MICR 271 Module 6
Griffith used two strains of S. pneumoniae to infect mice - a type III-S smooth strain which was ______________, and a type II-R rough strain which was _______________.
Virulent, nonvirulent
Key discovery: Discovery of a three-base code in T4 bacteriophage Scientific significance?
all forms of life use this principle to mediate protein syntheis
Key discovery: Virulent pathogens can be attenuated Scientific significance?
conceptual breakthrough for the development of vaccines
Key discovery: Transformation discovered in bacteria Scientific significance?
helped form the basis of molecular genetics
What does the iChip consist of?
-central plate that houses growing microorganisms -semi-permeable membranes on each side of the plate (separate plate from environment) -two supporting side panels
1957 - Microbial Production of Amino Acids
-discovers that bacteria can be used to produce monosodium glutamate -leads to a new industry
What is one mechanism that could be responsible for the development of autoimmune diseases:
-disruption of the barrier function in the GI tract -could occur through disruption of tight junctions —> allowing infiltration of microbes form the lumen into underlying tissue -this would result in chronic inflammation
How does studying cheese rinds overcome the challenges of studying complex microbial communities?
-facilitates moving from simply documenting diversity via metagenomics, which can be done with lots of microbial systems, to actually studying the microbial interactions that maintain diversity w/in microbial communities
Changes in immunogenicity and autoimmune disease:
-food additives could change the immunogenicity of a nutrient making antibodies that are cross-reactive with tight junction proteins
Changes in microbiota composition and autoimmune disease:
-food composition changes intestinal microbiota -e.g. abnormal microbiota composition in GI tract as a major driver of increased barrier permeability that amplifies the level of autoimmunity b/c of pro-inflammatory environment
1906 - Energy and Carbon Sources
-groundbreaking work on methane-using and methane-producing bacteria -first proof that methane can serve as an energy and carbon source
1988 - PCR Technology
-heat stable DNA polymerase from Thermus aquaticus to establish PCR technology
What is the fundamental property of CRISPR-Cas that makes it a useful tool for genome editing?
-highly precise tool which allows for the exploration (i.e., cleaving, mutation) of a specific region inside the genome to see the impact that this mutation will have on the cell
Cheese rind experiments - What was the scientist's main goal in this work?
-hoping to find a workable system that would allow them to study how microbes interact in a natural community
1973 - Recombinant DNA
-if DNA is broken into fragments & combined w/ plasmid DNA, such recombinant DNA molecules will reproduce if inserted into bacterial cells
What is the first known example of a living organism passing along an expanded genetic code to subsequent generations?
-in 2014, team of American scientists synthesized a plasmid containing natural T-A and C-G bps along with UBP's -they inserted the plasmid into E. coli where it was successfully replicated
Immune system (gut-brain axis)
-intestinal microbiome can prompt immune cells to produce cytokines that influence neurophysiology
1861 - The Pasteur Effect
-introduces terms aerobic and anaerobic -observes that alcohol was produced in absence of oxygen when sugar is fermented
Cheese rind experiments - What are the challenges in studying complex microbial communities?
-it is very difficult to culture all of the microbes in that system, bring them into the lab, and recreate those communities
Experiments published in 2006 took gut microbes from people with healthy and unhealthy weights and transplanted these microbes into humanize, germ-free mice. What 2 things were shown?
-it was shown that mice would gain weight or lose weight, depending upon the type of microbiome sample they had -it was also shown that the microbiome of obese people had adapted to being more efficient at converting nutrients into calories or 'harvesting energy'
Bacterial molecules (gut-brain axis)
-microbes produce metabolites such as butyrate, which can alter the activity of the cells in the blood-brain barrier
Enteric events also exert their effects through which systems?
Enteric endocrine, immune, and metabolic systems
Key discovery: Identification of Thermus aquaticus Scientific significance?
isolation of a heat stable polymerase, which is now used for PCR
Intestinal _______________ cells line the gut, while inside the _________ is home to trillions of microbes. A dense network of bloods vessels and neurons innervate the gut and connect to the ________ via the vagus nerve.
Epithelial, lumen, brain
What did the work with expanding the genetic code in 2012 open the door to?
Eventually designing new amino acids and proteins
TRUE OR FALSE: a study published in 2013 showed that giving ASD mice a specific bacterial strain, Bacteriodes fragilis, decreased gut epithelial barrier function and increased the ASD-associated behaviours.
FALSE - it INCREASED gut epithelial barrier function and DECREASED the ASD-associated behaviours.
What ratio is higher in IBS subjects?
Firmicutes:Bacteriodetes
Griffith discovers transformation and launches the field of molecular genetics:
Griffith was the first to show that bacteria can shuttle 'information' between cells in a process he called 'transformation'
What is a stress-related, functional brain-gut-microbiota profile associated with an altered gut microbiota profile and increased intestinal permeability?
IBS
Key discovery: Identification of Thermus aquatics Scientific significance?
Isolation of a heat stable polymerase which is now used for PCR
2010 Creation of a bacterial cell controlled by a chemically synthesized genome - They made the resulting 1.08-Mbp genome from a strain of Mycoplasma by synthesizing _______ fragments of DNA and including _______________ that allow them to differentiate the synthetic genome from a naturally occurring one.
Large, watermarks (inserted encrypted info into genome)
In obsess patients with asthma, _______ levels are often high and _________ levels are low, which results in system inflammation.
Leptin, adiopectin
When heat-killed smooth strain OR rough strain is injected in a mouse, it _______. If a mix of the two stains is injected into the same mouse, it _________.
Lives, dies
Who developed innovate laboratory glassware and experimental methods that conducted to the young field of bacteriology in the late 19th century?
Louis Pasteur
Another major role of the intestines is to facilitate the uptake of ______________, other microbial products, and _______ across this barrier.
Metabolites, water
Altered GI __________ and increased intestinal _____________ are often found along with ASD, pointing to the involvement of gut microbes in this disorder.
Motility, permeability
3 things that can lead to obesity? 3 things that obesity can cause?
Over-nutrition, low physical activity, genetic factors —-> obesity —> cardiovascular disease, type 2 diabetes, low grade systemic inflammation
Lipid II is a precursor for _____________. Lipid III is a precursor for _____________.
Peptidoglycan, teichoic acid
In 2012, researchers designed a totally new nucleotide base and then used it in a ____________ to show that DNA with his base could be _____________.
Plasmid, replicated
What two molecules are specifically linked with obesity and asthma?
Pro-inflammatory leptin & anti-inflammatory adiopectin
Key discovery: Lac region of E.coli was transported into another organism Scientific significance?
Realized chromosomes can be redesigned and genes moved
Lower fibre, high fat diets are often consumed by obese patients and this affects the microbiome and alters _______ production.
SCFA (short chain fatty acids; they are the breakdown product of fibre by microbes in GI tract)
Smooth vs rough strain?
Smooth —> presence of a protective capsule layer on the bacterium Rough —> absence of a protective capsule layer on the bacterium
Key discovery: Lac region of E.coli was transported into another organism Scientific significance?
realized chromosomes can be redesigned and genes moved
In 2010, the first example of a bacterium capable of replication with a totally _____________ genome was published.
Synthetic
There is a highly complex association between the incidence of obesity, asthma (autoimmunity), and the gut microflora that appears to be tied together by the control of...?
Systemic inflammation
In 2015, researchers reported the discovery of a new antibiotic called ______________. This new drug was found using a unique screening tool called the ________ that allows compounds to be isolated from soil microorganisms that have not been previously grown in the lab.
Teixobactin, iChip
The walls of the digestive system contain a "brain in your gut". What do scientists call this 'second brain'?
The enteric nervous system (ENS)
TRUE OR FALSE: ASD can be modelled in mice by injecting pregnant mice with a form of dsDNA that mimics viral infection. The offspring display ASD-like behaviours and GI dysfunction.
True
TRUE OR FALSE: animals studies have no conclusively shown that the composition of the gut microbiota has a significant effect on obesity.
True
TRUE OR FALSE: depression correlated highly with and discriminated between a high Firmicutes:Bacteriodetes ratio in IBS patients relative to patients with healthy microbiota signature.
True
TRUE OR FALSE: in the gut, it is critical to keep bacteria in the lumen of the intestine.
True
TRUE OR FALSE: many of the antibiotics in use today were originally isolated from soil organisms.
True
TRUE OR FALSE: the influenza pandemic of 1918-1919 had killed more people than WWI, at somewhere between 20 to 40 million people, and left both medical and scientific professionals at odds as to its cause.
True
TRUE OR FALSE: the microbes in the GI tract influence the quantity and types of signals going to the CNS.
True
What is the ENS composed of?
Two thin layers of more than 100 million nerve cells lining your GI tract from esophagus to rectum
It is thought that _____________ bacteria account for about 99% of all species in the external environment and these microbes represent an important source of new antibiotics.
Uncultured
The ENS is connected to the brain via what nerve? What is this nerve made up of?
Vagus nerve made up between 80% and 90% of afferent nerves
How does CRISPR-Cas protect the bacterium?
-by cleaving the phage genome
Peripheral serotonin (gut-brain axis)
-cells in the gut produce large quantities of serotonin, which may have an effect on signalling in the brain
1967 - Thermophile Bacterium Machinery
-Brock identifies a thermophile bacterium from which heat stable DNA polymerase is later isolated and used in PCR
1897 - Yeast Fementation
-Buchner publishes first evidence of a cell-free fermentation process using extracts isolated from yeast -this discovery refutes Pasteur's claim that fermentation requires presence of live cells
1977 - Sequencing DNA
-Gilbert uses technique to determine the sequence of an operon in a bacterial genome -Sanger use the technique to determine the sequence of all 5375 nucelotides of phage phi-X174
Is Teixobactin highly effective against Gram-neg or Gram-pos? Why?
-Gram-positive -it interferes with cell wall synthesis by binding two cell wall precursors (lipid II & lipid III)
1928 - Bacterial Transformation
-Griffith discovers transformation in bacteria and establishes the foundation of molecular genetics
Dysbiosis of gut microflora increases inflammation due to the leakage of microbial products, such as _______ into underlying tissues. Increased inflammation increases the probability of what?
-LPS -increases the probability of generation of antibodies to self-antigens and autoimmune disease such as asthma
1880 - Attenuated Vaccines
-Pasteur develops a method of attenuating a virulent pathogen, so it would immunize and not cause disease -conceptual breakthrough for establishing protection against disease by inoculation of a a weakened strain of causative agent
1965 - Molecular Systematics
-Pauling unlocked the key to the field of nucleic acid-based identification of microorganisms by intro of the concept of molecular systematics using proteins and nucleic acids
1970 - Restriction Enzymes & Reverse Transciptase
-Smith & Wilcox describe the action of restriction enzymes which quickly becomes tools for sizing DNA -Howard Temin & David Baltimore independently discover reverse transcriptase in RNA viruses
What is NSERC?
-The Natural Sciences and Engineering Council of Canada -government agency that provides grants for basic research in science and engineering
1890 - Bacterial Nitrification
-Windogradsky succeeds in isolating nitrifying bacteria from soil and preforms major definitive work on organisms responsible for process of nitrification in nature
1905 - Chemicals Produced by Bacteria
-aerobic bacilli isolated —> produces acetone, ethanol, and acetic acid
What do SCFAs influence (5 things)
-appetite suppression -gluconeogenisis -fat metabolism -modulation of serotonin synthesis -major anti inflammatory role (under normal conditions)
In obesity, what appears to be a fundamental problem related to the gut microbiome?
-bacterial composition of GI tract may contribute to weight gain by extracting more energy from the food consumed and the food consumed by alter gut flora —> shifting into dysbiosis —> altering serotonin levels
Molecular mimicry and autoimmune disease:
-molecular mimicry between food ingredients and tight junction proteins -e.g. antibodies against tight junction proteins would further degrade the intestinal barrier
What is one possible reason for why there is a difference in the way bacteria grow and multiply in a Petri dish versus in a natural environment?
-one hypothesis is that most bacteria do not like to grow in unfamiliar environments where they will probably be killed by predators or unfamiliar conditions
In a search for new medically relevant compounds, a research group screened the genomes of 10,000 bacterial strains for a gene involved in...? What did they identify?
-phosphonate biosynthesis -278 new stains containing the gene —> allowed them to identify further genes involved in phosphonate biosynthesis
In the late 1920's Frederick Griffith was studying the possibility of creating a vaccine for __________________ caused by what?
-pneumonia -S. pneumoniae
1924 - Oxidation, Fermentation, and Biosynthesis
-points out that life on earth would not be possible without microbes
1967 - Understanding Synthesis of Proteins Through Translation
-proposed the existence of transfer RNA that uses a three base code and mediates in the synthesis of proteins -triplet nature of genetic code was first found in T4 phage
1966 - Genetic Control
-realization that chromosomes could be redesigned and genes moved
How close are scientists to achieving the goal of synthesizing proteins with synthetic amino acids? What type of ethical issues does this research raise?
-scientists have created bacteria with synthetic nucleotides, but it has now yielded any new proteins yet -ethical issue: synthesis of new life forms poses many question, implications are poorly understood, hard to predict outcomes
1944 - DNA role in Bacterial Transformation
-shows that DNA is the transforming material in cells
What is the first class of growth factors discovered by Dr. Lewis' research? How do these growth factors facilitate the growth of normally unculturable bacteria?
-siderophores, which are secreted by some bacteria to pick up iron from the environment and bring it back to the cell -purified siderophores can be added to the media in Petri dishes; bacteria that do not make their own siderophores can now be grown independently of their siderophore-producing neighbours
iChip experiment:
-soil sample diluted and used to inoculate iChip which was then placed back in soil for a month -in some cases, colonies that grew in the iChip could subsequently be grown in the lab -extracts of 10,000 cultures grown in the lab were tested to see if they inhibit growth of Staphylococcus aureus -extracts of an isolate showing good activity were partially purified due and analyzed. The organism's genome also sequenced; genes in Teixobactin bio synthetic pathway were eventually identified
What are the two basic strategies used to develop new antimicrobial drugs?
-synthesize thousands of compounds in the lab and test them to see if they inhibit bacterial growth -search for pre-existing compounds
What is the "Great Plate Count Anomaly"?
-though bacteria will grow in their natural environment, bacteria will not grow in the same way, or not grow at all, in a Petri dish on artificial media -you will only see about 1% of cells that you counted forming colonies on the Petri dish
What is one way bacteria recognize a familiar environment and commit to division? What is the problem?
-to recognize neighbouring bacterial species -these bacteria produce growth factors which will be recognized by growing bacteria -since we do not know what these growth factors are, they are not included in the petri dishes
1901 - Growth Factors
-water-soluble compound of yeast, later found to be a B vitamin that is required for the growth of yeast
How probiotics contribute to ameliorating the symptoms of autism (4 steps):
1) alters the composition of gut microbiota 2) improves epithelial barrier integrity 3) reduces leakage of particular GI metabolites 4) restores serum metabolites
4 steps of synthetic genome synthesis via homologous recombination:
1) short, overlapping stretches of DNA in 1080-bp in size were synthesized 2) these were assembled in sets of 10 to produce 109 ~10kb pieces 3) these were assembled into 11 ~100kb fragments 4) the 11 fragments were joined to form the complete genome
In 2012, a group of American scientists published their design of an unnatural base pair (UBP). What is a UBP?
A designed nucleotide base of DNA which is created in a laboratory and does not occur in nature
The gut and brain are 'connected' in a _________________ manner, which serves to ________________ the functions of these organs under physiological conditions, or is ______________ in disease conditions.
Bidirectional, coordinate, deregulated
The intestinal neuronal networks serve as a sensing system, via messaging pathways, both distribution and delivery arrays, to the ______.
CNS
Major roles of ENS?
Controlling digestion: -swallowing -release of digestive enzymes -control of blood flow to facilitate nutrient absorption -elimination
Adipose tissue secretes ____________ and ____________ that contribute to inflammation.
Cytokines and adipokines
Is microbial diversity deceased or increased in patients with IBS?
Decreased
If bacteria cross into the underlying tissue of intestines, inflammation can occur, which can ultimately result in...?
Dysbiosis
Obesity and asthma are associated with a ___________ of the gut microflora that results in a breakdown of intestinal barrier function.
Dysbiosis
Key discovery: Isolation of aerobic bacilli Scientific significance?
used for the production of important industrial chemicals