20.106 Midterm 2 Review Session

Megan McBeeNovember 7th, 2006

Topics

• Nitrogen Cycle• Communities, symbiosis, genome reduction• Horizontal Gene Transfer• Biotechnology

Nitrogen Cycle

Important Reactions

Nitrogen Assimilation Incorporation of to NH3into organic molecules (R-NH2)

Deamination Deamination of organic molecules releasing NH3

NitrificationDenitrificationN2 Fixation

Nitrogen Cycle: NitrificationOxidation of NH3 to NO2

- then NO3 -

Only carried out by microbesSoil and aquatic bacteriaAerobic chemolithoautotrophic two step process

Ammonia oxidizers (Nitrosomonas, Nitrosocococcus, Nitrosospira, archaea)Nitrite oxidizers (Nitrobacter, Nitrospira)

Enzyme is ammonia monooxygenase

Nitrogen Cycle: DenitrificationNitrate reduction, conversion of NO3

- to N2 gas

Only carried out by microbesAnaerobic chemoheterotrophic processFacultative anaerobes in sediment and oxygen-limited waterAnaerobic respiration (using Nitrate and Nitrite as TEAP)Oxidation of organic matter for energySeveral enzyme systems--dissimilatory nitrate reductaseLoss of Nitrogen from systemNitrate to ammonia NOT denitrification

Plants perfom assimilatory nitrate reductionConvert NO3

- to organic nitrogen (R-NH2)

Nitrogen Cycle: N2 FixationMolecular conversion of N2 gas to NH3

(exist in solution as NH +4 )

• ANAEROBIC• Proteobacteria, Cyanobacteria, Ar

Requires nitrogenase enzyme– 21 genes– 8 subunits/accessory proteins– Molybdenum and iron cofactor

chaea

•

s

• High energy cost (16 ATP per N2)

ATP

ADP

Flavodoxin

Molybdo-ferredoxin

azoferredoxinacetyl-CoA + CO2

pyruvate + CoA

α β

β α

N=N

2NH3 + H2

FeSMoFeS

Flavodoxinreductase

Figure by MIT OCW.

Rhizobium• Free-living are aerobic, not N2 fixers• When symbiotic

– Rhizobium turn on plasmid-based Nod genes – Become anaerobic N2-fixing, bacteroid form– Legumes form nodules to control symbiotic

relationship

Images of free-living Rhizobium and bacterioids in nodule removed due to copyright restrictions.

Symbiosis and Genome Reduction

10.0

5.0

1.0

0.5

0.1100 500 1000 5000 10000

Nanoarchaeum equitansMycoplasma genitalium Wigglesworthia glossinidia

Rickettsia conoriiPelagibacter ubique

Ehrlichia ruminantiumBartonella quintanaThermoplasma acidophilum

Bartonella henselaeCoxiella burnetii

Prochlorococcus marinus MED4Prochlorococcus marinus SS120

Prochlorococcus marinus MIT9313

Synechococcus sp.WH8102

Silicibacter pomeroyi

Rhodopirellula baltica

Streptomyces coelicolor

Mesoplasma florum

Free-livingHost-associated

Obligate symbionts/parasitesPelagibacter ubique

Number of protein encoding genes

Gen

ome

size

(Mbp

)

Figure by MIT OCW.

Symbiosis and Genome Reduction• Buchnera aphidicola from two aphids Schizaphus

graminum (Sg) and Acyrthiosiphon pisum (Ap)• 70 million years

– No chromosomal rearrangements– Sequence divergence (9-9 synonymous substitutions/yr– 1.65-9 non-synonymous substitutions/yr

• E. coli and Salmonella spp. (closest free-living relatives) 2000x more liable

00

2000

4000

2000

Salmonella Typhimurium LT2

Esch

eric

hia

coli

K12

4000 00

200

400

200

Buchnera aphidicola str. APS (Acyrthosiphon pisum)

No inversions, translocations, duplications, or gene acquisitions !

Buc

hner

a ap

hidi

cola

str.

Sg

(Sch

izap

his

gram

inum

)

400 600

Figure by MIT OCW.

Genome Dynamics in BuchneraObligate endosymbiont

o Substantial sequence divergenceo Prominence of pseudogeneso Loss of DNA repair mechanismso Stable genome architecture HOW??

• Gene transfer elements reduced/eliminated– Reduced phage– Reduced exhange w/other genomes– Fewer repeat sequences– Fewer transposons

• Lack of recombination mechanisms (no recA, recF)• Lower frequency of recombination

Argobacterium

• Ti plasmid & crown gall disease– A portion of the Ti plasmid is

inserted into the plant chomosomecausing the formation of the tumor or gall.

Figure by MIT OCW.

Ti plasmidT-DNA Bacterial genome

Agrobacterium tumefaciens

Plant chromosomal DNA

T-DNA

Transformed plant cell

Crown gall

Figure by MIT OCW.

VirG

VirA

VirA

VirG

ADPATP Agrobacterium cell

Transcription of other vir genes

EE E E

E EE

E

T-DNAT-DNA Single-stranded nick

VirE(single-strand DNA bindingprotein)

Plant cell

Plant wound

VirB

Agrobacterium cell

Phenolic compounds

E EE

E

Vir D

P

Horizontal Gene Transfer: Transformation

Restriction

Degradation

Mutations,rerrangements

Selection

Negative Neutral Positive

Expression of competence

Stablization

Exposure to becteria

Inactivation Degradation

Uptake

Expression

Recombination Recircularization

Release of DNA by growing or decaying cells

Figure by MIT OCW.

Horizontal Gene Transfer: Conjugation and Transduction

Chromosome

Chromosome

Prophage

Mobilizableplasmid

Transposon

incX

incY

incY

IntegronConjugativeplasmid

Mobile genecassettes

1

3

3

2

Specialized

Transduction

Generalized

Pilus

incZ

Donor cell Recipient cellFigure by MIT OCW.

DNA Technologies• Basics of Sequencing

– Sanger method– Shotgun sequencing

• Cloning (cloning vectors)– Plasmids– Phage– Bacterial Artificial Chromosomes (BAC)– Yeast Artificial Chromosomes (YAC)