Lab on a chip 1. B cells exposed to bioagents in test sample 2. B-cell Abs bind agent 3. Cross-linking activates phosphorylation cascade 4. Ca²⁺ influx activates aequorin 5. Photons released hit photon detector

Fleming

Discovered penicillin

Outcome: Lysogeny

E. coli infected by multiple lambda ↓ CII transcription increases (protected from host protease FtsH by CIII - which is also higher when multiple infection) ↓ Activates PRE ↓ PRE → CI CI blocks PR (this leads to a stop in Cro production) CI binds to PRM which leads to more CI production

VRE - Vancomycin Resistant Enterococcus

E. faecalis - normal inhabitant of human gut; opportunistic pathogen Now major source of hospital bound infections Genome of resistant strain sequenced in 2003: -VanB gene complex -TEs -Predicted coding regions in + and - DNA strands -Bacteriophage genes (also involved in HGT)

Explain how marine viruses control the population of the marine alga E. huxleyi? How does this fit into carbon cycling in marine communities.

E. huxleyi is a phytoplankton that lives in the ocean and under the appropriate nutrient rich conditions can experience population booms. The algal blooms from this population explosion are so large that they can sometimes be seen by satellite because the high population density of the calcium rich coccolithophores reflects sunlight. During cocolitthophore blooms, the coccolithovirus will infect the population, resulting in lysis of many of the alga and releasing carbon into the marine environment.

Synchronized oscillating clock

Engineered E. coli LuxI gene from Vibrio fischeri aiiA gene from Bacillus thuringiensis

Emillania Huxleyi (EHUX)

Eukrayotic microbe (single cell alga) Cocolitthophore (calcite disks) Photosynthesizes plankton EHUX blooms Certain conditions → massive blooms

2 Types of Overall Toxins

Exotoxins Endotoxins

What type of environments are the most promising for bioprospecting?

Extreme environments with temperature and pH extremes.

How does food spoilage occur? Include descriptions of how breakdown of carbohydrates, proteins, and fats contributes to food spoilage.

Food spoilage occurs due to microbial changes that render a product unfit or unpalatable for consumption. Breakdown of carbohydrates occurs through hydrolysis of the bonds holding sugar monomers together in plant tissues. Carbohydrate degradation in vegetables/fruits is often caused by fungi - for instance Claviceps (ergot), responsible for the production of hallucinogens. In dairy products, bitter off-flavors may be produced by bacterial degradation of proteins. The release of amines causes a rise in pH. Protein degradation is most commonly caused by psychrophiles, species that grow well at cold temperatures, such as those of refrigeration. Meat and poultry are putrefied by decarboxylating bacteria, which produce amines with noxious odors. The breakdown of peptides and amino acids produces the undesirable odorants that define spoilage (for example, cadaverine and putrescine). Lipids (found in meats) commonly spoil via auto-oxidation (reaction with oxygen) of unsaturated fatty acids, independent of microbial activity. Seafoods especially spoil rapidly because their unsaturated fatty acids rapidly oxidize.

E. coli Pap system

For Type I pili assembly Pilin protein subunits secrete to periplasm by Sec System PapD - directs subunits to assembly site PapC - assembles pilins Assembly starts w/ PapG - binds to mannose on host membrane PapF & PapE added Several PapA pilin subunits strung together in series to form shaft PapH end piece (cap)

Given that efflux pumps cannot pump every single molecule of antibiotic out of bacterial cells, why are they so effective against the activity of antibiotics like vancomycin? How do these efflux pumps help generate multi-drug resistant bacteria?

For tetracycline to work, it must enter the cell cytoplasm and accumulate to a concentration that is sufficient to allow it to bind to the ribosome. A bacterial strategy that prevents antibiotics from reaching a high enough concentration in the cytoplasm is to pump the antibiotic out of the cytoplasm as it is taken up. This is done by protein pumps called efflux pumps that remove the antibiotic. The concern about these pumps is that they are often not specific to single antibiotics. The pump proteins will recognize multiple types of antibiotics and remove them from the cell, so this is another way bacteria can become simultaneously resistant to multiple drugs.

Briefly describe the life cycle of the T4 bacteriophage.

For the T4 bacteriophage, from injection to host cell lysis takes 22 minutes. Within two minutes of injection of DNA, the host RNA polymerase starts making viral mRNA. This is called early mRNA because it is made before the viral DNA is made. Early RNA directs the synthesis of proteins that take over the host cell machinery. Some early viral proteins include enzymes that degrade host DNA to nucleotides. This halts host gene expression and also provides the building blocks for viral gene expression. The next step in the T4 life cycle is the replication of the phage DNA in order to make the genetic material for production of more particles. Then, the late genes are expressed to produce the capsid components for virion formation, and eventually the enzymes needed for cell lysis are produced to release new virus particles into the environement. The virus achieves very specific temporal ordering of gene expression through: 1) Regulating the activity of the host RNA polymerase. Just after infection, a T4 enzyme adds an ADP group to the polymerase that modifies it to favor viral gene expression over host gene expression. Later it modifies it again by adding another ADP group, which turns off the expression of some early T4 genes and stimulates the transcription of later ones. 2) Organization of the T4 genome. Genes with related functions such as those involved in head and tail construction or DNA synthesis and replication are clustered together. This helps facilitate their expression at different times. They also get transcribed in different directions, with early genes going counter-clock wise and late genes going clock-wise.

Describe the differences in mode of action between beta-lactam antibiotics, quinolones/fluoroquinolones, tetracycline, and sulfa drugs. Why are some of these antibiotics considered bacteriostatic, while others are considered bacteriocidal?

Four key pathways by which antibiotics affect bacterial cells that cover the vast majority of how all antibiotics work: those that inhibit bacterial cell wall synthesis, those that interfere with DNA replication, those that inhibit protein production in the cell, and those that interfere with folid acid production (metabolic mechanisms). Penicillin and vancomycin inhibit bacterial cell wall synthesis. Penicillin is a beta-lactam antibiotics, meaning that it's beta-lactam ring binds to the transpeptidases that cross-link the peptides and inactivate them, so cross-linkage cannot happen. Vancomycin, on the other hand, actually binds to the peptides themselves. Specifically, they bind the two d-alanines at the end of the peptide chain together. It is energy in the cleaving of alanines that the transpeptidases use to make the cross links. Thus by attaching to the peptide and holding these subunits together, they stop the cross-linking reaction. These are both considered bactericidal, but they only can kill actively growing cells. These drugs do not affect cells in the stationary phase because in this state the cell has no need for new peptidoglycan. Quinolones target microbial topoisomerases like DNA gyrase. These enzymes catalyze supercoiling of bacteria DNA. So the question for tiny prokaryotes is: how do they pack all that DNA into their cells and still leave room for other cellular function? The way they do this is by wrapping their DNA into additional helices. This process is referred to as supercoiling. Quinolones this specific way bacteria that replicate their DNA. The way these antibiotics work is not by inhibiting the cutting action of the enzyme, but rather by binding the DNA gyrase to the DNA. In doing so, the complex can't get off when the cell is replicating and so they block the DNA replication machinery Because DNA is unable to be copied, the bacteria die. All quinolines can kill actively dividing bacteria, while there are some that can kill non-dividing bacteria. There are many drugs that inhibit protein synthesis by bacteria - a particularly important one is tetracycline. This drug works by affecting translation, although there are others that affect transcription as well. They do this by binding to the smaller of the two ribosomal subunits. This can happen because of differences between prokaryotic and eukaryotic ribosomes. In particular, they bind to the ribosome in a way that distorts the A site, which is the part that accepts the first tRNA. Because the site is distorted, this blocks the ribosome from building peptide chain. Tetracycline is a bacteriostatic drug, so it does not directly kill the bacteria, but instead does not let it grow. The fourth major pathway targets the synthesis of folic acids. Normally PABA, glutamic acid, and pteridine are complexed by an enzyme that sticks them all together and makes folic acid. When sulfanilamide is around, however, the enzyme grabs it and the two other molecules. Its side chain is different enough that it will not form a peptide bond to glutamic acid. It also will sterically hinder the binding of pteridine, so no folic acid is produced. These antibiotics are called sulfa drugs and they are also bacteriostatic.

Propagated epidemics

From introduction of single infected individual into susceptible population which is propagated to others

Common source epidemics

From single contaminated source i.e. food

Pathogenicity Islands (PI)

G+C content different from rest of genome Flanked by phage or plasmid genes Encode VFs (toxins, attachment proteins, capsules)

What is gene therapy? Describe the CRISPR-Cas system and how it has the potential to be used for gene therapy.

Gene therapy is the development of genetic treatments for disease that can replace drug or surgical treatments. Gene therapy works by replacing a mutated gene that causes disease with a healthy copy of the gene, inactivating, or "knocking out," a mutated gene that is functioning improperly, or introducing a new gene into the body to help fight a disease. The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. Invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA - CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity. In the CRISPR system from S. pyogenes, the protein Cas9 participates in the processing of crRNAs, and is responsible for the destruction of the target DNA. To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. During the destruction of target DNA, endonuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript. When the junction is fixed by the DNA repair machinery of the cell - it generates frame shifts that inactivate the gene leading to a gene "knock out". Alternately, if flanking DNA homologous to the damaged region is provided, the cellular recombination machinery can swap in a new gene or piece of DNA at the region of the break (thus replacing one gene with another).

Group 1: ds-DNA virus

Genes transcribed directly by RNA dependent polymerase Makes own DNA Ex: Herpes

ds-DNA viruses

Genes transcribed directly by RNA-d-polymerase either encoded by host or viral genome

Group V: (-) sense ss-RNA

Genomes of "template" or "negative sense" RNA Needs to pack viral RdRNAP for transcription of (-) RNA to (+) mRNA (-) strand RNA genomes often segmented Virus must provide machinery for translating antisense strand → + strand which is used as mRNA then Ex: Influenza

Describe how tuberculosis has re-emerged as a major pathogen. What are the epidemiological (disease in populations), human behavior, and microbiological factors that have contributed to its rise?

Incidence of tuberculosis dropped sharply in the 1950s, but the number of cases since 1980 has increased dramatically. One trigger for re-emergence was the AIDS pandemic (epidemiological). Immunocompromised aids patients are highly susceptible to infection by mycobacterium tuberculosis. Another was development of drug-resistant strains. Three antibiotics are typically administered, and patients usually don't take all 3 together (human behavior). This allows the organism to develop resistance to one drug at a time, until it becomes resistant to all of them. The highly drug-resistant strains are almost impossible to kill. Why does resistance occur so fast? The secret may lie in the diversity of growth phenotypes that arise from the unusual course of cell division in mycobacteria (microbiological). The diverse growth states cause different cells (alternators vs. accelerators) to resist different antibiotics. Now, all tuberculosis patients have to be in the presence of medical staff to take prescribed antibiotics.

What is transcription cap theft? Describe two ways in which it assists the virus replicate in the host cell.

Influenza has a negative strand genome, so it must make positive strand copies to serve as (1) mRNA for component virus proteins and (2) templates for replicase to produce more negative stranded copies of the genome. To separate these two groups, it uses a unique method that involves subverting the host cell's normal function. Normal mammalian mRNAs have a structure on their 5 prime ends called the 5 prime cap. This cap signals the mammalian cell translation machinery that this mRNA is targeted for translation. Instead of making such a cap for its own positive strand, the replicase of influenza acts as an endonuclease, once it has bound to the negative strand viral RNA. It cleaves the 5 prime cap plus some nucleotides from the host mRNA molecule. The captured cap then serves as a primer for the synthesis of a positive strand of viral RNA. This strategy accomplishes two goals. First, it targets certain viral + strands for translation. Those without the cap are used as templates for more - strand copies of the genome. The second part this theft strategy does is shut down translation of mammalian cell messages. This leaves the translation machinery of the infected cell devoted to synthesizing viral proteins

Briefly describe how intracellular pathogens evade the host cell defenses.

Intracellular pathogens are recognized by receptors on the host cell, and enter the cell by phagocytosis. However, there are three ways by which this type of pathogen can evade the host immune system. A subset of pathogens can mature in the acidic environment after the phagosome fuses with the low pH lysosomal compartment. Example pathogen: Coxiella Another group prevents the phusion of the phagosome by secreting proteins that prevent lysosomal fusion. Example pathogens from this group include Salmonella, Chlamydia, Mycobacterium, and Legionella The third mechanism is to use the enzyme hemolysin to escape the phagosome and then use motility to move from host cell to cell. Listeria and Shigella are pathogens that can achieve this.

Vancomycin resistance

Involves 3 enzyme pathway 1. VanH - catalyzes conversion of pyruvate → D-lactate 2. VanA or VanB - leads to formation of D-Ala-D-lactate; vancomycin can't bind D-Ala-D-lactate 3. VanX - cleaves any D-Ala-D-Ala formed by usual pathway back to D-Ala which prevents incorporation of D-Ala-D-Ala into cell wall synthesis

Bactericidal

Kills target microbe

Texiobactin

Kim Lewis From unculturable soil bacteria Eleftheria terrae Macrocylic peptide Active against gram + bacteria Binds cell wall precursor (lipids II & III)

You have a sore throat and are able to culture some bacteria from the back of your throat. You suspect that one species of Streptococcus is responsible for your sore throat, and you would like to test that hypothesis using laboratory mice in the mammal facility in LSEB. How would you employ Koch's postulate to test the hypothesis that your Streptococcus species is responsible for your sore throat?

Koch's postulates state that the organism must be present in every case of a disease, must be propagated in pure culture, must cause the same disease when inoculated into a naive host, and must be recovered from the newly diseased host. To determine if the Streptococcus species is responsible for the sore throat, we would first need to culture the strain from our throat tissue. Then, we would need to treat several mice (as biological replicates) with the strain (i.e. inoculate them with the strain) and observe some metric of a sore throat in the mice (e.g. a swollen and red throat). Finally, we would need to culture the Streptococcus species out of the infected mice once again. Ideally, we would include a Streptococcus control, such as a non-pathogenic Streptococcus strain (one that does not cause sore throat), and treat several different mice with this strain at the same time. If our Streptococcus species does cause sore throat, then 1) only mice in the treatment group would develop a sore throat, and 2) the pathogenic strain would only be isolated from the mice treated with the strain isolated from our infected throat.

CII

Lambda repressor Activates alternate promoter PRE which makes CI

Diacetyl acid & acetalaldehyde

Leads to undesirable flavor in beer & wine

High host cell density

Less lambda per host cell ↓ Less CI, CII, CIII production ↓ Favors Cro & lytic cycle

Pelagic zone

Low nutrients but high UV → lysis

Oligotrophic lake

Low nutrients → lysogeny

What sorts of environmental cues would initiate lysis by bacteriophages? Which cues would trigger lysogeny?

Lysis is triggered by low phage infection in individual bacterial host cells, which can happen when host cell abundance is high in the environment. Conditions that allow host cells to multiply rapidly include high nutrient (eutrophic) conditions. Temperate phages will enter lysogeny when the host cell abundance is low (because there are multiple phages infecting the same cell). This happens when nutrient availability is low (oligotrophic conditions). For the phage, the process of lysogeny allows a virus to remain viable within a dormant host during nutrient deprivation. In these situations, a prophage can survive while a virulent phage cannot, since the latter require active biosynthetic machinery. Other conditions that favor lysis are when the host cell is threatened. For example, chemical mutagens or intense UV light can induce a virus to engage in lysis.

Bacterial cell density is low during oligotrophic conditions and favors...

Lysogeny

Wildebeests & viruses

Major herbivore in Serengeti Population size law due to Mobillivirus (Rinderpest) Efforts ↑ beest as meat source → campaign to eliminate Mobillivirus via vaccination → eliminated → big increase in wildebeest population size Rinderpest linked to savannah development through fire frequency

Antigenic shift

Major ∆s → "killer flu"

Senior cells

Make drug-resistant mituberculosis Alternator: cycloserine/meropenem (B-lactam) Accelerator: Rifampcin (inhibition RNAP)

Ethanolic fermentation: beers & ales

Malt - germinated barley grains having activated enzymes Mash - malt after being mixed w/ H2O to hydrolyze starch to usable carbs Wort - clear liquid with fermentable carbs Wort heated with hops → flavor & assist in classification -Heating inactivates hydrolytic enzymes

What kinds of products can be made through industrial microbiology?

Many products can be made through industrial microbiology. Foods (beer, wine, cheese, other fermented foods) and preservation methods (production of ammonium, ethanol, organic acids) are generated by microbes and exploited commercially. Many medical products are also generated by microbes in industry: proteins (including enzymes), gene therapy, insulin, and vaccines. Industrial solvents and antibiotics are also made by microbes. Microbes can produce genetically modified plants and animals as well. We may distinguish between two fundamentally different sources of products: cloned genes from human, animal, or plant sources; and native microbial products, often from newly discovered species in extreme environments.

Pathogenic bacterial can use many different types of secretion systems to deploy their toxins into host cells. Compare and contrast four main types of secretion systems in these bacteria.

Many protein secretory systems evolved from, and bear structural resemblance to, other cellular structures that serve fundamental cellular functions. Type II protein secretion systems are homologous to Type IV pilus biogenesis. Proteins to be secreted first make their way to the periplasm via Sec-dependent pathway. In the periplasm, proteins are folded and ejected out of cell by pilus-like proteins that make up the type II secretion system. The pilus-like structure is used to ram the folded proteins through an outer membrane pore and out of the pathogen's cell. This occurs through constant assembly and disassembly of the pilus-like proteins. Type III protein secretion systems are homologous to flagellar synthesis. Unlike other secretion systems, the type III mechanism injects proteins directly from the bacterial cytoplasm into the host cytoplasm. This prevents dilution of the toxin by extracellular media. The proteins in these systems are related to flagellar assembly proteins, which export the flagellin proteins through the center of a growing flagellum. The first molecules that are ejected through the "needle" trigger creation of a pore complex in the host cell membrane. Then, effector proteins (or toxin proteins) are transported to base of needle by chaperone proteins. There, an ATPase unfolds the effector proteins so they can be transported through the needle. In the case of Salmonella bacteria, these toxins can cause rearrangements of the host cytoskeleton that cause engulfment of the microbe. Secretion is triggered by cell-cell contact between host and bacterial pathogen. Type IV protein secretion systems are homologous to conjugation. This system is employed by Bordetella pertussis, the bacterium that causes whooping cough in humans. This secretion system takes a toxin from the periplasm (moved there by SecA-dependent transport) or directly from the cytoplasm and transports it across the outer membrane of the bacterial cell. Type VI secretion systems (i.e. "poison darts) are homologous to phage tail parts normally used to deliver phage DNA into bacterial host cells. This secretion system is expressed from phage DNA that has integrated into the bacterial genome. The VipA/B proteins are homologous to the T4 phage tail sheath that contracts and drives the T4 phage needlelike core through the host cell wall. VgrG proteins are homologs of the T4 phage spike complex that initially punctures the target bacterial cell surface.

What sorts of environments would you look for a microbe with products that could be commercially important (i.e. bioprospecting)? What sorts of features might you look for in a microbe that you would like to use as an industrial strain? Describe the process of recombinant vaccine production in industrial strains of bacteria.

Most major sources of microbes for use in industrial microbiology have been natural environments. Unique ecosystems are the most promising sources of previously unknown microbial strains with valuable properties. Extreme environments, such as the hot springs of Yellowstone, are particularly promising because their products may tolerate conditions of temperature or pH required for industrial use. Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated. Psychrophiles from extremely cold environments, such as Antarctica, are a useful source for enzymes that do not require heating for activity, such as the Stainzyme laundry detergent enzyme. Unfortunately, many such unique ecosystems are endangered by pollution, human-introduced invasive species, and global climate change. Once a promising source strain is obtained, its genome is sequenced to investigate the genetic sequences that encode the useful product and regulate its expression. The operons encoding the product (or enzymes for its production) can then be cloned for optimal production An industrial strain is a strain whose growth characteristics are well studied and optimized for industrial production. The industrial strain must reproduce reliably, without major DNA rearrangements. It must also have an efficient gene transfer system by which vectors can introduce genes of interest into its genome. Industrial strains must be nonpathogenic and generate the desired product as a high proportion of its cell mass. Either the product must be secreted by the cell or, if the product is intracellular, the cells must be easily breakable. The production of DNA vaccines in an industrial strain (like E. coli) is very similar to the creation of clone libraries. First, the DNA that encodes the surface antigen of a virus or bacterium is inserted into a plasmid. Then, E. coli is transformed by taking up the plasmid with the foreign gene. The E. coli will then express the foreign gene, generating the surface protein. The protein can be collected from the E. coli cells and used in vaccine production.

Three important industrial microbial products or services are 1) pollutant degradation, 2) novel genomes, and 3) fuel. Describe an example of each product or service and the challenges or controversies associated with each.

Nicotine is found in high concentration in waste tobacco material from cigarette processing. Researchers in Shanghai characterized the pathway of nicotine degradation in Pseudomonas putida S16, a strain that efficiently degrades nicotine. The P. putida genes for nicotine catabolism were found to lie within a genomic island whose sequence analysis shows signs of horizontal transfer from other. The genomic island from P. putida was moved into E. coli, an organism unable to degrade nicotine. An E. coli strain containing the nicotine catabolism genes on a plasmid gained the ability to degrade nicotine. This finding is important because it points to the possibility of engineering an industrial strain for remediation of nicotine-contaminated waste. Scientists have taken the genome of one bacterial species, Mycoplasma mycoides (lung disease), moved it to yeast (they added a yeast centromere so it would replicate), modified the genome using yeast genetics, and then transplanted the modified genome into a different species, Mycoplasma capricolum, replacing the M. capricolum genome. This biotechnological advance is especially exciting because genetic manipulation of M. mycoides was previously impossible. It is estimated that the synthetic genome cost US$40 million to make and took 20 people more than a decade of work. Using this technique, we might redesign prokaryotic systems and even engineer new species. Green algae can also be used to generate biofuel. Because the cells grow in aqueous suspension, where they have more efficient access to water, CO2 and dissolved nutrients, microalgae are capable of producing large amounts of biomass and usable oil in either high rate algal ponds or photobioreactors.

What are nosocomial infections? What types of microbes are involved?

Nosocomial infections refer to hospital-acquired infections from pathogens that develop within a hospital or other clinical care facility and are acquired by patients while they are in the facility. These infections are often caused by bacteria that are members of normal microbiota, many of which have gained antibiotic resistance. Most nosocomial infections are caused by opportunistic pathogens (Enterobacter sp, Pseudomonas aeruginosa, Staphylococcus aureus). Enterobacterbacteria are nosocomial opportunistic pathogens that are causing more and more infections, including up to 5% of hospital-acquired septicemias, 5% of nosocomial pneumonias, 4% of nosocomial urinary tract infections, and 10% of surgical wound infections.

VIruses bust bloom, cycle carbon

Once have large host population virus attacks and kills EHUX Dead organisms fall to ocean floor, cycles carbon Heterotrophs who live further down can use material Leads to smaller EHUX population Process repeats

Describe the difference between a bacteriostatic antibiotic and a bacteriocidal antibiotic. Under what conditions might a bacteriocidal antibiotic have little to no effect on a pathogen?

One of the most common misconceptions about antibiotics is that they all kill bacteria. This is true for a number of antibiotics and those are called bactericidal. These kinds of antibiotics kill the bacteria through direct action, usually by causing the cells to lyse. Not all antibiotics work by killing bacteria and those are called bacteriostatic. Bacteriostatic antibiotics act on the internal workings of the bacterial cell to stop it from dividing. A bacterial population that divides more slowly, or that cannot divide at all is much more easily dealt with by the body's immune system. Bacteria that form biofilms change metabolically into a less active state. Such bacteria can resist killing by an antibiotic that would normally be bactericidal if the bacteria were dividing rapidly. Think about penicillin as an example. It's mode of action is to inhibit cell wall synthesis. The lack of strong cell walls causes the cells to rupture and die (hence it's classification as a bacteriocidal). In a biofilm where the bacteria are no longer growing, so they are not building new cell walls, this drug no longer works. This is the reason why medical biofilms that form on plastic implants or catheters are difficult to eliminate.

Describe some ways that we fight back against antibiotic resistance in bacteria, including an example of new technology and technologies from the ancient past.

One way we fight back against antibiotic resistance is by administering a second molecule along with treatments of beta-lactam antibiotics. In the case of Penicillin, the second molecule, clavulanic acid, is a beta-lactamase inhibitor, meaning it works against the beta-lactamase itself. What is does is mimic the beta-lactam, so the beta-lactamases cleave it and do not cleave the penicillin. If you add just penicillin and there is no beta-lactamase, that kills the bacteria. However, when beta-lactamase is around, if you add the beta-lactam it gets cleaves, and the bacteria is not affected. With the three molecules, beta-lactam, clavulanic acid, and B-lactamase, the activity of the beta-lactamase is inhibited enough that this restores the killing power of penicillin. There are of course also new ways that we may be able to treat bacteria to prevent them from growing where they shouldn't. Researchers have used tiny peptide molecules (nanotubes) that will self assemble into tubes that can insert themselves in the membranes of bacteria, which makes them unable to maintain ion balance and they die. The peptides are able to selectively target bacteria because their membranes have differences in phospholipid content relative to mammal cell membranes. While there are some other interesting future prospects, there is also growing interest in using past technologies to help deal with the resistance problem as well. One of the most promising is the use of copper, which has historically been known to have strong antimicrobial properties. The exact way in which copper kills microbes is not fully understood but it appears to involve multiple pathways it is known that copper can react with hydrogen peroxide to produce hydroxyl radicals that attack DNA and proteins. The levels of this kind of killing are truly impressive and they work against a wide range of microbes, including Vancomycin-resistant Enterococci (VRE) and C. difficile.

Describe the difference between outbreaks, epidemics, and pandemics of disease in populations.

Outbreaks refer to sudden, unexpected occurrence of disease in populations. The term "outbreak" is typically restricted to a small focal or segment of a population. In contrast, epidemics are sudden increases in frequency disease above an expected number in the population. Examples of epidemics are swine flu and avian flu, which reached high levels of disease in human populations around the world. Pandemics refer to increases in disease occurrence within large population over wide region (usually worldwide).

Endotoxins

Part of lipopolysaccharide of Gram (-) bacteria Released when bacteria die Immunopathogenesis: fever, hypotension, shock, death

What is a pathogenicity island? What are the three principle characteristics of pathogenicity islands?

Pathogenicity islands are regions that may be found in the chromosomal DNA, on plasmid DNA, or phage genomes and contain a genes that are important to the pathogenicity of the organism. Pathogenecity islands have the following characteristics: 1) They have G+C content different from rest of genome 2) They are often flanked by phage or plasmid genes 3) They encode "virulence factors": toxins, attachment proteins, capsules

Describe how we categorize pathogens. Include location of the pathogen, the type of host that is infected, the pathogenicity of the pathogen, and mode of transmission.

Pathogens are categorized based on their location relative to the host tissue. Ectoparasites live on the surface of the host (e.g. the fungus Trichophyton rubrum that causes athlete's foot), while endoparasites live inside the host's body (i.e. the flatworms that cause elephantitis). Pathogens can also be categorized based on the health of their host. Primary pathogens cause disease in healthy hosts, while opportunistic pathogens cause disease in immunocompromised individuals. Virulence is a measure of the degree or severity of a disease. It is measured via the infectious dose and/or the lethal dose. An infection dose refers to the dose of pathogen needed to colonize 50% of a host population. This metric is usually measured by infecting small groups of animals with increasing numbers of infectious agent (i.e. pathogen) and observing how many animals the pathogen successfully colonizes. The number of microbes that kill half the animals is called the lethal (LD50) dose. Pathogens that have a low LD50 are more virulent in the host. In terms of transmission, pathogens can be transmitted vertically (parent to offspring) or horizontally (from one member of a species to another). Simple infection cycles occur in pathogens that move from human to human. By contrast, complex cycles involve vectors as intermediaries.

Describe three types of toxins that pathogens can use to affect their hosts.

Pathogens can use exotoxins and endotoxins to affect their hosts. Exotoxins are released outside the pathogen cell, into the surrounding host cell tissue. Endotoxins can hyperactivate host immune systems to harmful levels. An example of this would be the lipopolysaccharide in the outer membrane of E. coli. There are many types of exotoxins that pathogens can use to cause disease in hosts. The classes of toxin are based on their mechanism of action. Exotoxins that target the host cell's cytoskeleton can stimulate either actin polymerization or actin depolymerization. Exotoxins that disrupt cell-cell adherence are proteases that cleave proteins binding host cells to one another. Exotoxins that promote exocytosis can alter the movement of nerve cell cytoplasmic vesicles to membranes where they release neurotransmitters. Other endotoxins can hyperactivate host immune systems to harmful levels. Pore-forming toxins assemble in target membranes and cause leakage of compounds into and out of cells. The A-subunit of the toxin forms a beta barrel pore in the target host cell membrane. The hydrophobic areas of each monomer face the lipids of the membrane, and the hydrophilic residues face the channel interior. Ribosome-disrupting exotoxins are produced by Shigella bacteria (i.e. the "Shiga toxin"). Shiga toxin attaches to a host cell receptor, enters the cell, and cleaves 28S rRNA in eukaryotic ribosomes to stop translation. The shiga toxin is a natural defense of Shigella bacteria against a ciliated protist that grazes on bacteria (Tetrahymena thermophila). However, it causes acute kidney failure in humans. Shigella is an emerging pathogen, because it lives undetected in cattle and can contaminate meat products. There was an outbreak of disease caused by Shigella bacteria at Jack in the Box in 1993. Another type of exotoxin can disrupt cell-cell signaling pathways in the host. Enterotoxigenic Escherichia coli heat-stable toxin affects cGMP production. Specifically, it causes runaway synthesis of cyclic guanosine monophosphate (cGMP) in target cells. Elevated cGMP levels, in turn, trigger critical changes in ion transport and fluid movement. The result is altered electrolyte transport—inhibition of Na+ uptake and stimulation of Cl- transport. In response to the resulting electrolyte imbalance, water leaves the cell. This causes diarrhea in the host.

Canning

Physical means of food preservation Food cooked under pressure in special containers to attain high enough temperature to destroy endospores (121 degrees) for 25-100 minutes Problems: Food slated for canning could be under-processed or could spoil before canning but main concern is contamination during transfer into final container via leakage of contaminated water into cans during cooling process

Cell membrane integrity antibiotics

Poke holes in bacterial cytoplasmic membrane

Group IV: (+) sense ss-RNA

Positive strand = coding strand - serves directly as mRNA for translation → proteins Replication of RNA genome requires synthesis of template (-) strand (complementary to + strand) by viral RdRNAP → ds-RNA intermediate Ex: Poliovirus

Food contamination "poisoning"

Presence of human pathogens

Bacteriostatic

Prevents growth/reproduction of microbes, no kill

A ____ is a compound that is harmless when it enters its target bacterial cell, but is converted to a toxic form by the specific enzymes/machinery of the cell.

Prodrug

During lysogenic infection phages remain within host cell as ______ by inserting their genome into host chromosomes

Prophage

Type II Secretion System

Protein secretion (homologous to Type IV & biogenesis) check on this BAt

Exotoxins

Proteins; kill host cell, unlock their nutrients 2 component toxins (AB) Subunits: A - toxic B - attachment 9 classes based on mechanisms

Stainyzme

Psychrophiles For bioprospecting

Florey & Chain

Purified & mass produced penicillin as drug

How does meat & poultry spoil

Putrefied by decarboxylating bacteria Produces amines with noxious odors

qPCR

Quenched fluorescent probe added to PCR reaction (degraded as amplification occurs)

Biosensors

Quorum sensing

Which basic microbial cell process can be utilized in the development of biosensors that use a synchronized clock mechanism?

Quorum sensing

Clavulanic acid

Used to fight back against antibiotic resistance B-lactamase inhibitor

Stage III: Replication

RNA virus → cytoplasm replication (Influenza is an exception, it replicates in nucleus) Influenza nucleoprotein core proteins - mimic host proteins to get through nuclear membrane pores Need to make mRNA that can be translated into protein = + mRNA (-) ss-RNA strand replication - Can't be directly translated by host ribosomes -Virus brings replicase with it RNA replicase - takes + stranded RNA, makes complementary - strand, then replicase can make more + strand RNA that can be genetic material for progeny of virions (virus comes replicase w/ it) Influenza - ssRNA processing - Some copies need to serve as mRNA to make viral proteins, some as templates for genome replication - Trick to process -ssRNA is Transcription cap theft/CAP snatching -- Steals 5' cap off mRNA of host cell ---Uses endonuclease

Describe the different mechanisms of viral genome replication, depending on the genome content of the virus. Compare and contrast RNA-based viruses and DNA-based viruses in terms of their replication cycles.

RNA-based viruses sometimes have to bring in their own machinery to replicate. DNA-based viruses can sometimes integrate into the host genome (i.e. retroviruses). Both RNA-based viruses and DNA-based viruses can co-opt the host cell machinery to replicate the virus genome and make proteins for new virion particles. The Herpes virus (a DNA-based virus) can do this so well that it overwhelms the host machinery with viral replication components.

Group VII: DNA Reverse transcribing viruses

Replication cycle requires reverse transcriptase but doesn't integrate into host genome Ex: Hep B (Pararetroviruses) 1st copies its ds-DNA → RNA then RNA → DNA

ss-RNA viruses

Require host DNAP to make complementary DNA strand which then transcribed into mRNA

Tetracycline resistance

Resistant bacteria: transport proteins - efflux pumps

PCR & RT-PCR

Reverse transcription PCR → to detect RNA expression

Once inside phagosome, pathogen might secrete protein to prevent fusion with lysosome

Salmonella, Chlamydia, Mycobacterium, Legionella Stay in phagosome, moves to host membrane, expels pathogen into extracellular space Pathogen engulfed by macrophage, can survive within phagosome Macrophage can travel to regional lymph nodes, disseminate organisms through circulation

Bioprospecting

Search for organisms w/ potential commercial applications Extreme environments

What is the purpose of preparing barley grains via the mash process during the brewing of beer?

Starch cannot be fermented directly (as a biopolymer) - this is why yeast can't ferment barley grains directly. Barley is mashed in order to generate these fermentable sugars. Barley grains are germinated, allowing enzymes to break down the starch to maltose for yeast fermentation. The germinating embryo makes enzymes needed to break down the barley starch to maltose (disaccharide) and glucose. These sugars can then be fermented by yeast to ethanol and carbon dioxide; they are called "fermentable sugars". While the grain is being mashed, the temperature is raised in steps, each of which optimizes the activity of a different hydrolase. At 52°C, the protein hydrolases are activated. Then at 68°C, the starch hydrolases convert long-chain sugars to the disaccharide maltose, which can support yeast fermentation. The final temperature (77°C) inactivates all enzymes; then the mash is cooled, pressed, and filtered. The liquid filtrate of the mash is called "wort."

Reverse genetics

Start with gene, want to know phenotype

Forward genetics

Start with phenotype, want to know gene

You are testing the lethal dosage of multiple strains of a bacterium in the laboratory in a mouse model. Strain A had a LD50 of 10 cells/mg body weight while Strain B has an LD50 of 200 cells/mg body weight. Which strain is more pathogenic?

Strain A

Selman Waksman and colleagues discovered this antibiotic from soil bacteria bearing a similar name, which was subsequently used to treat tuberculosis.

Streptomycin

Selman Waksman & Streptomycin

Systematically tested antibacterial property of Stretomyces vs. various pathogenic bacteria Discovered streptomycin - could be used to treat TB

Metabolism antibiotics

Tetrahydrofolic acid synthesis → sulfa drugs (bacteriostatic) Action of sulfa drugs: -Folic acid cofactor in pathway that makes NTs -Bacteria-specific --mammals don't make own folic acid

Plasma membrane toxins

Type of exotoxin Staphylococcus aureus Pore-forming toxins in target membranes Cause compound leakage into & out of cells Alpha-toxin - forms B-barrel pore in target cell membranes -Hydrophobic areas of each monomer face lipids of membrane -Hydrophilic residues face channel interior Damage cell membranes & matrices

Describe the roles of Cro proteins, CIII, CII, and CI proteins in lysis vs. lysogeny. What is the role of FtsH protein? How does it interact with CII and CIII to determine the fate of the bacterial host cell?

The events leading to either lysogeny or the lytic cycle involve a number of regulatory proteins that function as repressors or activators or both. There are two in particular that are important; lambda c1 protein, which is a product of the c1 gene, and Cro protein, which is the product of the cro gene. The Cro protein initiates the transcription of the late genes needed for the lytic cycle to go to completion. Both Cro and C1 are transcriptional regulators: activators (of their own gene transcription) and repressors (of the other's gene transcription). The decision to pursue either reproductive pathway involves a competition between the production of these two repressors. When many phages infect E. coli, another protein becomes important. It is the CII protein, which is an activator that recognizes a number of promoter sites. Only when there are multiple infections per cell does it really become important. This is because it is normally cleaved by host cell proteases (including FtsH). The lysogenic lifecycle begins once the cII protein reaches a high enough concentration to activate its promoters, after a small number of infections. When there are multiple infections it can build up in higher densities. This is mostly because multiple infections leads to greater production of CIII. The CIII protein acts as a bodyguard for CII and protects it from host cell degradation. So when CII is in higher abundance, it activates a promoter called PRE. This promoter is important because it leads to the production of the C1 protein. CI is important, because CI promotes its own gene's transcription and blocks transcription of Cro protein. At this point, lots of genes are getting described and the competition between the Cro protein and the CI repressor really starts to count. Both the Cro protein and the CI protein bind to the Or operator sites, which regulate the production of each protein. If the Cro protein binds, it inhibits synthesis of the C1 protein. If the C1 binds, it promotes its own synthesis and inhibits the synthesis of Cro. Cro gets synthesized before C1, so its initial concentrations in the cell are higher. However, Cro binds less tightly than the lambda repressor, so it takes a higher concentration of the Cro protein to inhibit the synthesis of the C1 protein. So if CII is high abundance (not being degraded by proteases), then amount of the C1 protein will win the race over the Or regulatory site. When this happens, the C1 repressor catalyzes it own transcription, but not that from PR which makes Cro. If CII is not plentiful, which would be when the cell is in nutrient rich conditions, Cro will outcompete the lambda repressor. This will block the genes needed for lysogeny and allow the lytic pathway to proceed.

Compare and contrast the way that phage virus genome enters a host bacterial cell, and the way that the genome of animal viruses (like influenza) enter animal host cells. What is similar about those processes for the two virus groups? What is different?

The first steps in viral life cycles involve attachment and uncoating. Most animal viruses, unlike bacteriophages, enter the host cell as whole virions. There are two main ways that animal viruses enter their host cells. Some viruses attach to the host cell receptor molecule they recognize. Then, they fuse with the host cell cytoplasmic membrane and introduce their nucleoprotein core into the cytoplasm of the host cell. For other viruses, the binding of the viral protein to the host receptor is stable enough to trigger cell to initiate endocytosis around the virus. Endocytosis is a normal pathway used by mammalian cells to ingest nutrients and the virus takes advantage of this pathway to facilitate entry into the cell. After the endocytic vesicle is formed, it fuses with a lysozome, and it begins to acidify. The acidification leads to a conformational change in the matrix proteins that underlie the envelope. It also effects the envelope proteins which mediate the fusion of the host and viral membranes so that the nucleoprotein core of the virus is released into the cytoplasm. Phages (viruses that infect bacteria) are no different than other viruses in that they attach to specific receptors on the cell surface of the bacteria they infect. Phages have a specialized tail that will clamp onto the host cell. The tail fibers bind to the proteins/lipopolysaccharides on the cell surface. Then, baseplate makes contact. Contact between the baseplate and cell outer membrane generates a conformational change in the sheath - which shortens and widens, in a way that injects a tube through the outer and inner membrane of the bacteria. The peptidoglycan surrounding the injector is digested by a lysozyme protein within the needle tip. The DNA in the head of the phage is then is released into the cell under high pressure, where it begins to interact with the host cell machinery to initiate replication.

Where do we find the highest incidence of EID on Earth? Describe where the majority of EIDs are reported in the world, as well as hotspots for EID events since the 1980s. What are some of the factors that contribute to the emergence of the infectious disease?

The highest incidence of EID is in the northern hemisphere (between 30-60 degrees North). Hotspots of EID events include the northeastern U.S.A, western Europe, Japan, and southeastern Australia. Zoonotic pathogens from wildlife: Risk of zoonoses from wildlife is calculated based on the correlation between wildlife zoonoses and human population size, as well as wildlife richness (or diversity) on the landscape. This is because our interactions with wildlife in their native habitats have been increasing over time, due to things like the bushmeat trade and disruption of natural habitats. Non-wildlife zoonotic pathogens: Risk of non-wildlife zonoses is predicted based on the correlation between non-wildlife zoonoses and human population size, human population growth, and latitude. This relationship likely reflects increased human interaction with farm animals in the developed and developing worlds. Drug resistant pathogens: Risk of drug-resistant pathogens has been predicted based on the correlation between drug resistant pathogen emergence and human population density, human population growth, and latitude. This relationship likely reflects development of antibiotics in the developed world, as well as increased misuse of antibiotics in the developed and developing worlds. Vector-borne pathogens: Risk of vector-borne diseases is predicted based on the correlation between vector-borne pathogens and human population density, as well as the relationship between vector growth and climate change. This could be because higher human population density provides more chances for vectors like mosquitoes to become infected by interacting with humans and transfer the disease. Other factors: global air travel, breakdown of public health measures.

Epidemics are categorized based on their source and mode of spread. Do common source epidemics (caused by pathogens with a "complex" infection cycle) spread more or less quickly through populations compared to propagated epidemics (caused by pathogens with a "simple infection cycle)?

The mode of disease transmission can affect the rate of epidemic development in a population. Pathogens with a complex infection cycle, such as those from a common source like contaminated food (i.e. Staphylococcus bacteria), can cause epidemics quickly. Pathogens with a simple infection cycle, such as those that are propagated from one person to another in the population (i.e. Streptococcus bacteria) can cause epidemics more slowly.

Describe the processes that drive succession of microbes over time during cocoa fermentation. Which species product acids? Which species consume acids? How does the process ensure that the cocoa seed (i.e. bean) does not sprout)?

The process of chocolate fermentation has been used for hundreds of years. At harvest, ripe pods of the cocoa plant are cut open and the pulp covered seed mass are wrapped in banana leaves is put into what are called sweat chambers in the ground. Fruit pulp ferments, liquefies, and drains away, while the beans acidify and turn brown. From there, a succession of microbes (indigenous to the beans themselves) begins the breakdown the plant material. (1) It begins with a series of yeasts, which hydrolyze the pectin that covers the seeds and ferments the sugars into ethanol, acetate, and CO2. The associated increase in temperature and alcohol concentration disfavors the growth the yeasts, so their populations decline. The yeast have also consumed citric acid, which raises the pH and favors the growth of lactobilli species. These species produce lactic acid from acetate (one of the byproducts of yeast metabolism), but eventually run out of acetate. (2) Air is then introduced into the mixture, which promotes the growth of Acetobacter species and oxidizes the phenols in the bean, making them brown. The rise in oxygen levels allows the Acetobacter species to oxidize ethanol to acetic acid, and also further oxidize the acetic acid to carbon dioxide and water. The acetic acid produced by the Acetobacter is key, because it kills the germinating sprout in the seed and releases enzymes that further breakdown protein and carbohydrates. (3) High temperatures and increase in pH along with increased aeration leads to the development of aerobic spore-forming bacteria of the genus Bacillus. Aerobic spore-forming bacteria form chemical compounds that cause acidity and sometimes off-flavoring if fermentation continues for too long. The entire process usually takes about 5-7 days. If it is stopped too early, the chocolate will be too bitter and if it goes too long, bacteria will grow in the seed instead in just the pulp around the seed. After fermentation, the seeds are called beans and are spread out to dry in the sun.

Ribosome activity toxins

Type of exotoxin Shiga Shigella dysenteriae Attach to ganglioside Gb3, enters cell, cleaves 28S rRNA in eukaryotic ribosomes to stop translation Gene encoding Shiga: Part of phage genome 5 subunits → Binding 1 subunit → Toxic (A1) Activity activated by low iron → Expression Shiga ↑ → kills host cells → release iron Inhibit protein synthesis

Signaling pathway toxins

Type of exotoxins E. coli ST Enterotoxigenic E. coli Heat-stable toxin affects cGMP production Runaway synthesis of cGMP in target cells ↑ cGMP → ∆s ion transport & fluid movement ∆s electrolyte transport Inhibits Na ⁺ uptake Stimulate Cl⁻ transport Imbalance → H₂O leaves cell → diarrhea

Describe the two main modes of pilus development in pathogenic bacteria.

The two main modes of pilus development in pathogenic bacteria include Type I pili and Type IV pili. In the synthesis of Type I pili, proteins that will form the pilus (i.e. "pilin" proteins) are secreted by the Sec system to the periplasm, where they are chaperoned by PapD to the site of assembly. PapC (an "usher" protein), assembles the individual proteins (pilins) in the proper order. Assembly starts with the tip protein, PapG, which ultimately binds to carbohydrates (mannoses) on host membranes. After ushers add PapF and PapE, identical PapA pilin subunits are strung together in a series to form the shaft. The subunits fit together like pieces of a jigsaw puzzle. The arrow at the head of the elongating pilus indicates the direction of pilus growth. In the synthesis of Type IV pili, PilA is the pilin protein, and PilC1 and Y1 form the attachment tip. Assembly and disassembly require the hydrolysis of nucleoside triphosphate (NTP) and take place at the inner membrane, not in the periplasm.

What is the role of the hemagglutinin trimer in attachment and entry of a virus into a host cell? Describe 1) how sialic acid is involved in host-specificity of hemagglutinin activity and 2) how fusion with a lysosome affects hemagglutinin protein conformation and activity.

The virus first attaches to its target mammalian cell by means of protein called hemagglutinin. Hemagglutinin proteins form trimers that project out from the viral surface, making it appear to be studded with spikes. The protein spikes bind very specifically to sialic acid residues of the surface of the host cell. Sialic acid is a sugar found widely on mammalian cells, so influenza can infect many types of cells. Cell-surface glycoproteins contain a terminal sialic acid. The sialic acid polysaccharide of the glycoprotein binds hemagglutinin, attaching the virion and enabling endocytosis. The way that some cells are infected by certain strains of influenza and others aren't has to do with the bond of the sialic acid to the rest of the host cell polysaccharide. If the sialic acid is bonded one way, one type of influenza will recognize the cell and infect it. If it is bonded another way then another type of influenza will recognize it but not the first one. This difference is important because we are protected from deadly avian influenza viruses that don't recognize the main human cells they interact with.

Describe the various mechanisms of antibiotic resistance.

There are four general ways that bacteria can become resistant to antibiotics. First, they can modify the target of the antibiotic so that it no longer binds to the antibiotic. This how bacteria become resistant to vancomycin. Vancomycin-resistance genes code for a suite of proteins that disrupt D-alanine-D-alanine bonds on the peptide portion of growing peptidoglycan chains. Another way bacteria can resist antibiotics is to simply pump the antibiotic out of the cell, so that it can't reach a level high enough to kill or inhibit growth (in the case of tetracycline). A third way is to modify the antibiotic itself by adding stuff to it that makes it no longer stick to its target (in the case of Chloramphenicol). The final way is if the drug is destroyed before it gets to its target (as in Penicillin resistance).

How has increased human contact with animals (wildlife and non-wildlife) promoted worldwide EID occurrence since 1940?

There are many ways in which human contact with animals has increased over time is through land use changes, agricultural intensification, food industry changes, and hunting for bushmeat. One important way in which humans have increased contact with wildlife is through land use changes. Essentially, humans have built residences, businesses, and other spaces we inhabit on top of wildlife habitat (i.e. habitat disruption). This has increased our interactions with wildlife like deer, raccoons, rodents, bats, opossums, etc. that can carry and/or transmit diseases. In addition, increasing the number of livestock managed through agricultural intensification has increased our interactions with farm animals worldwide. Demand for bushmeat has increased worldwide, putting pressure on local hunters to intensify their hunting activities. Because bushmeat hunting increases the risk for blood-blood contact between humans and live animals, there is increased risk of disease transmission.

Ultimate Immune Avoidance: Intracellular pathogens

Tricks to misdirect immune system Designed to buy time to overwhelm host -Molecular mimicry -∆ing cytokine profiles -Stopping programmed host cell death (mRNAs) -Interfering w/ autophagy (M2) -Redirecting ubiquitylation signals Invasion: 1. Bacterial pathogen attaches to host cell membrane 2. Pathogen induces phagocytosis 3. Once inside phagosome, pathogen has 1 of 3 fates A. Mature in acidic environment B. Secrete protein to prevent fusion w/ lysosome C. Use hemolysin to break out

Two Types of Pili

Type I Type IV

Vibrio cholera use this which secretion system to deliver Cholera toxin to host cells?

Type VI

Viral shunt

Viruses catalyze nutrient movement from organisms to organic particles & minerals Uninfected cells sink, carbon is removed from cycle Virus infection returns more C as CO2 to atmosphere

Which environmental factors may have led to the emergence of West Nile Virus in the U.S. in 1999?

West Nile Virus emerged in NYC in 1999. The virus, a (+) single-stranded RNA virus, comes from the flavivirus family, which includes the viruses that cause yellow fever and dengue (breakbone fever). These pathogens are all endemic to warmer environments, such as the Mediterranean and North Africa-hence the name "West Nile virus." The reason for the sudden appearance of West Nile virus in the United States is not known for certain, but some climatologists argue that West Nile and other emerging pathogens arise from unusual weather patterns related to global climate change. The unusually warm winter enabled a few mosquitoes carrying the virus to survive winter (until the spring.) The spring that followed was drier than usual, forcing birds to spend more time at dwindling water pools, where mosquitoes were concentrated. The July heat wave then increased the rate of viral replication in the mosquitoes. As the cycle continued, mosquitoes re-infected birds, which then infected more mosquitoes. Eventually, the cycle included mosquitoes infecting humans

Stages IV & V: Assembly & Departure

[Genomic fragment viral proteins] ↑ → Nucleoprotein core interacts with viral genome segments → Starts to assemble → Buds off Problem for influenza: - HA spikes stick virus to cell - NA cleaves bond b/w HA & sialic acid