BIOL 375 Exam 2

E: Chemotaxis in *E. coli*

Methyl accepting chemotaxis proteins (MCPs) are polarized to one side of the bacterium, bind to an attractant and signal for a change in flagellar rotation

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CheA and CheW autophosphorylate which leads to phosphorylation of CheYby CheZ which allows for random introductions of CW rotation (tumbles) otherwise the flagella defaults to CCW rotation (runs)

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when MCPs are bound outside the cell, they stop the autophosphoylation of CheA so CheY isn’t phosphoylated so the flagella stays in the run mode going CCW

E: Chemotaxis Capillary Assays

substance is in a suspension in a tt and placed into a container w/ bacteria that flow in more if attracted, evenly if neutral, less if repellent

other forms of taxis include: oxygen or ______taxis or lining up at certain wavelengths in__ ______

aerotaxis, phototaxis

Some water-borne bacteria have _______ __which confer__ ______ to the cell

gas vesicles, buoyancy

w/ gas vesicles, The bacterium is able to float up or down in response to ______________________________________________________

environmental changes (avail. of O2, light, etc)

what types of organisms can have a gas vesicle

Many phototrophs, some non-phototrophs, not euks

gas vesicle membrane is impermeable to ___ __but permeable to__ __

water, oxygen

what is the oriC

origin of replication on chromosome

what binds and opens up double helix

DNAa proteins

how does binary fission work

binding of DNAa to oriC initiates DNA replication

the cell elongates

the septum forms

the z-ring forms

the cells separate

the ____ Directs synthesis of the septum

divisome

Fts proteins: ----- – forms the dividing ring

Fts stands for __---------__

Other Fts proteins help form --------, etc.

FtsZ, __F__ilamentous __T__emperature __S__ensitive, peptidoglycan

anchors FtsZ ring to CM

zip A

min proteins do what

help guide FtsZ ring to the midpoint of the cell

mutants form minicells

what is the process of how the peptidoglycan wall forms

NEGs and NAMs are assembled in the cytoplasm but the pentapeptide attaches them to Lipid A which then enters the flippase which moves them across the CM where autolysin activity opens a hole in the existing structure and trans glycosylase activity adds it to the existing structure

transpeptidation then joins the NEGs and NAMs with peptide bonds

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antibiotics inhibit both ‘trans’ activities

in what environments could you find each of the discussed temperature loving categories

psychrophiles- fridge

mesophiles- human body

hyper/thermophiles- hot springs

what does the preferred temperature for each organism determine

their environmental niche

E: 3 cardinal temperatures & effect on bacterial growth/molecular integrity

range is us. 30 d C

at minimum temp, membrane gels and cellular transport happens too slowly for life to exist

at optimum, enzyme rxns are occurring at the max rate

at max, pplbl collapses in thermal lysis, protein denaturation

what causes food poisoning

enterotoxin

what are facultative psychrophiles aka psychrotolerants

they can survive cold temps like lysteria

how do psychrophiles adapt for cold environments

proteins have more alpha helices, more hydropob and less polar amino acids, fewer weak interactions, and cryoprotectants like glycerol

have a high \[\] of unsat FA to stay fluid

what is an example of a thermophile

methanopyrus

thermus aquaticus

what adaptions do thermophiles have

proteins: more charged amino acids, with lots of interactions with hydrophobic interiors, chaperone proteins, less Gly, and protective solutes like phosphate to increase the osmolarity to increase stability,

genome has more DNA binding proteins to keep it tightly coiled

membrane has more sat FA, more hydrophobic with longer chains, some archea have a lipid monolayer that has specific hydrocarbons instead of FA

what do extreme pH orgs have

acidophiles- need more H+ for membrane stability

alkyphiles- are used in detergents

what do extreme pH guys do to keep a normal cytoplasmic pH

import/ export ions, HSPs are made when pH drops, and differences in the cell wall/ membrane

what does water activity (Aw) indicate and what is the lower limit of life

how much pure water is available for use, 0 pure, 1 no water, shows osmolarity

aquaporins are channels that allow for the fast transport of water to reduce osmotic stress

.6

________: require high NaCl concentrations

—Marine organisms – 3.5% NaCl

—-------– 15-30% NaCl (salt crystals)

—________ (high sugar concentrations)

—Xerophiles (___ environments)

—_________: tolerate wide ranges of osmotic pressure

Halophiles, Extreme halophiles , Osmophiles, dry, Halotolerant

what is an example of a halotolerant organism

*Staphylococcus aureus*

**Strict/obligate aerobes**

require O2 to grow

**Microaerophiles**

only grow at low O2 levels

**Strict/obligate anaerobes**

die in the presence of O2

**Facultative anaerobes**

grow w/ or w/o O2 but grow better w/ it

**Aerotolerant anaerobes**

grow w/ or w/o O2 but use fermentation and are unaffected by O2

what is an example of an enzyme that destroys toxic oxygen species

catalase

what are the macronutrients needed for growth

CHNOPS

light for E, non-org for H donor, CO2 for C

Photolithoautotrophs

light for E, organic for H donor, organic for C

Photoorganoheterotrophs

chem E, non-org for H donor, CO2 for C

Chemolithoautotrophs

chem E, non-org for H donor, organic for C

Chemolithoheterotrophs

chem E, org for H donor, organic for C

Chemoorganoheterotrophs

what are the major micronutrients needed and what are they used for

Iron, zinc, nickel

making enzymes

fastidious need these supplied (like from tap water)

which micronutrient is the limiting thing in the human body

Fe

what are examples of organic growth factors

micronutrients, amino acids, purines, pyrimidines, vitamins

what are the uses of metabolism

—Bringing in nutrients

—Repair and maintenance

—Release of wastes

—Motility

—Generation of PMF

—Anabolism: building parts

—Biosynthesis

—Polymerization

—Assembly

what is an anerobic organism

one that does not use O as its final e- acceptor

what is respiration

Organic or inorganic compounds are oxidized with oxygen or some other compound to make ATP

what is the most common form of glycolysis and what are the alternatives

Embden-Meyerhoff-Parnas pathway

Pentose Phosphate shunt, ***Entner***–Doudoroff pathway

what is pyruvate used for

—Further processed in the CAC in respiration

—Reduced in fermentation

—Used for biosynthesis

E: Why respiration in prokaryotes is more efficient than in eukaryotes

DNA is more available and no energy is needed to move through membrane bound organelles

euks do ETC in inner mito membrane while prok do in cytoplasmic membrane

E: Uses of the PMF (Proton gradient) in prokaryotes

powering ATP synthesis through OP

powers uni/sym/anti port, drug efflux pump

flagellar rotation

E: Complete oxidation of glucose

glycolysis: need- 2 ATP, 1 glucose, 2 NAD+, get- 4 ATP 2 pyruvate, 2 NADH +2H+ 2e’s

intermediate step: 2 pyruvate, 2 NAD+ 2 GDP, get- 2 Acetyl CoA, 2 CO2, 2 NADH + 2H+, 2 GTP 4e-s

CAC: 2 acetyl CoA, 6 NAD+, 2 FAD, get- 6 NADH + 6H+, 2 FADH2, 3 CO2, 2 ATP 16e-s

ETC: 10 NADH + 10H+, 2 FADH2, 6 O2, get- 34 ATP (finish with 38)

C6H12O6 + 6 O2 + 38 ADP + 38 Pi  → 6 CO2 + 6 H2O + 38 ATP

E: How the proton motive force is generated

e- carriers are arranged so that H+ from water is separated from e-

e- carriers are arranged so that the next has a higher e- affinity than the last so the last carrier is the most pos reduction pot

carriers switch btwn reduced or oxidized forms

O is common final e- acceptor but there are some rare alternatives

meaning that NADH has more ATP gen potential than FADH2

happens through oxidation phosphorylation/ chemiosmotic theory

so ADP becomes ATP when H go down gradient

ATP synthase:

—A.k.a. _____

—Complex _ of ETC

—Site of ____________

—-------- per ATP produced

—_______ complex

—Catalyzes -------- phosphorylation of ADP to ATP

—Reversible (---------- need to generate a PMF for motility and transport)

has changed _____ evolutionarily

ATPase, V, oxidative phosphorylation, 3 to 4 protons, F1 /Fo, reversible, strict fermentors, little

what are alternative final e- acceptors

—nitrate, sulfate, carbonate

—Forms nitrite, hydrogen sulfide, methane

what do faculative areobes do when they run out of O2

glycolysis then fermentation

what occurs in fermentation

—No exogenous electron acceptor needed

—Organic molecules only partly oxidized

—Pyruvate converted to organic compounds

is inefficient w/ only 2 ATP per glucose molecule

NAD+ is restored for glycolysis

leads to a buildup of acid end products

E: Differences between prokaryotic and eukaryotic genetics

euk: nucleus, linear, haploid/diploid/poly, more protein encoding genes, histones, has introns, replicate DNA hours before mitosis, multi origins of rep

prok: nucleoid, circular, us haploid, less protein encoding genes, arch has histones bac has histone like, no introns in bac but introns in rRNA and tRNA of arch, replicate DNA just before BF, bac has one origin of rep but arch has multi

how are plasmids retained

must be transferred in conjugation to others or will be lost over time as they code for exogenous information, has an oriV center of replication

what do plasmids often code for

pilus on F-plasmid, enzymes for synthesis of unusual compounds, and/or genes for antibiotic resistance, virulence factors

what are segments of DNA that move from one location to another within other DNA molecules

transposable elements

where are transposable elements found

all 3 domains, in another DNA molecule like chromosomes or plasmids

how are transposable elements moved

transposition using transposase enzyme which moves DNA between inverted repeats

what are the 2 main types of transposable elements in bacteria

transposons and insertion sequences

both have genes to encode transposases and inverted repeated sequences to make them more recognized from multiple angles

what are transposons (Tn)

—Larger than insertion sequences & have IRs at ends; also encode for transposase

—May include genes for antibiotic resistance

—Examples are Tn5 and Tn10

what are insertion sequences

—Simplest transposable element with IRs at ends

—Ca. 1,000 nucleotides long

—Only gene is for the transposase

—Found in plasmids and chromosomes of *Bacteria* and *Archaea*

—Found in some bacteriophages

found in the chromosome and is basically a place to add genes

what is the physical exchange of DNA between genetic elements

recombination

what is the process that results in genetic exchange between homologous DNA from two different sources

homologous recombination

Recombination: A.k.a. _______________

—________ transfer

—Transfer of _ kinds of DNA

—found in _______

—----- forms (plasmids)

—Must be maintained in a ------

as -------

—Or --------- into chromosome, often via ----

Horizontal Gene Transfer (HGT), One-way, 2, Fragments, Stable, recipient cell, plasmids, recombined, IS’s

Homologous Recombination in prokaryotes is facilitated by letting strand invasion occur via _____

recA proteins, promotes base pairing btwn the 2 strands

E: Processes of, and differences between, transformation, conjugation and transduction

—Transformation= Uptake of “free” DNA

donor cell is lysed and new cell can take up DNA, but new cell must have competence proteins in order to take it up, was used to determine that DNA is the vehicle of heredity, is often used as a genetic tool, virulence factor is transferred via a capsule, transferred as a plasmid or as a fragment, can artificially induce this, transformasomes allows for competent proteins to let a pillus form

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—Conjugation= 2 cells exchanging DNA

occurs through cell to cell contact through a pillus, conjugated or transferable plasmids have the genes for conjugation; F plasmid in e. coli codes for fertility factor that allows conjugation that sometimes also brings in host’s genome, tra genes encode for gene transfer that encode for the pillus and has multi IS sites and 2 origins of replication (oriT (rep in DNA transfer, everything behind this arrow is what is trans first) and oriV (rep of non-conjugating plasmid), transfer begins at the oriC), pillus only brings together the 2 cells, a conjugation bridge forms, DNA goes through tube where it is replicated on both sides, the cell then separates and both are now F+

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—Transduction= Via bacteriophages

bacteriophages takes up DNA from one host and inserts it into a new host

auxotroph vs prototroph

—Auxotroph: Mutant strain requiring a growth factor (vitamin, AA, etc.)

—Prototroph: Wt – typically does not require any growth factors

how are plasmids transferred in conjugation

Conjugation -Replication and Transfer Process

what differentiates F+ and F- cells

—F+ cells mate __only__ with F- cells

—F-: No F plasmid

—F+ cell: *unintegrated* F plasmid

—F- becomes F+ after mating

—F+ cannot mate with F+

—Surface receptors altered so that cell can no longer act as a recipient in conjugation

what is the most common way antibiotic resistance is transferred between species

conjugation

E: Experiment of how transformation was discovered

Fred Griffith in 1928 used *Streptococcus pneumoniae* called it the transforming principle

had non-encapsulated bacteria injected in mouse- lived w/ encapsulated bac recovered

had encapsulated bac injected- died w/ a few non-en found

had heat killed encapsulated bac injected- lived w/ no colonies

added non-encapsulated to the dead encapsulated- died w/ encapsulated

E: Experiments of how conjugation was discovered

Joshua Lederberg and Edward Tatum 1946 saw that mixing 2 auxotrophs can make a prototroph

had tt of 2 opposite mixes of proto and auxos and a combo of both and plated on minimal media and only the combo grew

Bernard Davis 1950 saw that physical contact was needed for conjugation

put in tube that physically separated and didn’t happen so couldn’t be transformation