CHARACTERIZING THE RESPONSE OF
   Vibrio tubiashii TO CHANGES IN
  ENVIRONMENTAL CONDITIONS
           a funomic approach
...
Vibrio tubiashii




RE98
How do changes in the environment
influence Vibrio tubiashii physiology?
How do we assess alterations in
     Vibrio tubiashii physiology?



changes in expression of genes and proteins

    basi...
Experiments


   • Simple
    • Low oxygen conditions
   • Little more complex
    • Presence of oysters
Low oxygen conditions




                        OD@600
Approach - Protein Expression

Protein Extraction

                         2D gels


                                   I...
Low oxygen conditions
Low oxygen conditions




         for audience orientation
Differential Expression
Low oxygen conditions
1/3 Anoxic, 6hours
1.                                                                1/3 Anoxic, 6ho...
Low oxygen conditions
Control                                                                                                                   ...
Control
GO Annotations                                                     Chart 6

                                      biosynth...
V. tubiashii

V. tubiashii +
 live oysters

V. tubiashii +
sterile oysters

Oysters
                  Experiment Two: Oyst...
Approach - Gene Expression

RNA Extraction


                 Select Genes


                                qPCR
Approach - Protein Expression

Protein Extraction

                         2D gels


                                   I...
Gene Discovery using NGS
            Library Prep




       Cluster Generation




              Sequencing
Gene Discovery using NGS




                      226 million bp
Genes
flagellar biosynthesis protein FlhG        outer membrane protein W                    flagellin
6                        ...
flagellar biosynthesis protein FlhG        outer membrane protein W                    flagellin
6                        ...
Approach - Protein Expression

Protein Extraction

                         2D gels


                                   I...
24 hour
exposure
Putative replication protein
HTH-type transcriptional regulator
fructose repressor
Thermostable 8-oxoguanine DNA glycosyla...
peptide ABC transporter
bacterial sugar transferase
Dna-J like membrane chaperone protein
putative ABC transporter substra...
Summary - Low oxygen


   • Low oxygen conditions alter protein
     expression patterns
   • Comparative genome analysis ...
Summary - Oyster presence

   • NGS is viable option for gene discovery
     and expression
   • Flagella associated genes...
Future Direction

   • Comparative microbial analysis to
     determine species specific responses to
     environmental ch...
V tubiashii - NSA 2009
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V tubiashii - NSA 2009

  1. 1. CHARACTERIZING THE RESPONSE OF Vibrio tubiashii TO CHANGES IN ENVIRONMENTAL CONDITIONS a funomic approach Tatyana Marushchak, Sam White, Steven Roberts School of Aquatic and Fishery Sciences | University of Washington
  2. 2. Vibrio tubiashii RE98
  3. 3. How do changes in the environment influence Vibrio tubiashii physiology?
  4. 4. How do we assess alterations in Vibrio tubiashii physiology? changes in expression of genes and proteins basic biological information complementing experimental and environmental studies
  5. 5. Experiments • Simple • Low oxygen conditions • Little more complex • Presence of oysters
  6. 6. Low oxygen conditions OD@600
  7. 7. Approach - Protein Expression Protein Extraction 2D gels ID differences
  8. 8. Low oxygen conditions
  9. 9. Low oxygen conditions for audience orientation
  10. 10. Differential Expression
  11. 11. Low oxygen conditions 1/3 Anoxic, 6hours 1. 1/3 Anoxic, 6hours Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase. 13. Glycogen synthase. Vibriobactin-specific 2,3- dihydroxybenzoate-AMP ligase. Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase. Regulatory protein luxO. Peptide chain release factor 3. NAD-dependent malic enzyme 1. 2. NAD-dependent malic enzyme. DNA gyrase subunit B. CTP synthase. 3. 14. Fatty acid oxidation complex subunit alpha. Thiamine biosynthesis protein thiI. 4. GTP-binding protein engA. Beta-hexosaminidase. Soluble pyridine nucleotide transhydrogenase. DNA mismatch repair protein mutS. Thiamine biosynthesis protein thiI. DNA mismatch repair protein mutS. 15. Penicillin-binding protein 1A. Probable endonuclease 4. DNA gyrase subunit B. Probable endonuclease 4. 5. RNA polymerase sigma-32 factor. Penicillin-binding protein 1A. Probable endonuclease 4. DNA gyrase subunit B. Probable inorganic polyphosphate/ATP-NAD kinase. 6. S-adenosyl-L-methionine-dependent methyltransferase mraW. 5-methyltetrahydropteroyltriglutamate-- homocysteine meth... 16. 7. Fatty acid metabolism regulator protein. UvrABC system protein A. ATP synthase a chain. UvrABC system protein A. Lipase. UvrABC system protein A. Fatty acid metabolism regulator protein. HTH-type transcriptional regulator malT. 17. UvrABC system protein A. 2-dehydro-3-deoxyphosphooctonate aldolase. 8. Diaminopimelate epimerase. ToxR-activated gene A lipoprotein. Glucosamine-6-phosphate deaminase. ToxR-activated gene A lipoprotein. Glucosamine-6-phosphate deaminase. HTH-type transcriptional regulator malT. Glucosamine-6-phosphate deaminase. 9. N-acetylmannosamine kinase. UvrABC system protein A. 2-dehydropantoate 2-reductase. UvrABC system protein A. 2-dehydro-3-deoxyphosphooctonate aldolase. UvrABC system protein A. 18. UvrABC system protein A. UvrABC system protein A. 10. UvrABC system protein A. ~ 23 upregulated tRNA uridine 5-carboxymethylaminomethyl modification enzy... 19. tRNA 5-methylaminomethyl-2-thiouridine biosynthesis bifun... 11. 20. Threonyl-tRNA synthetase. Probable O-sialoglycoprotein endopeptidase. proteins 1-deoxy-D-xylulose-5-phosphate synthase. Formimidoylglutamase. tRNA uridine 5-carboxymethylaminomethyl modification enzy... tRNA pseudouridine synthase D. Macrolide export ATP-binding/permease protein macB. Homoserine O-succinyltransferase. 1-deoxy-D-xylulose-5-phosphate synthase. Succinylglutamate desuccinylase. tRNA uridine 5-carboxymethylaminomethyl modification enzy... Putative ribosome biogenesis GTPase rsgA 2. Threonyl-tRNA synthetase. Formimidoylglutamase. 1-deoxy-D-xylulose-5-phosphate synthase. tRNA pseudouridine synthase D. Acetyl-coenzyme A synthetase. 21. Urocanate hydratase. Probable sucrose-6-phosphate hydrolase. Choline dehydrogenase. DNA repair protein recN. Choline dehydrogenase. Urocanate hydratase. NAD-dependent malic enzyme 1. DNA repair protein recN. 2-succinyl-5-enolpyruvyl-6-hydroxy-3- cyclohexene-1-carbo... Thiol:disulfide interchange protein dsbD. Formate--tetrahydrofolate ligase. Thermostable direct hemolysin 2. Choline dehydrogenase. Peptide deformylase 2. 2-succinyl-5-enolpyruvyl-6-hydroxy-3- cyclohexene-1-carbo... 23. NAD-dependent malic enzyme. Ferric uptake regulation protein. Urocanate hydratase.
  12. 12. Low oxygen conditions
  13. 13. Control 1/3 Aerobic, 6 hours 37. Aspartate carbamoyltransferase catalytic chain. UPF0276 protein VP3015. 1/3 Aerobic, 6 hours 1/3 Aerobic, 6 hours Recombination-associated protein rdgC. 1. Porphobilinogen deaminase. Nuclease sbcCD subunit C. 19. 38. 2. Phosphoenolpyruvate carboxylase. Toxin coregulated pilus biosynthesis protein F. Autoinducer 2 sensor kinase/phosphatase luxQ. 20. tRNA-modifying protein ygfZ. 3. Regulatory protein luxO. tRNA 2-thiocytidine biosynthesis protein ttcA. Microbial collagenase. 21. Toxin coregulated pilus biosynthesis protein F. 4. Lipid-A-disaccharide synthase. tRNA 2-thiocytidine biosynthesis protein ttcA. Glutamate-ammonia-ligase adenylyltransferase. Lipid-A-disaccharide synthase. tRNA 2-thiocytidine biosynthesis protein ttcA. Glutamate-ammonia-ligase adenylyltransferase. Putative dioxygenase VC_1345. tRNA 2-thiocytidine biosynthesis protein ttcA. 5. 22. 39. Chaperone protein hscA homolog. D-erythrose-4-phosphate dehydrogenase. Probable O-sialoglycoprotein endopeptidase. Vitamin B12 transporter btuB. 23. Homoserine O-succinyltransferase. 6. Beta-hexosaminidase. 4-hydroxythreonine-4-phosphate dehydrogenase. DNA polymerase III subunit alpha. N-acetyl-gamma-glutamyl-phosphate reductase. 7. UPF0176 protein VC_1259. 40. Methionyl-tRNA synthetase. 24. Autoinducer 2-binding periplasmic protein luxP. Methionyl-tRNA synthetase. Phosphate import ATP-binding protein pstB 2. 3-ketoacyl-CoA thiolase. UvrABC system protein B. Diaminopimelate epimerase. 3-ketoacyl-CoA thiolase. Methionyl-tRNA synthetase. Glutamate racemase. 41. Methionyl-tRNA synthetase. Pirin-like protein VC_A0969. N-acetylglucosamine-6-phosphate deacetylase. 8. Lipid-A-disaccharide synthase. Glutamyl-tRNA synthetase. 25. Probable cysteine desulfurase. GMP synthase [glutamine-hydrolyzing]. Carboxylesterase bioH. Sulfate/thiosulfate import ATP-binding protein cysA. Membrane-bound lytic murein transglycosylase F. Vitamin B12 import ATP-binding protein btuD. Tryptophan synthase beta chain. GMP synthase [glutamine-hydrolyzing]. Shikimate dehydrogenase. 42. 9. Uncharacterized exonuclease xni. Glucans biosynthesis protein G. Aldehyde dehydrogenase. 26. ~ 47 upregulated Inner membrane protein oxaA. ATP synthase subunit alpha. Transaldolase. CTP synthase. UDP-N-acetylmuramate--L-alanine ligase. Dihydrodipicolinate synthase. Inner membrane protein oxaA. Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase. 27. UPF0061 protein VC_1931. Aldehyde dehydrogenase. Leucyl/phenylalanyl-tRNA--protein transferase. Glucans biosynthesis protein G. proteins ATP synthase subunit alpha. UPF0271 protein VV2_0273. 43. UDP-N-acetylmuramate--L-alanine ligase. UPF0135 protein VC_2093. Catalase-peroxidase. 10. 28. GTP pyrophosphokinase. Dihydrolipoyl dehydrogenase. Anthranilate synthase component 1. 1,4-alpha-glucan-branching enzyme. Regulatory protein luxO. Glucose-6-phosphate isomerase. Catalase-peroxidase. 3-isopropylmalate dehydratase large subunit. Glucose-6-phosphate isomerase. 44. Regulatory protein luxO. 29. 1-deoxy-D-xylulose-5-phosphate synthase. 11. 30. PTS system N-acetylmuramic acid-specific EIIBC component. 3-octaprenyl-4-hydroxybenzoate carboxy- lyase. Regulatory protein luxO. 1-deoxy-D-xylulose-5-phosphate synthase. Cytochrome c-552. L-arabinose isomerase. 45. Nicotinate phosphoribosyltransferase. Histidine ammonia-lyase. DNA mismatch repair protein mutS. 12. Regulatory protein luxO. DNA mismatch repair protein mutS. Lipid-A-disaccharide synthase. 31. DNA mismatch repair protein mutS. 3-oxoacyl-[acyl-carrier-protein] synthase 2. Glucose-6-phosphate isomerase. Autoinducer 2 sensor kinase/phosphatase luxQ. 13. Phosphoenolpyruvate carboxykinase [ATP]. DNA mismatch repair protein mutS. Nuclease sbcCD subunit C. Phosphoenolpyruvate carboxykinase [ATP]. DNA gyrase subunit B. 14. Peptide chain release factor 3. 46. HTH-type transcriptional regulator malT. 32. Autoinducer 2 sensor kinase/phosphatase luxQ. 15. Peptide chain release factor 3. 47. DNA-directed RNA polymerase subunit beta'. 33. PTS system N-acetylmuramic acid-specific EIIBC component. Outer membrane protein U. Glutamate-ammonia-ligase adenylyltransferase. Cell division protein zipA homolog. 16. Regulatory protein luxO. Outer membrane protein U. Glycine dehydrogenase [decarboxylating]. 34. Cell division protein zipA homolog. HTH-type transcriptional regulator malT. GTP-binding protein era homolog. Outer membrane protein U. HTH-type transcriptional regulator malT. 35. Cell division protein zipA homolog. HTH-type transcriptional regulator malT. UPF0229 protein VV1_2091. 17. UPF0229 protein VV2350. ToxR-activated gene A lipoprotein.
  14. 14. Control
  15. 15. GO Annotations Chart 6 biosynthesis response to endogenous stimulus transport Control ion transport signal transduction cell communication transcription protein metabolism cell cycle carbohydrate metabolism protein biosynthesis cell organization and biogenesis response to external stimulus amino acid and derivative metabolism morphogenesis protein modification generation of precursor metabolites and energy Low Oxygen death response to abiotic stimulus cell death cell differentiation cell homeostasis nucleic acid metabolism secondary metabolism catabolism lipid metabolism DNA metabolism response to stress 0 0.025 0.050 0.075 0.100
  16. 16. V. tubiashii V. tubiashii + live oysters V. tubiashii + sterile oysters Oysters Experiment Two: Oysters
  17. 17. Approach - Gene Expression RNA Extraction Select Genes qPCR
  18. 18. Approach - Protein Expression Protein Extraction 2D gels ID differences
  19. 19. Gene Discovery using NGS Library Prep Cluster Generation Sequencing
  20. 20. Gene Discovery using NGS 226 million bp
  21. 21. Genes
  22. 22. flagellar biosynthesis protein FlhG outer membrane protein W flagellin 6 6 6 FlhG OMP flag 4 4 4 2 2 2 0 0 0 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour sigma-E factor negative chemotaxis protein alkaline serine protease regulatory protein RseA 8 15 15 CheW ASP Rse 6 10 10 4 5 5 2 0 0 0 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour
  23. 23. flagellar biosynthesis protein FlhG outer membrane protein W flagellin 6 6 6 FlhG OMP flag 4 4 4 2 2 2 0 0 0 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour sigma-E factor negative chemotaxis protein alkaline serine protease regulatory protein RseA 8 15 15 CheW ASP Rse 6 10 10 4 5 5 2 0 0 0 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour 0 hour 1 hour 24 hour
  24. 24. Approach - Protein Expression Protein Extraction 2D gels ID differences
  25. 25. 24 hour exposure
  26. 26. Putative replication protein HTH-type transcriptional regulator fructose repressor Thermostable 8-oxoguanine DNA glycosylase TrbL/VirB6 plasmid conjugal transfer protein glutamyl-tRNA synthetase glycine betaine-binding protein transcriptional regulator, AraC family protein peptide ABC transporter, Na+/proline symporter putative lipase activator protein RecF/RecN/SMC N domain protein putative exopolysaccharide biosynthesis protein signal transduction histidine kinase ribosomal protein L15 lipase chaperone putative transposase putative extracellular serine protease putative solute/DNA competence effector translation initiation factor IF-2 putative ABC transporter msha biogenesis protein mshf cobalamin biosynthesis protein ATP-dependent helicase HrpA ribose ABC transporter, periplasmic ribose- binding protein isocitrate dehydrogenase nitric oxide reductase putative anti-sigma B factor antagonist ABC-type tungstate transport system, cytochrome c4 Transcriptional regulator, SorC family arsenate reductase outer membrane lipoprotein blc
  27. 27. peptide ABC transporter bacterial sugar transferase Dna-J like membrane chaperone protein putative ABC transporter substrate-binding protein translation intiation factor Sui1 putative glycosyltransferase glycine dehydrogenase methyl-accepting chemotaxis protein glycine cleavage system protein T2 fructose 1,6-bisphosphatase II transposase OrfAB, subunit B membrane carboxypeptidase transcriptional regulator, AraC family protein gp5 Putative replication protein gamma-glutamyltranspeptidase 30S ribosomal protein S1 HTH-type transcriptional regulator glutamyl-tRNA synthetase N-6 DNA Methylase family
  28. 28. Summary - Low oxygen • Low oxygen conditions alter protein expression patterns • Comparative genome analysis is effective to assess physiological response in V. tubiashii
  29. 29. Summary - Oyster presence • NGS is viable option for gene discovery and expression • Flagella associated genes are not expressed at higher levels when oysters are present • Cellular stress response regulated in V. tubiashii upon oyster exposure (RSE) • Protease expression increases at 24 hours of exposure
  30. 30. Future Direction • Comparative microbial analysis to determine species specific responses to environmental change • Coordinate with experimental and environmental sampling - relevance • Characterize the host response in relation to pathogen physiology

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