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85 Cards in this Set

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What can a microarray do?


Microarray experiments measure what?
Probe the genome of an organism.. due to improved technology and sequenced genome for the organism

Microarrays indirectly measure mRNA levels
What are the two types of microarray experiments?
Affymetrix and cDNA glass slide arrays
Affymetrix Array involves what?
Short DNA strands ~25u arrayed onto glass surfaces on computer chips at about 25 strands per spot. Allow specific hybridization. Up to 65,000-400,000 genes per chip.
cDNA glass slide arrays involve what?
DNA copies of mRNA(cDNA) arrayed onto glass microscope slides ~10,000per slide that are annotated with sequences. Add control cDNA with cDNA from treatment organism (two different dyes) and quantify expression levels based on color and intensity.
Example of comparative genomic hybridization... Pseudomonas aeruginosa how conserved is the genome between 3 different isolates?
Made labled cDNA from chromosomal DNA from 3 different strains. Found over 90% conservation and genes related to phage and transposons and plasmids were not highly conserved. Weakness know gene is there but it might not be functional or expressed.
What is Proteomics? Why proteomics?
The study of the proteome which is the protein complement of the genome similar to transcriptomics. Just because you have high level of mRNA doesn't mean have high level of protein expression.. so analysis is complementary.
What four steps are involved in Proteomics?
1. Separate protein mixture by 2D-SDS-PAGE gel
2. Isolate spots with individual proteins and digest with specific protease like trypsin
3. Take peptides from digested protein and run through Mass spectrometer MALDI-TOF tofind the peptide mass.
4. Run a database search on what you have found to identify protein.
How do you assay the genomic content of environmental samples?
Metagenomics
How does metagenomics work?
Take environmental pieces of DNA and clone into plasmids to get a metagenome library and then put into e-coli for sequence step.
Can you mine environmental DNA for new enzymes/functions?
Yes, once pieces of DNA are cloned and put into E-coli, can run assays to detect protease activity like a screen.
What did metagenomic analysis reveal when the worm Olavius algarevensis was looked at?
Indicated the presence of 4 genomes of symbionts within worm. One is a sulfur oxidizing [S->SO4] gamma3symbiont and another a sulfate reducing [SO4 ->H2S] bacteria delta1symbiont.
What clustering characteristics did they use to distinguish sequences?
GC content, dinucelotide abundance, absence/presence of DNA repeats
What does worm use as nutritional source?
Worm internalizes and lyses the symbionts as a nutritional source.. no digestive system
How do symbionts help worm get rid of nitrogenous waste and organic osmolytes?
Nitrogen containing compounds like ammonia and urea 3 transport mechanisms (3 found) that are taken up by 3 symbionts. the gamma3 symbiont has genes for denitrification
As worm moves up and down redox gradients, what do the symbionts do?
Symbionts maximize nutritional efficiency by sulfur oxidation, aerobic respiration (gamma) and heterotrophy (delta) and sulfur oxidation nitrate respiration (gamma) and heterotrophy, sulphate respiration (delta) and sulfur oxidation, anaerobic respiration (gamma) and hydrogen oxidation, sulfate respiration (delta)
What are 5 key aspects to structured microbial communities?
They are 5 key GRADIENTS
1. Nutrients
2. Signaling molecules
3. Secondary metabolites
4. Toxic compounds
5. Chemical gradients
What influences gradients? (4)
1. Microbial metabolic activity (allows microbes to grow inside biofilms)
2. Spacial distribution of populations
3. Biofilm extracellular matrix
4. Physical factors (high flow vs low flow)
Example of gradients in biofilms.. wastewater treatment and nitrogen cycle. there were three gradients tested by FISH with colored probes and then using a microelectrode going inwards towards anaerobic conditions
Closer to the oxygenic surface, found ammonia oxidizing bacteria [NH4 -> NO2], then Nitrite oxidizers [NO4 -> NO3], then Denitrifiers [NO2 -> N2]
What was shown to be upregulated/downregulated in P.aeruginosa during the conversion to the biofilm lifestyle?
Bacteriophages upregulated
Flagella Downregulated

Possible that over 35% of proteins are differentially regulated in biofilms.
What are the five stages of biofilm development?
1. reversible attachment
2. irreversible attachment
3. maturation-1
4. Maturation-2
5. dispersion
Whiteley study paradox
Transcriptional profile will represent the sstem representing the most producing RNA communities..
Heterogeneity within system.. genotypic diversification... what does that mean?
Certain genetic variants may be better suited for different parts of the biofilm
What does dispersion involve?
Dispersion is a regulated process induced by environmental cues (important for industry because inducing dispersal could overcome biofilm-mediated resistance)
Involves cavitiy rupture
How many infections are caused by biofilm forming microbes?
~60-80%
(CF patients have cilia that don't beat) Over time Pesudomonas becomes the dominant bacteria in the respiratory tract do to ability to outcompete by:
Loss of motility functions
Loss of LPS 0-antigens
Alginate overproduction
Amino acid auxotrophy
AND
wspF mutations or overproduction of cyclic-di-GMP = super adhesive
Example 2 - Bacterial Endocarditis
Biofilm complex formed of both bacterial and ost components located on an injured cardiac valve
What V.cholerae variants are hyper-biofilm formers?
Rugose mutants can autoaggregate in liquid culture and protect Vc from low pH
Bdellovibrio bacteriovorous, translation?
Bdello "leech"
vibrio "bent rod"
bacteriovorous "bacteria-eating"
Characteristics of Bdellovibrios
Physiologically diverse depending on environment ~1/5 volume of E.coli, 1.25µm. Fast Fast Fast, Cytoplasmic membrane sheathed around flagellum, obligate aerobes, high respiration high catabolism
Life cycle of Bdellovibrios
Find prey by accidentally bumping into them.
1. Attack phase cells
2. "Bdello plast" growth chamber (within periplasm)
3. "Growth phase"
Bdellovibrio "Attachment"
Localized pili (bunch of pilus like structures) on the nose may be required for predation. Bv finds prey by accidentally bumping into them and it swivels around for ~2 minutes and gets firm attachment (tight junction) and then cell death.
Bdellovibrio "invasion"
Bv sheds flagellum and penetrates into cell. Glycinase gets it through the cell wall, Lipase removes LPS, Peptidase (removes lipoprotein from cell wall and lack of connection between cell wall and membrane leads to rounding up becoming a sphere)
Bdelloplast "stabilization"
Produce Acetylase that counteracts Glycenase and removes acetyl groups from peptidoglycan making it no longer susceptible to glycinase... Protease activity attachment of fatty acids to peptidoglycan crosslinking glycogens
Bdellovibrio "Growth phase"
Starts producing evenly dispersed chromosomes 1chromosome/unit cell length. It elongates without dividing.
Key signal involved that s ays it's time to call it quits. Differentiation and Release... Glyconase to get out?
Who is susceptible? Specific host ranges?
All Gram-neg bacteria are susceptible and isolates show unique and also specific host ranges.. receptors remain unknown. Development of prey cell resistance is by acclimation.. it's a very complex system for binding.
Bdellovibrio : Lifestyle? Genome size? Anabolism? Catabolism?
Lifestyle: Obligate predator except mutants can be faclultatative predators and have anexic growth
Genome size: 3,783kb
Anabolism: Precursors from prey
Catabolism: Glycolysis, TCA, oxidative phosphorylation
Agrobacterium tumefaciens : Lifestyle? Genome size? Anabolism? Catabolism?
Lifestyle : Opportunistic plant pathogen (has free living form)
Genome size: 5,670kb
Anabolism: Can grow on minimal medium
Catabolism: Glycolysis, Entner-Duododroff, TCA, Oxidative phosphorylation
Rickettsia Lifestyle? Genome size? Anabolism? Catabolism?
Lifestyle: Obligate intracellular pathogen insect vector inside insect gut epithelial cells and becomes human pathogen...blood meal insect
Genome size: 1,112kb
Anabolism: Limited
Catabolism: TCA, Oxidative phosphorylation and ATP from host!!!
Chlamydia Lifestyle? Genome size? Anabolism? Catabolism?
Lifestyle: Obligate intracellular pathogen elementary body resistant to dessication, is dormant and airborne, reticulate body is the form growing inside host cell
Genome size: 1,0043kb
Anabolism: Limited
Catabolism: Aerobic respiration of glutamate also ATP from host
Mycoplasma genitalium Lifestyle? Genome size? Anabolism? Catabolism?
Lifestyle: Urogenital pathogen. Grows in mucous membranes and joints
Genome size: 580kb only 480kb used
Anabolism: Limited
Catabolism: Glycolysis
Carsonella Lifestyle? Genome size? Anabolism? Catabolism?
Lifestyle: Obligate intracellular symbiont of aphids that can't get some aa from sap they feed on
Genome size: 160kb
Anabolism: Liimited synthesizes essential amino acids for aphid
Catabolism: Glycolysis
Nanoarchaeum equitans Lifestyle? Genome size? Anabolism? Catabolism?
Lifestyle: Extracellular parasite of certain crenarchaeota. It is so deep branching, the 16s primers don'nt work. Very limited metabolic No pseudogenes
Genome size: 491kb
Anaboism: Limited
Catabolism: Limited Simple ATPase
MitochondriaLifestyle? Genome size? Anabolism? Catabolism?
Lifestyle: Organelles
Genome size: 16kb human several hundred kb plant
Anabolism: no
Catabolism: no
Agrobacterium have genes responsible for crown gall tumorslocated where?
TI plasmid
TI plasmid is composed of which active areas for forming crown gall tumors?
T-DNA and vir genes areas
T-DNA (transfer DNA) after wound attachment
This is integrated into the plant's genome and it carries the genes for tumor formation (oncogenes) and the production of a number of modified amino acids called opines.. Opines (Pyruvate + argenine = Octopine) are a source of carbon and nitrogen for Agrobacterium cells.
vir genes (different from nod factors)
The vir genes on the Ti plasmid encode proteins that are essential for T-DNA transfer Expression of vir genes is induced by plant signal molecules "Phenolics" from wounded tissues
vir A,B,G,C,D,E
virA recognizesplant phenolics and phosphylates virG induces virB encodes pilis like conjugation genes for transferring DNA virG also induces virC, virD (encodes endonucelase that will cleave on strand of T-DNA) and virE encodes SSDNA Binding proteins
Myxococcus
"Myxo" slime. Spores are coccus, vegatative are rod shaped Every aspect of life cycle involves cell-cell interactions.
Myxococcus feed on what?
What's the purpose of cell density?
Feed on polymers, cellulose or proteins extracellular enzyme lysozyme relaesed that can cleave polymer but needs high cell density to break up polymer.
Myxococcus Motility:
Adventurous and Social
Adventurous = one single -> slime jet. jet propultion moves fwd. others will follow.
Social= go together retractile pili. Cells come into contact with eachother stimulate eachother by passing proteins from one another.
Illistration of cell-cell motility interaction: Donor gives what to mutant?
Donor transferred tgl+ (outer membrane lipoprotein) a kind of tissue that develops between organisms
What happens when you starve cells?
Starve -> aggregation -> fruiting body -> spores... gene expression at certain stages in development.
Myxococcus experiment that proves signaling is going on: 5 steps
1. Mutagenize and get 3 mutants and a wild type, a morphogenesis mutant, a signal reception mutant and a signal production mutant.
2. Starve culture so it enters developmental pathway. (morphogenesis and signal reception mutants don't form spores)
3. Heat to kill still veg mutants
4. Isolate and streak out what you have left.
5. Only isolated wild types will recover... Isolated signal production mutants need a donor signal in order to become viable again.
5 different signals: How do we know? What are the Signals we have studied?
We know because groups of organisms mutants can rescue eachother count how many gorups we have (no growth) and can figure how many signals groups there are.
A signal
Acts early and is an indication of cell density smaller amino acids diffuse through liquid medium
C signal
Trancieved by end to end contact it rescues a protein that's bound to the surface used for cell-cell contact communication only end to end. and then you get transcription of c signal and formation of spores
Bacterial adaptions
motility, chemotaxis, cell division
Magnetotactic bacteria... why?
To get to optimum O2 concentration s swim along magnetic lines of course. Theyre also aerotactic.
Spirochete... why?
What can induce change in movements?
To swim around in viscous material (corkscrew) by smooth, flexing and reverse movements. Flagellum in between inner and outer membrane any where between 2 flagella to a whole tuft. Chemotaxis can induce flexing/reversal/smooth movements
How do you enrich for spirochetes?
Take mud from pond, place on a filter on top of agar containing Rifampicin that targets RNA polymerase.
Gas Vacuoles...why?
Bouyancy. without flagella or gliding motility need someway to position. If gas moves out, bacteria sink to bottom. GvpA and GvpC interaact to form a watertight but gas-permeable structure.
Magnetosome
Actin like filaments reminiscent of protein filaments in eukarkyotes.
Caulobacter - prosthecate bacteria why?
Take flagellated cell and before it will divide, it drops the flagellulm and grows a stalk. Their stalk (since prostheca s attached we call it a stalk) end is attached and they bud from cell end, not from hyphae type end. Can tell haw many times it's divided by the number of marks in stalk.
Bacillus
Endospore septum grows around protoplast ( engulfment) and you have forespore formation with exosporium and a primordial cortex
Symbiosis:
Mutualism, Commensalism, parasitism.
Riftia pachyptila
have sulfide oxidizers that live there and no where else and they have phagocytes that digest as a regulation to keep everything from getting out of hand.
In nutrient recycling, the symbiont ___________ the waste of it's host and returns it as ___________ .
In nutrient recycling, the symbiont detoxifies the nitrogenous waste of it's host, returnin it as usable organic nitrogen.
In symbiosis animal/algal relationships carbon flows from where to where in what form?
Carbon flows in the form of amino acids from the symbiont to the host animal tissue C:N 6:4, or even 9:1
In symbosis animal/algal relationships nitrogen flows from where to where and in what form?
Nitrogen flows from Animal host in the form of uric acid C:N 5:4.. broken to ammonia and used by the algal symbiont Corals ability to control symbiont decreases when their stressed like sunscreen.
Mycorrhizal associations?
create a nutritional internet that links an entire ecological community of plants together taking up carbon compounds and giving back inorganic nutrients.
Convoluta?
Photosynthetic flatworm with an obligate association for the host because it's completely lost mouth and digestive tract.
Symbionts can be sources of what?
Carbon/food, fixated nitrogen, inorganic nutrients, venom (Frogs, ampibians, anelids produce TTX [Gram + bacteria])
Enzymes that catalyze light emission are: ____
and their oxidized substrates are _______ .
Enzymes that catalyze light emission are called luciferase (only they're not always related) and their oxidized substrates are called luciferin.
What is the phylogeny of bacterial luminescers?
All Gram negative proteobacteria
Why luminescence?
To attract favored environment 10x11/ml
Photorhabdus luminescens is a commensal to _____ and a pathogen to ______ .
A symbiont of insect pathogenic nematodes (roundworms)
1. Insect invasion by worm
2. expulsion of bacterial symbionts into haemocoel bloodstream of insect
3. Exoenzymes that turn insect to gell and produce antibiotics and pigments
4. Insect glows from bacteria and proliferated worms leaving insect carcass re-ingest bacteria.
Symbiotic bacterial light organs in marine animals:
Are species specific, can either be obligate or facultative, product light continuously... most have some sort of symbiosis ligt organ. majority of light organs are internal
Bobtail squid and Vibrio fisheri... what is the progression of light-organ colonization?
1. Bacterial peptidoglycan signal causes cells of ciliated squid surface epithelium to secrete mucus.
2. Only viable gram-negative bacteria form dense aggregations.
3. Motile or non-motile V.fischeri out-compete other Gram-negative bacteria for space and become dominant in aggregations.
4. Vf migrate through the pores ind into the ducts to colonize host tissue
5. Following colonization, bacterial cells become non-motile and induce host epithelial cell swelling.
6. Squid vent 90% every day (Vf can become motile again)
Halide peroxidases
Host produced potent form of oxygenated stress released into crypt with Gram- bacteria as a selective measure to get rid of non ideal symbionts. Vf is one of few that can tolerate it
What may be another reason Luciferase is used?
It has a high affinity for O2 and it could be used to get more ATP
Functions of light organs in the bobtail squid. Reflector, Lens
Reflector to reflect light of bacteria below downward. Lens to diffuse light.
Examples of cell functions regulated by quorum sensing:6
1. Luminescence Vf
2. Antibiotic synthesis Wrwinia cartovora
3. Plasmid conjugation Agrobacterium tumefaciens
4. Exoenzyme/pigment synthesis (Chromobacterium violacein)
5. Virulence functions (Pesudomonas aeruginosa)
6. Cellular organiztion/biofilm formation (Pseudomonas arigunosa)
The lux operon of Vibrio fischeri luxR,I,C,D,A,B,E,G
1. luxC, luxD and luxE are fatty acid reductases (luxCDE)
2. luxA,B make Luciferase
3. luxG makes FMN Reductase
This pattern is conserved throughout every luminescent organism suggesting it was invented only once.
What is the hormone that induces density dependent luminescence?
AHL acyl homoserine lactone... inhibits luxR and promotes luxI