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Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation
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Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation

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  • 1. Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation NIELS-ULRIK FRIGAARD* AND DONALD A. BRYANT Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania Journal Article Review Heather Jordan BMMB 507 April 8, 2003
  • 2. What is Chlorobium tepidum ? <ul><li>Moderately thermophilic green sulfur bacterium </li></ul><ul><ul><li>Temperature: 47 o C </li></ul></ul>
  • 3. What is Chlorobium tepidum ? <ul><li>Moderately thermophilic green sulfur bacterium </li></ul><ul><ul><li>Temperature: 47 o C </li></ul></ul><ul><li>Anaerobic, obligate autotrophs </li></ul>
  • 4. What is Chlorobium tepidum ? <ul><li>Moderately thermophilic green sulfur bacterium </li></ul><ul><ul><li>Temperature: 47 o C </li></ul></ul><ul><li>Anaerobic, obligate autotrophs </li></ul><ul><li>Live in sulfide-rich aquatic environments </li></ul><ul><ul><li>Photo-oxidize reduced sulfur compounds (i.e., sulfide & sulfur) </li></ul></ul><ul><ul><li>Found in sediments, muds, microbial mats and anoxic & sulfide-rich waters </li></ul></ul>
  • 5. What is Chlorobium tepidum ? <ul><li>Moderately thermophilic green sulfur bacterium </li></ul><ul><ul><li>Temperature: 47 o C </li></ul></ul><ul><li>Anaerobic, obligate autotrophs </li></ul><ul><li>Live in sulfide-rich aquatic environments </li></ul><ul><ul><li>Photo-oxidize reduced sulfur compounds (i.e., sulfide & sulfur) </li></ul></ul><ul><ul><li>Found in sediments, muds, microbial mats and anoxic & sulfide-rich waters </li></ul></ul><ul><li>Distinct phylogeny (not closely related to other bacteria) </li></ul>
  • 6. Current Research Interests <ul><li>Lithotrophic oxidation of sulfur compounds </li></ul><ul><li>CO 2 fixation </li></ul><ul><li>Photosynthetic electron transport </li></ul><ul><li>Energy transfer and organization of the chlorosomes </li></ul><ul><li>Biosynthesis and function of chlorophylls </li></ul><ul><li>Nitrogen fixation </li></ul>
  • 7. 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>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><ul><li>pAQ1 has been sequenced </li></ul>
  • 8. Transformation of C. tepidum <ul><li>Methods for natural transformation allow for targeted gene inactivation by homologous recombination </li></ul>
  • 9. Transformation 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>
  • 10. Transformation 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>
  • 11. Transformation of C. tepidum <ul><li>Antibiotic resistance used as marker </li></ul><ul><ul><li>Spectinomycin, streptomycin, Ampicillin & chloramphenicol </li></ul></ul>
  • 12. 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>
  • 13. 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>
  • 14. 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>The only genes targeted for inactivation encode: </li></ul><ul><ul><li>CsmC & CsmA (chlorosomal proteins) </li></ul></ul><ul><ul><li>Reaction center cytochrome c551 Prc </li></ul></ul><ul><ul><li>rbcL (Rubisco subunit) </li></ul></ul><ul><ul><ul><li>Only CsmC & rbcL fully segregated </li></ul></ul></ul>
  • 15. Nitrogen Fixation <ul><ul><li>Major Nutrient </li></ul></ul><ul><ul><ul><li>Accounts for 11% of dry weight of bacterial cells </li></ul></ul></ul><ul><ul><li>Nitrate reduction & assimilation is a high energy process </li></ul></ul><ul><ul><ul><li>Up to 30 % of the electrons generated by photosynthetic H 2 O oxidation are consumed during the reduction of nitrate to ammonia </li></ul></ul></ul><ul><ul><li>Cyanobacterial cells preferentially use reduced nitrogen sources </li></ul></ul><ul><ul><ul><li>Ammonia & Urea </li></ul></ul></ul>
  • 16. Nitrogen Fixation <ul><li>Nitrogen is needed for the synthesis of amino acids & nucleotides </li></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>N fixation is an energetically costly process </li></ul></ul></ul><ul><li>Nitrogen fixation related ( nif ) genes are expressed under anaerobic conditions </li></ul><ul><li>nifD gene : encodes a subunit of nitrogenase </li></ul>
  • 17. Study Objective <ul><li>To form a foundation for the systematic targeted inactivation of genes in C. tepidum (the genome for which had been recently sequenced). </li></ul><ul><ul><li>nifD gene used to formulate the general model </li></ul></ul><ul><ul><li>Inactivation of nifD expressed phenotypically (inability to grow diazetrophically) </li></ul></ul><ul><ul><ul><li>Markers used include Spectinomycin-Streptomycin, Gentamicin & Erythromycin </li></ul></ul></ul>
  • 18. Maps <ul><li>NifHDK operon </li></ul>
  • 19. Maps <ul><li>NifHDK operon </li></ul><ul><li>Streptomycin-Spectinomycin Resistance Cassette </li></ul>
  • 20. Maps <ul><li>NifHDK operon </li></ul><ul><li>Streptomycin-Spectinomycin Resistance Cassette </li></ul><ul><li>Gentamicin Resistance Cassette </li></ul>
  • 21. Maps <ul><li>NifHDK operon </li></ul><ul><li>Streptomycin-Spectinomycin Resistance Cassette </li></ul><ul><li>Gentamicin Resistance Cassette </li></ul><ul><li>Erythromycin-Chloramphenicol Resistance Cassette </li></ul>
  • 22. 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>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>
  • 23. Antibiotic Sensitivity <ul><li>Temperature: </li></ul><ul><ul><li>Lower than optimum (48 o C): 40 o C </li></ul></ul><ul><ul><li>Antibiotic resistance markers originate from mesophiles </li></ul></ul><ul><li>Concentrations That Inhibit Growth: </li></ul><ul><ul><li>Gentamicin, 100 μ g ml -1 </li></ul></ul><ul><ul><li>Erythromycin, 2 μ g ml -1 </li></ul></ul><ul><ul><li>Chloramphenicol, 30 μ g ml -1 </li></ul></ul><ul><ul><li>Tetracycline, 1 μ g ml -1 </li></ul></ul><ul><ul><li>Streptomycin & Spectinomycin, 300 μ g ml -1 & 150 μ g ml -1 (combined) </li></ul></ul><ul><ul><li>Kanamycin (100 μ g ml -1 +) </li></ul></ul><ul><ul><li>Ampicillin (100 μ g ml -1 +) </li></ul></ul><ul><li>aadA Cassette: </li></ul><ul><ul><li>Confers resistance to Streptomycin & Spectinomycin </li></ul></ul><ul><ul><li>Antibiotics Not Tested: </li></ul></ul><ul><ul><li>Amoxicillin, nalidixic acid, vancomycin, mitomycin C and colistin. </li></ul></ul>
  • 24. Optimization of Transformation <ul><li>Transformation Frequency : </li></ul><ul><ul><li>100 μl late exponential-phase culture </li></ul></ul><ul><ul><li>Incubated with 1 μg of Ahd I-digested pTN1G4 </li></ul></ul><ul><ul><li>In 10 hours, frequency reached 2E-7 to 3E-7 </li></ul></ul><ul><ul><ul><li>Corresponds to 100 transformants per μg DNA </li></ul></ul></ul><ul><li>:. Most transformation events occurred at the beginning of the experiment and were stable. </li></ul>• Gentamicin-resistant transformants ° Transformation Frequency
  • 25. Optimization of Transformation <ul><li>Liquid Suspensions & Transformation : </li></ul><ul><ul><li>Same method as for plates </li></ul></ul><ul><ul><li>Incubated with 1 μg DNA </li></ul></ul><ul><ul><li>Then plated on selective plates </li></ul></ul><ul><ul><li>Highest transformation frequency 1 order of magnitude lower than transformation frequencies on agar plates </li></ul></ul><ul><ul><li>Why? </li></ul></ul><ul><ul><ul><li>“ DNA may interact differently than in liquid suspension… allow for increased uptake of DNA by the cells.” </li></ul></ul></ul>• Gentamicin-resistant transformants ° Transformation Frequency
  • 26. Optimization of Transformation <ul><ul><li>Stationary vs. Late Log Phase Cells : </li></ul></ul><ul><ul><ul><li>Cells are competent in both phases </li></ul></ul></ul><ul><ul><ul><li>Stationary cells gave ½ as many transformants as the late-exponential-phase cells </li></ul></ul></ul><ul><ul><li>Linearized DNA </li></ul></ul><ul><ul><ul><li>Increasing amounts of DNA yielded an increased transformation frequency </li></ul></ul></ul><ul><ul><ul><li>Increasing the DNA from 0.1-10 μg increased the frequency only 3-fold </li></ul></ul></ul><ul><ul><ul><li>Suggests that 10 μg of DNA is close to the saturation amount </li></ul></ul></ul><ul><li>Transformation frequency with linear plasmid was an order of magnitude higher than with circular plasmid. </li></ul><ul><ul><li>Difference is probably due to DNA binding and uptake mechanisms of the cell. </li></ul></ul>
  • 27. Effect of Variation in Length of Homologous Flanking DNA <ul><li>When a plasmid construct is made for gene inactivation by homologous recombination, it is typically advantageous to include a large region of the homologous DNA to increase the probability of homologous recombination. </li></ul><ul><li>Restriction endonuclease sites and toxic gene products may impose limits on the length of homologous DNA that can be cloned. </li></ul>
  • 28. Effect of Variation in Length of Homologous Flanking DNA <ul><li>To determine the length of homologous flanking DNA on transformation, 3 constructs for inactivation were made. </li></ul>{ { {
  • 29. Effect of Variation in Length of Homologous Flanking DNA <ul><li>To determine the length of homologous flanking DNA on transformation, 3 constructs for inactivation were made. </li></ul><ul><li>Gentamicin resistance marker is inserted in the middle </li></ul>{ { {
  • 30. Effect of Variation in Length of Homologous Flanking DNA <ul><li>Plasmids were digested with : </li></ul><ul><ul><li>Ahd I, which cuts only once </li></ul></ul><ul><ul><li>Eco RI, which cuts twice. </li></ul></ul>{ { {
  • 31. Effect of Variation in Length of Homologous Flanking DNA <ul><li>Transformation frequencies with : </li></ul><ul><ul><li>2.93 kb homologous DNA </li></ul></ul><ul><ul><ul><li>Similar regardless of enzyme used for linearization </li></ul></ul></ul><ul><ul><li>1.08 kb homologous DNA </li></ul></ul><ul><ul><ul><li>Transformation frequency was 1 order of magnitude lower when the plasmid was digested with Eco RI (as oppsed to Ahd I) </li></ul></ul></ul><ul><ul><li>0.29 kb homologous DNA </li></ul></ul><ul><ul><ul><li>No transformation observed regardless of enzyme </li></ul></ul></ul><ul><li>Why? </li></ul><ul><ul><li>Some bacteria partially degrade absorbed DNA via exonuclease activity. </li></ul></ul>
  • 32. Effect of Variation in Length of Homologous Flanking DNA <ul><li>Given this: </li></ul><ul><ul><li>A homologous flanking region of about 1 kb should be used in transformation </li></ul></ul><ul><ul><li>Linearize with a plasmid that leaves dispensible flanking DNA at the ends of the fragment </li></ul></ul><ul><ul><li>Separate nontransforming DNA from transforming (produced by digest) </li></ul></ul><ul><ul><ul><li>Nontransforming may compete with transforming DNA for uptake into the cells </li></ul></ul></ul><ul><ul><ul><li>Supported by side experiment in which 20 μg of sonicated chromosomal DNA from Synechococcus to a C. tepidum transformation mixture (containing 1 μg of linearlized DNA) decreased the transformation frequency an order of magnitude. </li></ul></ul></ul>
  • 33. Various Selection Markers <ul><li>3 constructs for nifD inactivation were made with different antibiotic resistance markers </li></ul><ul><li>Transformation was about the same when the Gentamicin and erythromycin-chloramphenicol resistance markers were used </li></ul><ul><li>Oddly, only resistant to erythromicin (even though both genes were present). Confirmed by southern hybridization. </li></ul>
  • 34. Various Selection Markers <ul><li>Chloramphenicol marker didn’t function because: </li></ul><ul><ul><li>Either the expressed protein is not functional in C. tepidum </li></ul></ul><ul><ul><li>Or the promoter is too weak in C. tepidum </li></ul></ul><ul><li>Neither of these possibilities were investigated further </li></ul>
  • 35. Various Selection Markers <ul><li>Transformation efficiency was 4 orders of magnitude higher with the aadA marker than the other two ( aaC1 & ermC ) </li></ul><ul><li>The reason for this may lie in the genomic sequence </li></ul><ul><ul><li>aadA marker contains a 59-bp “recombinational hot spot” </li></ul></ul><ul><li>All 22 mutants created were first screened for antibiotic resistance and then for the expression of the desired phenotype. </li></ul>
  • 36. Test of Transformants <ul><li>Expected phenotype of transformants is the inability to reduce dinitrogen. </li></ul><ul><li>Results confirmed that mutants had lost nitrogen fixation ability and that mutations were fully segregated. </li></ul><ul><li>PCR analysis amplified a 0.41 kb fragment in WT and 1.46 kb fragment in the mutants. </li></ul><ul><li>PCR with primers specific for the aaC1 did not produce a product in WT but amplified a 0.75 kb product in the mutants. </li></ul>  
  • 37. Conclusions <ul><li>Genes in C. tepidum can be insertionally inactivated by natural transformation & homologous recombination </li></ul><ul><li>The following markers were used successfully: </li></ul><ul><ul><li>Gentamicin ( aacC1 ) </li></ul></ul><ul><ul><li>Erythromycin ( ermC ) </li></ul></ul><ul><ul><li>Streptomycin-Spectinomycin ( aadA ) </li></ul></ul><ul><ul><ul><li>This marker gave significantly higher transformation than the others </li></ul></ul></ul>
  • 38. Guidelines for Routine Gene Inactivation by Natural Transformation <ul><li>Use cells from at least 100 μl of a late-exponential liquid culture </li></ul><ul><li>Use linearized DNA (1-10μg) with sequences of at least 0.5 kb of flanking homologous DNA </li></ul><ul><li>Transforming cells should be spotted on agar surface & incubated for 10-20 hours at 40 o C </li></ul><ul><ul><li>Shorten incubation time for higher temperatures </li></ul></ul><ul><li>Transformation may be done by scraping cells off of a plate and incubating the tranforming mixture overnight on a nonselective plate. </li></ul><ul><ul><li>The cells should then be re-streaked the following day . </li></ul></ul>
  • 39. 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>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>Sakamoto, T., Inoue-Sakamoto, K. and Bryant, D.A. (1999) A Novel Nitrate/Nitrite Permease in the Marine Cyanobacterium Synechococcus sp. Strain PCC 7002. Journal of Bacteriology. 7363-7372. </li></ul>
  • 40. ??Questions??

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