Genomic Analysis of Nitrogen fixation in  Synechococcus  sp. PCC 7002 and  Chlorobium tepidum <ul><li>Heather Jordan </li>...
Bryant Lab Research Interests <ul><li>Physiology of growth at low temperature in cyanobacteria </li></ul><ul><li>The role ...
Bryant Lab Research Interests <ul><li>Chlorobium tepidum </li></ul><ul><li>Synechococcus sp. PCC 7002 </li></ul>
What is cyanobacteria? <ul><li>7500 species </li></ul><ul><ul><li>Greatest diversity in fresh water </li></ul></ul><ul><li...
What is cyanobacteria? <ul><li>Morphological Hierarchy: </li></ul><ul><ul><li>Single cells </li></ul></ul><ul><ul><li>Colo...
Evolutionary Significance <ul><li>Important source for fixed nitrogen </li></ul><ul><li>Cyanobacteria most likely origin o...
Diversity of Cyanobacteria <ul><li>Differences probably due to adaptation to different environments </li></ul><ul><li>Sequ...
The Ecological Significance of Cyanobacteria <ul><li>Can lead to erosion in limestone & coral reefs </li></ul><ul><ul><li>...
Nitrogen Fixation <ul><ul><li>Nitrogen is needed for the synthesis of amino acids & nucleotides </li></ul></ul><ul><ul><li...
Nitrogen Fixation <ul><li>Often have symbiotic relationships with other organisms </li></ul><ul><ul><li>Lichens, Hornworts...
Nitrogen Fixation <ul><ul><li>In non-heterocystous cyanobacteria (i.e.,  Synechococcus ), N is fixed aerobically </li></ul...
Nitrogen Fixation Genes <ul><li>NAR1 ( Synechococcus ) </li></ul><ul><li>nifD ( C. tepidum ) </li></ul><ul><li>Nitrogen fi...
Project 1: Genomic Analysis of Nitrogen Fixation in  Synechococcus  sp. PCC 7002
Optimal Growth Conditions for Synechoccus <ul><li>Temperature:  38 o C </li></ul><ul><li>Likes brackish water </li></ul><u...
Other Characteristics of  Synechococcus <ul><li>Also known as  Agmenellum quadrupiplicatum  strain PR-6 </li></ul><ul><li>...
Other Characteristics of  Synechococcus <ul><li>Foundation of the marine food web </li></ul><ul><li>Primary producers on a...
Hypothesis: <ul><li>Knocking out NAR1 should result in physiologically interesting phenotypes </li></ul>  <ul><li>Do thi...
Checking for segregation & orientation <ul><li>Segregation:  Is the insert present? </li></ul><ul><ul><li>YES! </li></ul><...
Project 2: Genomic Analysis of Nitrogen Fixation in  Chlorobium tepidum
Optimal Growth Conditions for C. tepidum <ul><li>Found in sediments, muds, microbial mats and anoxic & sulfide-rich waters...
Other Characteristics of  C. tepidum <ul><li>Methods for natural transformation allow for targeted gene inactivation by ho...
Transformation of  C. tepidum <ul><li>Antibiotic resistance used as marker </li></ul><ul><ul><li>Spectinomycin, streptomyc...
Genome   Highlights <ul><li>1 circular DNA molecule </li></ul><ul><li>2,154,946 bp </li></ul><ul><li>G+C content 49.1% </l...
nifD Details <ul><li>nifD gene encodes a subunit of nitrogenase </li></ul><ul><li>Located in the middle of the nifHDK oper...
Creating a nifD knockout <ul><li>Making pTN1CX </li></ul><ul><ul><li>nifD knock-out construct for C. tepidum </li></ul></u...
Hypothesis: <ul><li>Since targeted inactivation of nifD using antibiotics as markers has worked successfully in the past, ...
nifD Project Outline ——————  I am here. ——————  Previous End
References <ul><li>Frigaard, N.U., and Bryant, D.A. (2001) Chromosomal Gene Inactivation in the Green Sulfur Baterium Chlo...
Acknowledgements <ul><li>Joel Graham </li></ul>Niels-Ulrik Frigaard
?? Questions??
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Genomic Analysis Of Nitrogen Fixation In Synechococcus Sp. PCC 7002 and Chlorobium tepidum

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Genomic Analysis Of Nitrogen Fixation In Synechococcus Sp. PCC 7002 and Chlorobium tepidum

  1. 1. Genomic Analysis of Nitrogen fixation in Synechococcus sp. PCC 7002 and Chlorobium tepidum <ul><li>Heather Jordan </li></ul><ul><li>Department of Biochemistry and Molecular Biology </li></ul><ul><li>The Pennsylvania State University </li></ul><ul><li>University Park, Pennsylvania 16802 </li></ul><ul><li>March 18, 2003 </li></ul>
  2. 2. Bryant Lab Research Interests <ul><li>Physiology of growth at low temperature in cyanobacteria </li></ul><ul><li>The role of multiple sigma factors in gene expression in cyanobacteria </li></ul><ul><li>The structure, function & biogenesis of PI rxn center </li></ul><ul><li>Alternative electron transport pathways in cyanobacteria </li></ul><ul><li>Photosynthetic apparatus in green sulfur bacteria (chlorosome) </li></ul><ul><li>Physiology & genetics of green sulfur bacteria C. tepidum & C. vibrioforme </li></ul><ul><li>Bacteriochlorophyll biosynthesis in green sulfur bacterium C. tepidum </li></ul>
  3. 3. Bryant Lab Research Interests <ul><li>Chlorobium tepidum </li></ul><ul><li>Synechococcus sp. PCC 7002 </li></ul>
  4. 4. What is cyanobacteria? <ul><li>7500 species </li></ul><ul><ul><li>Greatest diversity in fresh water </li></ul></ul><ul><li>Pigmentation </li></ul><ul><ul><li>Chlorophyll </li></ul></ul><ul><ul><li>Carotenoids </li></ul></ul><ul><ul><li>Phycobillins </li></ul></ul><ul><li>Add biomass & nitrogen to barren areas </li></ul><ul><li>Found in a wide variety of environments ranging from hot springs to glacier ice </li></ul><ul><ul><li>Recently found in the most barren area of Antarctica where no other life is found. </li></ul></ul><ul><ul><li>May also be endolithic </li></ul></ul>
  5. 5. What is cyanobacteria? <ul><li>Morphological Hierarchy: </li></ul><ul><ul><li>Single cells </li></ul></ul><ul><ul><li>Colonies </li></ul></ul><ul><ul><li>Filaments </li></ul></ul><ul><ul><li>Branched, unbranched or clustered </li></ul></ul>
  6. 6. Evolutionary Significance <ul><li>Important source for fixed nitrogen </li></ul><ul><li>Cyanobacteria most likely origin of chloroplasts </li></ul><ul><ul><li>Endosymbiont Theory </li></ul></ul><ul><ul><li>Supported through analysis of photosynthetic apparatus & C assimilation </li></ul></ul><ul><li>Components (LHC) of C. tepidum chlorosome resembles plant chloroplasts </li></ul><ul><li>Mechanism of CO 2 fixation distinct from Calvin cycle found in high plants. </li></ul><ul><li>Prominent role in global carbon cycling </li></ul><ul><li>Constitute some of the oldest fossils (over 2.7 billion years old) </li></ul><ul><ul><li>Oldest oxygen-producing organism </li></ul></ul><ul><ul><li>Profoundly changed the earth’s atmosphere. </li></ul></ul>
  7. 7. Diversity of Cyanobacteria <ul><li>Differences probably due to adaptation to different environments </li></ul><ul><li>Sequencing projects seek to establish </li></ul><ul><ul><li>Which genes are common to all </li></ul></ul><ul><ul><li>Which genes are specific to ecological niches </li></ul></ul>
  8. 8. The Ecological Significance of Cyanobacteria <ul><li>Can lead to erosion in limestone & coral reefs </li></ul><ul><ul><li>Grazing animals eat the cyanobacteria and subsequently reduce the amount of limestone present, </li></ul></ul><ul><li>Can lead to limestone </li></ul><ul><li>deposits </li></ul><ul><li>Some marine species </li></ul><ul><li>precipitate CaCO 3 </li></ul>
  9. 9. Nitrogen Fixation <ul><ul><li>Nitrogen is needed for the synthesis of amino acids & nucleotides </li></ul></ul><ul><ul><li>Organic Route: Breakdown of proteins </li></ul></ul><ul><ul><li>Inorganic Route: Nitrate Reduction </li></ul></ul><ul><ul><ul><li>Nitrate reduced to nitrite via nitrate reductase </li></ul></ul></ul><ul><ul><ul><li>Nitrite reduced to ammonia via nitrite reductase </li></ul></ul></ul><ul><ul><ul><li>Ammonia incorporated into amino acids </li></ul></ul></ul><ul><ul><li>N fixation is an energetically costly process </li></ul></ul>
  10. 10. Nitrogen Fixation <ul><li>Often have symbiotic relationships with other organisms </li></ul><ul><ul><li>Lichens, Hornworts, Azolla (fern), Cyads, Gunnera (flowering plant) </li></ul></ul><ul><li>Heterocysts are sites for N fixation </li></ul><ul><ul><li>Provide an anaerobic environment </li></ul></ul><ul><ul><li>Oxygen inhibits nitrogenase </li></ul></ul><ul><ul><li>Internal membranes are thylakoids that have lost chlorophyll (sites for nitrogenase) </li></ul></ul><ul><ul><li>Connected to vegetative cells via microplasmodesmata (pore in end wall) </li></ul></ul>
  11. 11. Nitrogen Fixation <ul><ul><li>In non-heterocystous cyanobacteria (i.e., Synechococcus ), N is fixed aerobically </li></ul></ul><ul><ul><li>Cyanobacteria unique in that they can perform both N fixation & oxygenic photosynthesis </li></ul></ul><ul><ul><ul><li>N-fixation occurs when PSII is not oxidizing H 2 O to O 2 . </li></ul></ul></ul>
  12. 12. Nitrogen Fixation Genes <ul><li>NAR1 ( Synechococcus ) </li></ul><ul><li>nifD ( C. tepidum ) </li></ul><ul><li>Nitrogen fixation related ( nif ) genes are expressed under anaerobic conditions </li></ul><ul><li>Nitrate Assimilation Related ( NAR ) genes </li></ul><ul><ul><li>NAR1 gene encodes a chloroplast membrane protein involved in nitrite transport </li></ul></ul><ul><ul><li>Nitrite reduced to ammonia </li></ul></ul>
  13. 13. Project 1: Genomic Analysis of Nitrogen Fixation in Synechococcus sp. PCC 7002
  14. 14. Optimal Growth Conditions for Synechoccus <ul><li>Temperature: 38 o C </li></ul><ul><li>Likes brackish water </li></ul><ul><li>Obligate requirement for B12 </li></ul><ul><li>Facultative photoheterotroph </li></ul><ul><li>Tolerant of high light intensities (up to 5000  E m -2 s -1 ) </li></ul>
  15. 15. Other Characteristics of Synechococcus <ul><li>Also known as Agmenellum quadrupiplicatum strain PR-6 </li></ul><ul><li>Isolated by Chase Van Baalen in 1961 from a marine mud sample in Puerto Rico </li></ul><ul><li>Naturally transformable </li></ul><ul><li>Among fastest-growing cyanobacteria (doubling time under opt. conditions = 3.5 hours) </li></ul><ul><li>Cells 1.5-2.5  m in size. </li></ul><ul><li>Usually occur as single cells but sometimes in clusters </li></ul><ul><li>Gram negative </li></ul>
  16. 16. Other Characteristics of Synechococcus <ul><li>Foundation of the marine food web </li></ul><ul><li>Primary producers on a global scale </li></ul><ul><li>One of the most numerous genomes on Earth </li></ul><ul><li>Obligately marine </li></ul><ul><li>1/3 of open ocean isolates possess a type of swimming motility not seen in any other type of microbe (propel at speeds of up to 25 mm/sec) </li></ul><ul><li>Motility in response to small gradients of nitrogenous compounds </li></ul>
  17. 17. Hypothesis: <ul><li>Knocking out NAR1 should result in physiologically interesting phenotypes </li></ul>  <ul><li>Do this by inserting a Sp r cassette </li></ul>
  18. 18. Checking for segregation & orientation <ul><li>Segregation: Is the insert present? </li></ul><ul><ul><li>YES! </li></ul></ul><ul><li>Orientation: Is the insert going in the proper direction? </li></ul><ul><ul><li>Will find out tomorrow. </li></ul></ul>
  19. 19. Project 2: Genomic Analysis of Nitrogen Fixation in Chlorobium tepidum
  20. 20. Optimal Growth Conditions for C. tepidum <ul><li>Found in sediments, muds, microbial mats and anoxic & sulfide-rich waters </li></ul><ul><li>Anaerobic, obligate autotrophs </li></ul><ul><li>Photo-oxidize reduced sulfur compounds (i.e., sulfide & sulfur) </li></ul><ul><li>Temperature: 47 o C </li></ul>
  21. 21. Other Characteristics of C. tepidum <ul><li>Methods for natural transformation allow for targeted gene inactivation by homologous recombination </li></ul><ul><li>More than 30 mutants have been created with specifically inactivated genes </li></ul><ul><li>Revealed information about processes pertinent to biosynthetic pathways of carotenoids and bacteriochlorophylls to chlorosome proteins </li></ul>
  22. 22. Transformation of C. tepidum <ul><li>Antibiotic resistance used as marker </li></ul><ul><ul><li>Spectinomycin, streptomycin, Ampicillin & chloramphenicol </li></ul></ul><ul><li>Can use natural transformation, chemical transformation & electroporation. </li></ul><ul><li>Most genes targeted for inactivation were chlorosomal proteins </li></ul><ul><li>Inactivation of nifD expressed phenotypically (inability to grow diazetrophically) </li></ul><ul><ul><li>Markers used to date include Spectinomycin-Streptomycin, Gentamicin & Erythromycin </li></ul></ul>
  23. 23. Genome Highlights <ul><li>1 circular DNA molecule </li></ul><ul><li>2,154,946 bp </li></ul><ul><li>G+C content 49.1% </li></ul><ul><li>2,284 ORFs </li></ul><ul><ul><li>50% have been assigned a known function </li></ul></ul><ul><li>pAQ1 has been sequenced </li></ul><ul><li>Six plasmids (pAQ1-pAQ6) </li></ul><ul><ul><li>4.6, 10.0,15.9, 31.0, 38.6 and 115.6 kb respectively </li></ul></ul>
  24. 24. nifD Details <ul><li>nifD gene encodes a subunit of nitrogenase </li></ul><ul><li>Located in the middle of the nifHDK operon </li></ul>
  25. 25. Creating a nifD knockout <ul><li>Making pTN1CX </li></ul><ul><ul><li>nifD knock-out construct for C. tepidum </li></ul></ul><ul><li>6,553 bp </li></ul><ul><li>nifD (~1100  2550) </li></ul><ul><li>Restriction Sites: </li></ul><ul><ul><li>AhdI (6553) </li></ul></ul><ul><ul><li>HindIII (4018, 1639, 896) </li></ul></ul><ul><ul><li>ScaI (3139, 1570, 1560, 289) </li></ul></ul><ul><ul><li>Sty I (3378, 1390, 1377 339, 69) </li></ul></ul><ul><ul><li>SspI (2747, 2701, 1105) </li></ul></ul>
  26. 26. Hypothesis: <ul><li>Since targeted inactivation of nifD using antibiotics as markers has worked successfully in the past, use of a slightly altered cassette containing the same markers should work as well. </li></ul>
  27. 27. nifD Project Outline —————— I am here. —————— Previous End
  28. 28. References <ul><li>Frigaard, N.U., and Bryant, D.A. (2001) Chromosomal Gene Inactivation in the Green Sulfur Baterium Chloroboum tepidum by Natural Transformation. App. & Env. Microbiol. 2538-2544. </li></ul><ul><li>Herdman, M., Janvier, M. Waterbury, J.B., Ripka, R., Stanier, R.Y., and Mandel, M. (1979a) Deoxyribonucleic acid base composition of cyanobacteria. J. Gen. Microbiol. 111, 63-75. </li></ul><ul><li>http://geoweb.princeton.edu/research/biocomplexity/index.html </li></ul><ul><li>http://www.bact.wisc.edu/microtextbook/Metabolism/NitrogenAssim.html </li></ul><ul><li>http://www.bigelow.org/cytometry/Image_gallery/SYN.html </li></ul><ul><li>http://www.biologie.uni-hamburg.de/b-online/library/webb/BOT311/Cyanobacteria/Cyano.html </li></ul><ul><li>http://www.bmb.psu.edu/deptpage/faculty/bryant/bryant.html </li></ul><ul><li>http:// www.bmb.psu.edu/faculty/bryant/lab/index.htm </li></ul><ul><li>http://www.bom.hik.se/~njasv/disp.html </li></ul><ul><li>http://www.cbs.dtu.dk/services/GenomeAtlas/Bacteria/Chlorobium/tepidum/TLS/Ctepidum.htm </li></ul><ul><li>http://www.dsmz.de/strains/no012025.htm </li></ul><ul><li>http://www.er.doe.gov/production/ober/gc/omp.html </li></ul><ul><li>http://www.jgi.doe.gov/JGI_microbial/html/synechococcus/synech_content.html </li></ul><ul><li>http://www.ornl.gov/TechResources/Human_Genome/publicat/99santa/158.html </li></ul><ul><li>Roberts, T.M. and Koths, K.E. (1976) The blue-green alga Agmenellum quadriplicatum contains covalently closed DNA circles. Cell 9, 551-557. </li></ul><ul><li>Van Baalen, C. (1962) Studies on marine blue-green algae. Bot. Mar. 4, 129-139. </li></ul>
  29. 29. Acknowledgements <ul><li>Joel Graham </li></ul>Niels-Ulrik Frigaard
  30. 30. ?? Questions??

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