General Microbiology - Final Exam

You have discovered a new single celled organism with a nucleus, no rigid cell wall, and multiple motile flagella. Its average diameter is 30 micrometers. The new microorganism is likely….?

Protozoan

What kind of bacteria contain lipopolysaccharides in their cell wall? And what is its effect?

Found in gram-negative bacteria. It acts as a key structural component and endotoxin which stimulates the immune system.

You are working with a bacterium that was previously shown to twitch swim and swarm and glide. Which of the following best describes how a mutant lacking the gene encoding for the flagella filament would move differently compared to a wild type strain that does not have any mutations?

The mutant would be unable to swim and swarm

How does the bacterium utilize the twitching motility?

A form of surface translocation that occurs through the extension and retraction of type IV pili and is independent of flagella.

You are studying motility patterns of live bacterial cells in a liquid medium. Which type of microscopy allows you to see the cells without staining?

Dark-Field Microscopy or Phase-Contrast

What is the composition of the flagellum, and how does it harness its power?

The filament is composed of repeating peptide assembled into a hollow tube. The flagella hook attached the filament to the basal body. The basal body has four rings in gram-negative and two rings in gram-positive bacteria. The flagellum gets its power through a proton-motive force.

What is the role of the replisome?

The replisome is a molecular machine that copies DNA; its job is to unwind the DNA double helix and build two new, identical DNA strands using the originals as templates, ensuring accurate genetic inheritance during cell division. Includes but not limited to helicase and DNA polymerase.

What is the role of the parS-ParA system?

The ParS-ParA system works to ensure that bacteria and archaea accurately sort their genetic material during cell division.

ParS: A specific DNA sequence on the chromosome or plasmid that acts like a centromere (a "handle" for moving DNA).

ParB: A protein that binds to the parS sequence and forms a complex, marking the DNA to be moved.

ParA: An enzyme (ATPase) that binds the inner wall of the cell and uses energy (ATP) to dynamically push or pull the ParB-parS complex toward the correct cell location.

What is the role of the MIn system?

The Min system is a dynamic protein system in bacteria that ensures cell division occurs accurately at the cell's midpoint. It acts as a negative regulator, preventing the formation of the essential Z-ring (made of FtsZ protein) at the cell poles.

What is the role of the divisome?

The divisome is a large, multi-protein machine in bacteria responsible for orchestrating cell division (cytokinesis). It coordinates the final steps of splitting one mother cell into two genetically identical daughter cells

What is the role of FtsZ?

FtsZ protein is a key component of bacterial cell division, where it forms a "Z ring" structure at the future division site. This ring is crucial for recruiting other proteins, constricting the cell, and initiating the formation of a new cell wall (septum) to separate the two daughter cells.

What is the role of the elongasome?

The elongasome is a multi-protein complex responsible for bacterial cell elongation and maintaining the rod shape in non-spherical bacteria. It directs the synthesis and insertion of new peptidoglycan (PG) material into the cylindrical sidewalls of the cell, effectively causing the cell to grow in length before dividing.

Which microbiologist developed a set of postulates to establish a causal relationship between a microorganism and a specific disease?

Robert Koch

What type of transport involves powering the transport process by transferring a phosphoryl group from a donor onto a transporter and then onto the molecule being transported?

Group Translocation

What is the mechanism for the β-lactam antibiotics, and an example?

B-lactam antibiotics, like Penicillin, work by inhibiting bacterial cell wall synthesis, specifically targeting penicillin-binding proteins (PBPs) that cross-link peptidoglycan strands, crucial for cell wall integrity, leading to weakened walls, osmotic lysis, and bacterial death

What is the mechanism for glycopeptides, and an example?

Glycopeptide antibiotics work binding to peptide side chains blocking the ability of enzymes to catalyze cross bridges formation during peptidoglycan synthesis. The most common example is vancomycin.

What is the mechanism for macrolides, and an example?

The antibiotics bind reversibly to the P site on the 50S ribosomal subunit of the bacterial ribosome interfering with protein synthesis. An example is Erythromycin.

What is the mechanism for aminoglycosides, and an example?

Binding to the 30S subunits interfering with protein elongation. An example is streptomycin.

What is the mechanism for fluoroquinolones?

Binding to DNA gyrase/topoisomerase interfering with DNA replication.

What is the mechanism for rifamycin?

Binding to RNA polymerase interfering with transcription.

How does the mechanism of moist heat (autoclaving, boiling, pasteurization) impact bacteria?

Denatures proteins, degrades nucleic acids, and disrupts cell membranes

How does the mechanism of UV radiation impact bacteria?

Results in the production of pyrimidine dimers

How does the mechanism of ionizing radiation impact bacteria?

Dislodges electrons producing ions, which then cause damage to DNA and other molecules.

How does the mechanism of ethylene oxide gas impact bacteria?

Alkylates proteins, DNA, and other molecules

How does the mechanism of metals and aldehydes impact bacteria?

Denatures proteins

What are the main characteristics of proteobacteria?

• Similar in size to mitochondria

• cell type is a diderm, has a double membrane, like mitochondria

• The 16s rRNA gene sequence of mitochondria is most similar to this cell type

• Gram-negative

What are the characteristics of lokiarchaeum?

a group of single-celled microorganisms (archaea) considered a crucial evolutionary "missing link" between simple prokaryotic cells and complex eukaryotic cells (which make up all animals, plants, and fungi). Their primary characteristic is the possession of a unique set of genes that were previously thought to be exclusive to eukaryotes.

What is the role of the lysozyme?

Serves as a crucial component of the innate immune system by providing protection against bacterial, and to some extent, viral and fungal infections. It acts as a natural antimicrobial agent.

What is the role of a neutrophil?

The primary role of a neutrophil is to serve as the first line of defense in the innate immune system, rapidly migrating to sites of infection or inflammation to destroy invading microorganisms like bacteria and fungi.

• NETs

• Phagocytosis

What is the role of a B-cell?

It is a component of the adaptive immune system that identifies specific pathogens (antigens) and produces large quantities of highly specific antibodies. These antibodies circulate in the bloodstream to neutralize toxins, flag invaders for destruction by other immune cells, and provide long-lasting immunity through the formation of memory B-cells.

• Neutralize pathogens by binding to them

• Facilitating phagocytosis through opsonization

• Activating the complement system

What is the role of the microbiota?

trillions of microorganisms (including bacteria, fungi, viruses, and archaea) that inhabit the body, primarily in the gut, skin, and mucosal surfaces. They form a largely symbiotic relationship with the host, performing vital functions essential for overall health and apart of the innate immunity.

What is the role of T-cells?

T-cells (T lymphocytes) are essential components of the adaptive immune system responsible for cell-mediated immunity. They coordinate the entire immune response and directly kill infected or cancerous cells.

What is the role of an antibody?

The primary role of an antibody (also known as an immunoglobulin) is to act as a highly specific targeting and elimination tool within the adaptive immune system. Produced by B-cells, antibodies circulate in body fluids and function in multiple ways to identify and neutralize foreign invaders (antigens).

What is the role of complement?

The complement system is a vital part of the innate immune system that enhances (or "complements") the ability of antibodies and phagocytic cells to clear pathogens and damaged cells from the body. It acts as a rapid, cascading defense system in the blood and tissues.

What is the role of the mucous membranes in the immune system?

The mucous membranes function as a critical part of the innate immune system, serving as the body's largest and most active interface for defense against external threats. They line surfaces exposed to the environment, such as the respiratory, gastrointestinal, and urogenital tracts.

The structure on the surface of our cells that displays potential antigens to T-cell receptors is call the _______.

Major Histocompatibility Complex (MHC)

What type of immune response is most important in controlling extracellular bacterial infections?

The most crucial immune response for extracellular bacteria is the Humoral Immune Response, involving antibodies from B cells and the complement system, which neutralize bacteria and tag them for destruction

What are the outcomes of complement activation?

• recruitment of inflammatory cells via anaphylatoxins (e.g. C3a, C5a)

• formation of the membrane attack complex (MAC)

• opsonization of pathogens by complement components like C3b

The most common cause of bacterial pharyngitis in children is…?

Group A Streptococcus (GAS)

What diseases are caused by Group A Streptococcus (GAS)?

• strep throat

• scarlet fever

• necrotizing fasciitis

Virulence factors for H. pylori?

• Shiga-like toxin

• Urease

• VacA

What is ID₅₀?

the dose that will cause an infection in 50 percent of the population

True or False: Complement activation can lead to inflammation?

True

When does acquisition of the human microbiome normally begin?

During childbirth

In what way does the microbiome contribute to innate immunity?

By physically blocking pathogen adhesion sites

Which of the following molecules recognized pathogen-associated molecular patterns (PAMPs)?

Toll-like receptors: important mediators of inflammatory pathways in the gut which play a major role in mediating the immune responses.

What is the crucial co-stimulatory molecule on T cells that binds to B7 on antigen-presenting cells (APCs)?

CD28, provides a vital second signal for full T cell activation, proliferation, and differentiation, crucial for launching adaptive immune responses.

Unlike biochemical or genetic tests, MALDI-TOF identifies microorganisms by:

Comparing protein mass spectra from the sample to a database of known organisms

What patient group is at highest risk of disseminated histoplasmosis?

Immunocompromised individuals

Eukaryotes use the membranes of organelles for the electron transport chain involved in respiration and photosynthesis, while prokaryotes…?

use the cytoplasmic membrane for respiration and photosynthesis associated electron transport

Strains of lactobacilli are used to make cheese because they ferment. If a bacterium uses fermentation as its only source of metabolism it will grow:

at the same rate in either the absence or presence of oxygen.

Bacillus subtillis uses the Embden Meyerhof and Pentose Phosphate Pathways. Which pathway produces NADPH?

Pentose Phosphate Pathway

What are the six steps of cloning?

1. Isolate DNA to be cloned

2. Amplify (use PCR)

3. Digestion of a vector

4. Ligate DNA into vector

5. Introduce the recombinant vector into cell

6. Select for cells containing DNA

What are the basics of PCR

• Denaturing, Annealing, Extension, and Cycling

• Buffer is needed to produce correct conditions for polymerase to function

• dNTPs (free nucleotides)

• Primers, DNA Polymerase, DNA template, and thermocycler needed

What are the different used for End Point and Real-Time PCR

• End point = used for cloning and diagnostics

• Real-time = used for diagnostics and gene identification

What is the purpose of gel electrophoresis?

Separates macromolecules based on size and charge. Helps to determine if PCR was done correctly & vector digestion has occurred.

Define Restriction Enzymes:

Derived from bacteria, recognize specific sequence of DNA and cut

• ligase is used to combine target DNA with the vector

• product of basic science research

• cleaves unmethylated DNA at specific nucleotide sequences to defend against phages

Define cloning vectors:

plasmids, circular extrachromosomal genetic elements. Selectable marker (antibiotic resistance). Contains origin of replication.

Is transformation a rare or common process?

Rare!

How do you make a bacteria competent?

Chemical competence = exposure to CaCl₂ in presence of heat shock

Electrical competence = exposure to cold water washed makes it take up DNA when zapped with electricity

Where do viruses come from?

• Viruses have independently evolved at least 20 times

• 3 main hypothesis:

  • Viruses predate or coevolved with cells

  • Reduced forms of cellular life

  • Arose from genetic elements that gained the ability to move between cells

What are the reasons for and against if bacteria are alive?

• No: viruses lack properties shared by all life and cannot replicate without a host

• Yes: Have DNA/RNA, they evolve, and they engineer their environment leading to the construction of unique organelles

Define host range, permissibility, and susceptibility…

• Spectrum of host cells that a pathogen can infect.

• Does the host cell have everything the virus needs

• can the virus bind

How do viruses impact ecosystems…

• Bacteriophage specific to microbes release dissolved organic material into aquatic material

• allows some animals/species advantages over the other

• can lead to ecosystem shifts or famines (ex: rinderpest pandemic of 1890)

Define viron:

Mature virus particle

Which are more hardy: naked capsids or enveloped viruses?

Naked capsids because the envelopes are more sensitive to changes in the environment.

Define enveloped virus:

Virus includes nucleocapsid and envelope, this is required for pathogenicity

What are the roles of enzymes packaged in the virion?

Lysozyme = makes entry hole for the phage

Neuraminidase = untethers virus from host cell to continue replicating elsewhere

What is the Baltimore scheme?

Developed by David Baltimore.

• Groups based on their genome and mRNA

• Should not be used to direct lineage/relationships in the same class

• demonstrates class specific host preference

Explain the latent, eclipse, and maturation phases in the viral replication cycle:

• Latent Phase

  • eclipse phase: time when no virions are detected

  • maturation phase: packaging of nucleic acids in capsids (first virions are detected)

    • burst size = number of virions released

Steps in the viral replication cycle

1. Attachment: to receptor on host

2. Entry: either virus or part

3. Synthesis: viral nucleic acids produced

4. Assembly: nucleic acids in capsids

5. Release: host lysis or budding

define virulent phage:

lytic cycle leads to phage replication and lysis of cell

define temperate phage:

capable of lytic and lysogenic stages (viral DNA is integrated forming a prophage but will undergo lysis under stress conditions)

Example of a latent virus that undergoes induction

Herpes is an example as it can reoccur in times of stress

Basics of T-4 Phage

• model phage that infects E.coli

• T-4 attaches to LPS

  • tail fibers are particular to their target

• Entry/Penetration

  • tail fibers retract and pins with cell wall

  • T4 lysozyme forms small pore in peptidoglycan

  • tail sheet contracts

• Synthesis

  • chops up bacterial genome and inhibits transcription and translational factors

  • starts to make proteins (early and middle/late stage)

• Assembly

  • puts everything together

  • occurs essentially at the same time as synthesis

• Release

  • two late stage enzymes disrupt the membrane and peptidoglycan

Define prophage:

Phage genome integrated into host. Mediated by recombinase/integrase (phage encoded).

Generalized vs Specialized transduction

Generalized = any gene in the bacterial genome is packaged in viron

Specialized = genes near insertion site are repeatedly packaged in the viron with phage DNA

What are the three main bacteria defenses against phages?

• CRISPR; cuts out specific DNA seq and chops. Specific, needs previous exposure.

• Absorptive infection; induce cell suicide through inactivation of the antitoxin, for the benefit of the community

• Restriction nucleases; cleaves unmethylated DNA and is degraded. Prevents it from chopping up its own DNA.

What is an adjuvant?

It is an ingredient added to a vaccine that is used to create a stronger immune response. Stimulate inflammation at the site of the vaccine, ensuring that the 3 required steps for T-cell activation are achieved

Why is it harder to achieve herd immunity with diseases that have a high R_o?

More people need to be vaccinated due to higher transmission rates, and not everyone can get vaccinated.

What characteristics are shared by all vaccine types?

they elicit an immune response that allows the recipients immune response to remember the antigen it encountered

compare and contrast the uses of vaccines, antibiotics, and antivirals

vaccines are given prior to exposure and promote the activity of our immune system. antivirals and antibiotics are typically given after exposure inhibiting or destroying the pathogen allowing our immune response the time and opportunity to successfully clear the pathogen.

What are three reasons why vaccines fail?

inadequate immune responses, excessive side effects, and limited long-term immunity.

Which type of vaccine is likely to promote the most effective and long-lasting immune response?

Whole agent attenuated vaccines elicit the longest and most effective immune response. This is because it stimulates the production of long-lasting memory T and B cells. They also have many antigens that are recognized and are “remembered” by the immune system.

Describe how the live attenuated polio vaccine was produces and how the virus used in this vaccine is distinct from wild-type polio virus?

The polio virus passes through a non-human primate host 24 times and then through monkey kidney cell cultures 42 times, those plaques were then purified and mutation that let to attenuation were identified in the 5’ end that resulted in a loss of specificity of the virus to neurons. Instead when the attenuated virus is injected, it replicates only in intestinal cells which leads to high mucosal immunity and protection from the pathogen at its initial site of replication. 

Why it is inappropriate to suggest that the SARS-CoV-2 mRNA vaccine modifies the recipients genome?

the mRNA mediates the production of viral protein (specifically COVID spike protein) and then is degraded. It is in no way integrated into the host genome.

What are some characteristics of following vaccine type: while agent, live attenuated

• whole living virus 

• obtained by directly mutating the virus or putting in conditions that promote mutation

• pros: they elicit a strong memory response, vaccine escape is unlikely because of many antigenic sites

• cons: potential reversion, risk to immunocompromised, some spread of attenuated strain

• example: Polio vaccine

What are some characteristics of following vaccine type: whole agent, killed

• whole killed virus

• pathogen is inactivated by physical or chemical methods

• Pros: elicits a strong memory response, vaccine escape is unlikely because of many antigenic sites, no risk of infection, can be used in immunocompromised

• Cons: higher dosages required compared to live attenuated due to lack of replication, boosters are often required

• example: Flu vaccine

What are some characteristics of following vaccine type: subunit

• purified antigen that elicits an immune response

• Antigens are purified from cultures of the pathogen, or antigens are produced in mass using recombinant DNA technology

• Pros: no risk of infection or side effects on immunocompromised, stable, and easier to store.

• Cons: time-consuming and research-intensive, requires boosters, a lot of protein is needed, or an adjuvant to make potent

• example: RSV vaccine

What are some characteristics of following vaccine type: conjugated subunit

• purified non-protein antigen that elicits an immune response attached to a protein

• antigens are purified from cultures of the pathogen, or antigens are produced en mass using recombinant DNA tech

• pros: no risk of infection/side effect, stable and easy to store

• cons: time consuming and research intensive, requires boosters, a lot of protein 

• example: Hib vaccine

What are some characteristics of following vaccine type: nucleic acid subunit

• DNA or RNA encoding for a protein antigen

• nucleic acid is synthesized and then packaged in a liposome

• pros: no risk of infection/side effects, stable but liposome requires cold chain (-80 freezers), as soon as genetic info is identified that encodes for an antigen the nucleic acid can be quickly synthesized

• cons: requires frequent boosters, public misinformation that they cause genetic modifications (they do not)

• example: Covid

What are some characteristics of the following vaccine type: vector

• non-pathogenic virus expressing an antigen that elicits an immune response from the pathogen you want to offer protection against

• genetic modifications of the non-pathogenic vector virus to ensure expression of the antigen

• pros: vector viruses approved to the safe can be used, minimal reversions, well studied, elicit a strong immune response

• cons: not safe for immunocompromised, antibodies against vector can clear it before an immune response to the antigen is stimulated, laborious

• example: EBOV vaccine

what is vaccine escape?

the phenomenon where a virus mutates to a point where it is no longer recognized and effectively counteracted by the immune response generated by existing vaccines. This results in reduced vaccine efficacy and a higher risk of infection, re-infection, or disease even in vaccinated individuals, which is known as a breakthrough infection

How is microbial metabolism relevant to ecosystems?

It helps with the process of nutrient cycling and provides nutrients and oxygen to other organisms. For example, cyanobacteria are primary producers that contribute to dissolved organic material in aquatic environments. They also produce oxygen which is essential for any aerobe in the ecosystem.

How are microbial communities relevant to climate change?

Microorganisms contribute heavily to carbon cycling. Decomposition leads to the release of CO₂, carbon fixation converts CO₂ to organic matter, and the production and oxidation of methane. Methane and CO₂ are greenhouse gases, so the contribution of microbes to this pool has a direct impact on climate change by removing these strong greenhouse gases.

How do microbial metabolism relate to agriculture?

Agriculture is an economically relevant ecosystem, so many of the points above apply. Importantly, microorganisms contribute to mineralization and nitrogen fixation, which helps plants grow b/c they need nutrients.

How does microbial metabolism relate to food microbiology?

Fermentation plays a major role in creating many foods we eat (kimchi, cheese, etc.) as well as the prevention of food spoilage.

How does microbial metabolism relate to infection?

Microbial metabolism impacts the growth of microorganisms, so understanding it is important for control of pathogens. Furthermore, unique metabolic pathways can be detected for diagnostics.

Why is ATP such a good energy currency for cells?

ATP hydrolysis is exergonic and can easily be coupled with endergonic reactions because of ATP’s high phosphate transfer potential.

What is substrate level phosphorylation?

a metabolic process where a phosphate group is directly transferred from a high-energy substrate molecule to ADP, forming ATP. Occurs in glycolysis