20.106 Midterm 2 Review Session

Megan McBee November 7th, 2006 Topics

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

Important Reactions

9Nitrogen Assimilation Incorporation of to NH3

into organic molecules (R-NH2) 9Deamination Deamination of organic molecules releasing NH3 9Nitrification 9Denitrification

9N2 Fixation Nitrogen Cycle: Nitrification - - Oxidation of NH3 to NO2 then NO3

Only carried out by microbes ƒ Soil and aquatic bacteria ƒ Aerobic chemolithoautotrophic two step process ƒ Ammonia oxidizers (Nitrosomonas, Nitrosocococcus, Nitrosospira, ) ƒ Nitrite oxidizers (Nitrobacter, Nitrospira) ƒ Enzyme is ammonia monooxygenase Nitrogen Cycle: Denitrification - Nitrate reduction, conversion of NO3 to N2 gas

Only carried out by microbes ƒ Anaerobic chemoheterotrophic process ƒ Facultative anaerobes in sediment and oxygen-limited water ƒ Anaerobic respiration (using Nitrate and Nitrite as TEAP) ƒ Oxidation of organic matter for energy ƒ Several enzyme systems--dissimilatory nitrate reductase ƒ Loss of Nitrogen from system ƒ Nitrate to ammonia NOT denitrification Plants perfom assimilatory nitrate reduction - 9 Convert NO3 to organic nitrogen (R-NH2) Nitrogen Cycle: N2 Fixation Molecular conversion of N2 gas to NH3 + (exist in solution as NH4 )

• ANAEROBIC • , Cyanobacteria, Archaea

ATP acetyl-CoA + CO • Requires nitrogenase enzyme 2 azoferredoxin Molybdo- N=N ferredoxin

– 21 genes α β Flavodoxin Flavodoxin Mo reductase FeS FeS – 8 subunits/accessory proteins β α

– Molybdenum and iron cofactors pyruvate + CoA 2NH3 + H2 ADP

Figure by MIT OCW. • High energy cost (16 ATP per N2) 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 Streptomyces coelicolor

5.0 Rhodopirellula baltica

Silicibacter pomeroyi Coxiella burnetii Bartonella henselae Synechococcus sp.WH8102 Thermoplasma acidophilum Prochlorococcus marinus MIT9313 Bartonella quintana Prochlorococcus marinus SS120 Ehrlichia ruminantium Prochlorococcus marinus MED4 Pelagibacter ubique 1.0 conorii

Mesoplasma florum genitalium

Genome size (Mbp) Wigglesworthia glossinidia 0.5

Host-associated Free-living Obligate symbionts/parasites Pelagibacter ubique

0.1 100 500 1000 5000 10000 Number of protein encoding genes 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

No inversions, translocations, duplications, or gene acquisitions !

4000

400

2000 200 Buchnera aphidicola str. Escherichia coli K12 Sg (Schizaphis graminum)

0 0 0 2000 4000 0 200 400 600 Buchnera aphidicola str. Salmonella Typhimurium LT2 APS (Acyrthosiphon pisum) Figure by MIT OCW. Genome Dynamics in Buchnera Obligate endosymbiont o Substantial sequence divergence o Prominence of pseudogenes o Loss of DNA repair mechanisms o 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 Plant wound Phenolic compounds

VirA

VirA Agrobacterium cell ATP ADP

Plant chromosomal DNA VirG of other VirG P vir genes T-DNA Bacterial genome Ti plasmid T-DNA T-DNA T-DNA Single- stranded Crown gall nick

Vir D Transformed plant cell Agrobacterium tumefaciens E E E E VirE E E (single-strand E E DNA binding Figure by MIT OCW. protein)

• Ti plasmid & crown gall disease Plant cell

– A portion of the Ti plasmid is VirB inserted into the plant chomosome Agrobacterium E E E causing the formation of the tumor cell E or gall. Figure by MIT OCW. Horizontal Gene Transfer: Transformation

Release of DNA by growing or decaying cells

Exposure to becteria Stablization Inactivation Degradation

Expression of competence

Uptake

Restriction

Degradation Recombination Recircularization

Mutations, rerrangements

Expression

Selection

Negative Neutral Positive Figure by MIT OCW. Horizontal Gene Transfer: Conjugation and Transduction

Chromosome

Chromosome

1 Specialized Prophage Transduction

Mobilizable plasmid Generalized 3 incX Transposon Pilus incY

Integron Conjugative plasmid 2 Mobile gene incZ incY cassettes 3

Figure by MIT OCW. Donor cell Recipient cell DNA Technologies • Basics of Sequencing – Sanger method – Shotgun sequencing • Cloning (cloning vectors) – Plasmids –Phage – Bacterial Artificial Chromosomes (BAC) – Yeast Artificial Chromosomes (YAC)