Midterm 2 Review Session. Megan McBee November 7th, 2006

Transcription

1 Midterm 2 Review Session Megan McBee November 7th, 2006

2 Topics Nitrogen Cycle Communities, symbiosis, genome reduction Horizontal Gene Transfer Biotechnology

3 Nitrogen Cycle Important Reactions Nitrogen Assimilation Incorporation of to NH 3 into organic molecules (R-NH 2 ) Deamination Deamination of organic molecules releasing NH 3 Nitrification Denitrification N 2 Fixation

4 Nitrogen Cycle: Nitrification Oxidation of NH 3 to NO 2- then NO 3 - Only carried out by microbes Soil and aquatic bacteria Aerobic chemolithoautotrophic two step process Ammonia oxidizers (Nitrosomonas, Nitrosocococcus, Nitrosospira, archaea) Nitrite oxidizers (Nitrobacter, Nitrospira) nzyme is ammonia monooxygenase

5 Nitrogen Cycle: Denitrification Nitrate reduction, conversion of NO 3 - to N 2 gas Only carried out by microbes Anaerobic chemoheterotrophic process Facultative anaerobes in sediment and oxygen-limited water Anaerobic respiration (using Nitrate and Nitrite as TAP) Oxidation of organic matter for energy Several enzyme systems--dissimilatory nitrate reductase Loss of Nitrogen from system Nitrate to ammonia NOT denitrification Plants perfom assimilatory nitrate reduction Convert NO 3 - to organic nitrogen (R-NH 2 )

6 Nitrogen Cycle: N 2 Fixation Molecular conversion of N 2 gas to NH 3 (exist in solution as NH + 4 ) ANAROBIC Proteobacteria, Cyanobacteria, Archaea Requires nitrogenase enzyme 21 genes 8 subunits/accessory proteins Molybdenum and iron cofactors High energy cost (16 ATP per N 2 ) acetyl-coa + CO 2 Flavodoxin reductase pyruvate + CoA ATP azoferredoxin Flavodoxin FeS ADP Molybdoferredoxin α β Mo FeS β α N=N 2NH 3 + H 2 Figure by MIT OCW.

7 Rhizobium Free-living are aerobic, not N 2 fixers When symbiotic Rhizobium turn on plasmid-based Nod genes Become anaerobic N 2 -fixing, bacteroid form Legumes form nodules to control symbiotic relationship Images of free-living Rhizobium and bacterioids in nodule removed due to copyright restrictions.

8 Symbiosis and Genome Reduction 10.0 Streptomyces coelicolor 5.0 Rhodopirellula baltica Genome size (Mbp) Mycoplasma genitalium Coxiella burnetii Bartonella henselae Thermoplasma acidophilum Bartonella quintana hrlichia ruminantium Pelagibacter ubique Rickettsia conorii Mesoplasma florum Wigglesworthia glossinidia Nanoarchaeum equitans Silicibacter pomeroyi Synechococcus sp.wh8102 Prochlorococcus marinus MIT9313 Prochlorococcus marinus SS120 Prochlorococcus marinus MD4 Host-associated Free-living Obligate symbionts/parasites Pelagibacter ubique Number of protein encoding genes Figure by MIT OCW.

9 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 non-synonymous substitutions/yr. coli and Salmonella spp. (closest free-living relatives) 2000x more liable No inversions, translocations, duplications, or gene acquisitions! 4000 scherichia coli K Buchnera aphidicola str. Sg (Schizaphis graminum) Salmonella Typhimurium LT2 Buchnera aphidicola str. APS (Acyrthosiphon pisum) Figure by MIT OCW.

10 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

11 Argobacterium Plant wound Phenolic compounds VirA VirA ATP Plant chromosomal DNA Ti plasmid T-DNA Bacterial genome VirG P VirG T-DNA T-DNA Agrobacterium cell ADP Transcription of other vir genes T-DNA Singlestranded nick Crown gall Vir D Agrobacterium tumefaciens Transformed plant cell Figure by MIT OCW. Ti plasmid & crown gall disease A portion of the Ti plasmid is inserted into the plant chomosome causing the formation of the tumor or gall. Figure by MIT OCW. Vir (single-strand DNA binding protein) Plant cell VirB Agrobacterium cell

12 Horizontal Gene Transfer: Transformation Release of DNA by growing or decaying cells Stablization xposure to becteria Inactivation Degradation xpression of competence Uptake Restriction Recombination Degradation Recircularization Mutations, rerrangements xpression Selection Negative Neutral Positive Figure by MIT OCW.

13 Horizontal Gene Transfer: Conjugation and Transduction Chromosome Chromosome Prophage 3 1 Mobilizable plasmid incx Specialized Transduction Generalized Transposon incy Pilus incy Integron Conjugative plasmid 2 3 Mobile gene cassettes incz Donor cell Recipient cell Figure by MIT OCW.

14 DNA Technologies Basics of Sequencing Sanger method Shotgun sequencing Cloning (cloning vectors) Plasmids Phage Bacterial Artificial Chromosomes (BAC) Yeast Artificial Chromosomes (YAC)