NCSU MB 414 - exam 1

"what is bacterium made out of?" and "how it is assembled" (macromolecular synthesis), "genomics"

What percent of protein is in bacterium?

55%

What percent of DNA/RNA is in bacterium?

24%

What percent of lipids is a bacterium?

9%

What percent of Carbohydrates is in bacterium?

6%

What percent of other organics in in bacterium?

3%

What percent of inorganics is in bacterium?

1%

What is the internal cytoskeleton made up of?

MreB

What does Hydrogenase do?

It is an enzyme that splits protons or hydrogens, and it can also go backwards

Hydrogenase is a ______ dimer

Heterodimer

The flagellum is made out of?

Protein

Proteins get information from

methyl-accepting chemotaxis proteins

methyl-accepting chemotaxis proteins or MCPs are …. and what do they detect

transmembrane sensors in bacteria and archea. They detetct food and toxins

DNA and RNA are used as

information

RNA provides

• informaiton

• regulation

• catalysis

Lipids are in the….

membrane as phospholipid

Bacteria would make ______ and it put it in granules. It is used as storage and degrade very easily

PHB

Lipids include ______ and ______ for energy

Fatty acids and glycerol

Carbohydrates are _____ for energy

glucose

They provide sturcture with ____ ,_____ and ______

NAG, NAM, lipopolysaccharide

Regarding other organics, reductant transfer molecules include… these are invovled in transfer of electrons

• Nicotinamide

• Flavin

• Heme

inorganics inclue the metals…

• iron

• sulfur

• phosphate

• nickel

• magnesium

The 2nd law of thermodynamics is

An increase in disorder is spontaneous in the absence of an input of energy

autotrophs get energy from

• sun

• inorganic chemicals

Heterotrophs get their energy from

• eat (steal) energy in the form of reduced carbon (steal electrons)

Rank Methanol, formaldehyde, methane, Carbon dioxide, and formate from most energy to least energy

Methane, methanol, formaldehyde, formate, carbon dioxide (MMFFC)

Rank Methanol, formaldehyde, methane, Carbon dioxide, and formate from more reduced to least reduced

Methane, methanol, formaldehyde, formate, carbon dioxide (MMFFC)

The most reduced form will have the most _____

energy

Classical genetics involves..

• Random mutagenesis (stop swimming) and see what genes are involved

• Complementation (start giving back little chunks of DNA to the mutant until it starts working again)

Reverse Genetics invovles..

• Having sequence of ALL genes

• Looking through the genome and guessing what genes are involved in motility (in this case)

• Gene-directed mutagenesis

Sanger sequencing is when… and what happens

• DNA sequenceing done by synthesis sanger nucleotide chain termination rxns

• when replicating a chromosome, its done 5’-3’.

  • so adding a nucleotide to 3’ hydroxyl of deoxy ribose sugar

What did sanger think to add to the 3’ and what happens?

Dideoxy. We won’t have a 3’ hydroxyl and cant add. (chain terminator)

How did sanger use his new synthesis?

• Utilizes nucleotide chain termination reactions

• diDeoxy nucleotides

• Nucleotide adds until one without 3' OH stops reaction

• Fragments are run on gel (lengths are different)

• Read bottom/up (5' to 3')

Ex of sanger sequencing

diDeoxy A in a tube which stops synthesizing

—You know each strand with specific length ends an A (Run all A's on a gel)

—Smallest molecule runs to the end (bottom to top)

—Repeat process for T, G, and C's (each with different tube)

In sanger sequencing Agrose gels, there is _____ sized products. Each product will only be ____ smaller than the previous.

different sized products; 1nt smaller

What is invovled in sanger sequencing

1) DNA Polymerase
2) All dNTP's ATCG
3) 1 ddNTP A
4) DNA
5) Labeled primer

In fluorescent sequencing, all reactions can be one lane and why?

Is the sequence read from 5’ to 3’ or 3’ to 5’?

up to how many bps can be read?

• One lane because each base has its own color

• 5’ to 3’ prime

• up to 1000 pb read length.

So you have a fluorescent sequence, using what type of sequencing? Explain how it works

• Capillary sequencing.

• The DNA is sequenced using dideoxy chain termination

  • Instead of gels, the bands are separated into capillary tubes by size

  • The sample is read as it leaves the tube.

454 or pyrosequencing during mid 2000s. What happens?

• sequencing by synthesis

• using DNA polymerase to add nucleotides

• detecting what was the last nucleotide

• Add nucleotides one at a time

  --Brightness correlates to number of nucleotides added

  (Can only tell up to 6 then can't tell difference)

Shotgun pyrosequencing is

200-400 bp fragments of genomic DNA
1) Adaptor added to each bead
2) Each bead has DNA sequence
3) Each hole fits one single bead
4) Run beads through PCR (Each bead has same sequence)
5) DNA bead has millions of copies of same fragment
6) Light vs. No light —> Camera

Illumina-solexa

1) Shear DNA around 200-300 bp
2) DNA sticks to plate
3) PCR creates DNA clusters of same sequences
4) Fluorescent (color) light
--Can add multiple nucleotides since color coded

How Illumina-Solexa Works

--Each nucleotide has a fluorescent tag
--Also keeps other nucleotide from adding
--Provides color and signals A,T,C, or G
--Get rid of Fluorescent tag with wash
(Do not have to worry about multiple nucleotide additions)

Ion torrent

• flood chip with nucleotides

• if nucleotide is incorporated, a proton is released --> pH decreases

• proton is read by the sensor

• If multiple nucleotides, the H+ level is multiplied

  --2x drop in pH = 2 nucleotides in a row (similar to 454)

Nanopore

• Does not rely on sequencing

• enables much longer reads

• data is shown real time

• data about nucleotide modification and DNA damage is available

• HAS VERY HIGH ERROR RATE

• 10-100 kB pieces.

How nanopore works

• nanopore instered into membrane with electrical potential

• motor proteins loads DNA into channel

• Neg. charged DNA is driven from neg. side to pos. side of membrane.

• DNA travels thoruhg pore, different bases induce differences in electrical current

• Current is measured within the pinch points.

Nanapore work flow

• DNA is digested into 10-100 kB pieces

• ends are repaired for ligation

• adaptors are added that are bound to the motor and pore anchors

• DNA/motor/anchor complex is added to the nanopore.

Nanopore uses base calling to

how much current is going through. The pinhc points allow a certain ammount of current and it know the difference between A. c, g, and T.

Nanopore error rate at first was.. now its..

15%; 1% but its still way high

• not allowed for genome sequencing

Ordered clones

• Clone very large pieces of DNA

• sequences both ends

• puts clones in order

• design primers to sequence further

• repeat until whole genome is done

• Time-consuming and expensive

Shotgun sequencing

• Generates lots of little pieces of DNA

• Sequence them all (small)

• have a computer put the pieces in order

• disadvantage: have to sequence 6- 100 times as much DNA to ensure complete genome coverage ( long time)

How ordered clone sequences work

• make a very large clone

• cosmids (DNA viruses with cohesive ends)

• BAC (bacterial artificial chromosomes)

• Cosmids keep the BACs

• Have all these clones and slowly sequence around them

• Order the clones - pick overlapping clones that give complete coverage of whole genome

• Mycobacterium tuberculosis

• Human genome (20,000 ordered clones)

Guest lecture content:

• M. tuberculosis has unusally high PGRS sequences, whats it for

• Crazy high G + C content; what does that indicate?

• 
  

• Surface protein that helps with cell wall integrity, nad contributes to host:pathogen interaction, immune invasion, and virulence.

• High GC content indicated that Mtb has not undergone horizontal gene transfer

• Increases sequencing difficulty because DNA is hard to access and separate the strands of DNA.

Shotgun sequencing (1st generation)

• Clone lots of small (1000bp or less) fragments

• sequence a lot of them

  • rule: sequence 6 times the ammount of DNA u think is in genome

• All inserts can be sequenec with a primer that is complementary to the vector (dont have to keep making new ones)

Shotgun sequencing (2nd generation)

• Don’t bother cloning

• sequence DNA fragments but need 30- 100-fold genome coverage

• sequence 100-300 mbp for a 1 mbp bug

• align fragments (various folds of coverage)

• ends don’t match anything

• create contiguous sequences

• 1 contiguous sequence = 1 gap

How to fix gaps

1) Can PCR sequence to find gaps
2) Use other bacteria as scaffold if working with other species
3) Use two different methods, i.e. illumina and ion torrent, to yield different gaps

How to find genes from raw sequence?

• For prokaryotes, look for start and stop codons In frame and mroe than 100 bp apart (and a multiple of 3)

• Translates the genes and search the data base for homologous genes

• If yes, likely a good gene, annotate

• if no, likely a novel gene

Novel sequence, things to take into account before deciding if novel sequence

1. context: does it overlap another open reading frame? is it in an operon?

2. is there a recognizable promoter or ribosome binding site?

3. does it use the same codon bias as the rest of the genome?

codon bias

• Bacteria tend to use the same triplet to encode an amino acid enven though the code is redudant.

• ex. isoleucine; 3 codons, dif usage

Conserved vs. hypothetical

Hypothetical: "I think it is a protein"
Conserved Hypothetical: "Found another hypothetical protein that is homologous but no known function"

Cytoskeleton - MreB

• MreB is the skeleton that gives the bacterium its shape (baccili; MreB is absent in coccoid bacteria)

• As MreB polymerizes, the cell elongates

• Resemble eukaryotic actin filament (structural level)

The capsule

• outermost structures of G (+) and (-) cells

• physically attched to bacterium and not easily washed off

• most are branched polysaccharides

• exception is poly glutamate capsule of B. anthracis

• Many pathogenic bacteria produce capsules to evade immune surveillence and to inhibit complement fixation

Guest lecture:

S. pyogenes has a hyaluronic acid capsule

• hasBAC operon; hasA encodes the hyaluronan synthase

• Deleting hasA removes the capsule

• hasA promotes s. pyogenes during nasal infection

• Loss of HA capsule reduces bacterial persistence, which is associated with a weaker host inflammatory response

• Delta hasA gene is responsible for the phenotype

Cell surface - membrane

• Gram type pos and gram type neg.

• Gram + stain very well due to exposed thick peptidoglycan (no outer membrane)

• Gram (-) thin peptidoglycan layer (strength)

• 
  

peptidoglycan (exoskeleton)

• peptide cross-links in a carbohydrate

• Large (Gram-positive) and small (Gram-negative)

• Made up of DAP

DAP

• diaminopimelic acid

• made from lysine and then added another carbocylic acid

• amino acid nucleus on both sides- helpful for crosslink

NAG-NAM

Alternating sequences that make up the peptidoglycan exoskeleton (transglycosidation reactions)

--Crosslinking occurs between NAM residues

Peptidoglycan synthesis - inside

Cytoplasm:

1. NAG is coupled iwth UDP

2. ½ is converted to UDP-n-acetylmuramic acid

3. amino acids added sequentially to NAM

the coupled subunits then cross the cytoplasmic membrane

UDP

• gets coupled with NAG

• helps shuttle cell wall components across cell membrane

Peptidoglycan synthesis - outside

cell wall extension (outside cytoplasm):

• the sugar (NAG, NAM) subunits are polymerized via transglycosidation reactions

• The peptides are cross-linked via transpeptidases

How is integrity of peptidoglycan maintianed

• a large multi-enzyme complex consisting of both murein hydrolases and murein synthases

  • add new polymers to expand cell wall

• Complex moves along guide strand that will be hydrolyzed

• hydrolyzed via a lytic transglycosylase and endopeptidase

• a meurin triplet is made below the guide strand via transpeptidase and transglycosylase

• Net result: one strand is safely replaced by 3 new strands

Teichoic acid (TA)

• TA strenghtens the gram(+) cell wall

• links layers of polypetidoglycan together via colvalent bonds to NAM

LTA

• attach the cell wall to the lipid by layer via a lipid anchor into the membrane

R groups on glycerol teichoic acid

• can be alanine, glucose, or glucosamine

Teichoic acid in the wall

• Layers of petidoglycan arent connected except through teichoic acid

• lipid teichoic aicd connects the entire structure of the membrane so it doesnt shift

The cytoplasmic membrane

• is a permeability barrier

• no solutes can pass thorugh without specific transport proteins

• Bacterial membrane composed of a phospholipid bilayer

• self assamble

• Hydrophilic heads and hydrophobic tails

Bacterial and archaeal lipids

• bacterial use unbrached isoprene, fatty acids, ester

• archaea use branched isoprene chains

outer membrane proteins

• The outermembrance is not as restrictive as cytoplasmic membrane

• Low molecular weight compouns are allowed free access across the membrane thorugh porins

• Other porins are specific for iron siderophores, B12, carbohydrates, and phosphate

Guest lecture:

Serratia marcescens

• has pigment prodigiosin

• clinical strains lack pigment (37degree celisus)

• Outer membrane vesicles (OMVs)

  • only produced by G- bacteria

  • bud off cell envelope

  • allow bacteria to interact with the environment, mediate mult. functions

• OMV in s. marcescens

  • formed in response to stress (temp)

  • selectively package cargo (toxic to certain species- pathogencity and virulence factor)

• OMV as virolence factor

  • degrade host proteins, indcue inflamatory, damage host tissue

  • neuromuscular action

Gram (-) outer membrane

• can form OMV

• outer leaflet has lipopolysaccharide

lipopolysaccharides

• Long chains found on the outside membrane of Gram (-) bacteria

• Made up of (outside to inside): O-polysaccharide, Core polysaccharide, and lipid A

The nucleoid

• The region of the cell where chromosomes are located

• most bacterial - circular but GREATLY compacted to fit in nucleoid space

DNA Packaging calculation

1 genome (circular) = 1.7 × 10^-3m or 1.7mm

Nuclear Packaging - super coils

1. compact DNA

2. Bacterial DNA is made with negative supercoiling

Supercoiling - two types of winding proteins

• Topisomerase I:

  • can relieve negative supercoilds by “nicking” on strand of DNA.

  • “nick” is sealed without ATP

• Gyrase or Topoisomerase II:

  • introduces negative supercoils dependent on ATP

  • requires breaking and resealing both strands of ATP

How Topoisomerase I works:

1. nick (break phosphodiester bonds)

2. allow DNA to untwist from torsion

3. creates phosphotyrosine bond

4. DNA re-anneals (energy in P-bond is conserved)

How Gyrase (Topoisomerase II) works:

1. breaks back segment (must be held by protein)

2. reseal break on front side

3. can only reseal with proper protein (lethal)

Catenation Vs. Decatenation

After replication, two circles are linked together
Catenation: The process of linking two DNA segments together
Decatenation: Process of unlinking two DNA segments, post replication

Nuclear packaging - DNA bendning proteins

• bacteria lack histones

• have histone like proteins

  • compact the DNA further

  • ex IHF, HU, H-NS

• Help compact DNA in the correct way (Genes highly transcribed on outside

Locomotion

• used for finding nutrients, avoiding toxins, and colonizing favorable ecological niches

• bacteria use flagella to swim thorugh aqueous solutions

• to move on solid surfaces - glide, swarm, and twitch.

Myxococcus motility

• genetically determined(at least 50 mutants in discrete genes)

• motor and PMF driven

• MreB-dependent

motors

localized focal adhesions on one side of gliding cells

Proton ionophore

pokes hole in cell membrane→ PMF collapses

Mycoplasma mobile

• tiny mixed

• driven by ATP

• two proteins

• basically walking

• no PMF

• walker motif —— ATP binding motif

Guest lecture:

Listeria monocytogenes

• two modes of motility

  • flagella - mainly inbetween host

    • stops expressing flagella @30 degree celsius (human temp)

  • Actin

    • push molecules together. spring in and out → propels cell forward

• PLC and ActA allow degradation of the autophagosome

• ActA motility allows the movement into the cytosol where replication takes place

Twitching motility

• mediated by pili or fimbrae

• twitching is used by bacteria to move over solid or semi-solid media

• twitching cells hyperpilated

• extensions and retraction of pili

Guest lecture:

pseudomonas aeruginosa

• mechanotaxis - cell movement guided by physical or mechanical cues

• retractile type IV pili for twitching

  • extension, attachment, and retraction

• Chp- controls balance of forward and reverse twitching

• WT - collisions stimulate reversals

• chp mutant - always or never reversed

Monotrichous

one flagella on one end

lophotrichous

six to eight flagella on one end

amphitrichous

one flagella on each end

peritrichous

Flagella all over the cell

Flagella structure

• powered by proton gradient set up by electron transport chain

• Basal body: subunits get through entire membrane

  • motor protein, motor switch, etc

• Hook: creates propeller motion (for motion)

• filament: makes up the shaft of the flagella