V tubiashii - NSA 2009

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

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

basic biological information complementingexperimental and environmental studies

Experiments

• Simple

• Low oxygen conditions

• Little more complex

• Presence of oysters

Low oxygen conditions

OD

@60

0

Approach - Protein Expression

Protein Extraction

2D gels

ID differences


for audience orientation

Differential Expression

1/3 Anoxic, 6hours1. Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase. Glycogen synthase. Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase. Peptide chain release factor 3.2. DNA gyrase subunit B.3. Fatty acid oxidation complex subunit alpha.4. Beta-hexosaminidase. DNA mismatch repair protein mutS. DNA mismatch repair protein mutS. Penicillin-binding protein 1A. DNA gyrase subunit B.5. Penicillin-binding protein 1A. DNA gyrase subunit B.6. 5-methyltetrahydropteroyltriglutamate-- homocysteine meth...7. UvrABC system protein A. UvrABC system protein A. UvrABC system protein A. HTH-type transcriptional regulator malT. UvrABC system protein A.8. ToxR-activated gene A lipoprotein. ToxR-activated gene A lipoprotein. HTH-type transcriptional regulator malT.9. UvrABC system protein A. UvrABC system protein A. UvrABC system protein A. UvrABC system protein A.10. tRNA uridine 5-carboxymethylaminomethyl modification enzy... tRNA 5-methylaminomethyl-2-thiouridine biosynthesis bifun...11. Threonyl-tRNA synthetase. 1-deoxy-D-xylulose-5-phosphate synthase. tRNA uridine 5-carboxymethylaminomethyl modification enzy... Macrolide export ATP-binding/permease protein macB. 1-deoxy-D-xylulose-5-phosphate synthase. tRNA uridine 5-carboxymethylaminomethyl modification enzy... Threonyl-tRNA synthetase. 1-deoxy-D-xylulose-5-phosphate synthase. Acetyl-coenzyme A synthetase. Urocanate hydratase. Choline dehydrogenase. Choline dehydrogenase. NAD-dependent malic enzyme 1. 2-succinyl-5-enolpyruvyl-6-hydroxy-3- cyclohexene-1-carbo... Formate--tetrahydrofolate ligase. Choline dehydrogenase. 2-succinyl-5-enolpyruvyl-6-hydroxy-3- cyclohexene-1-carbo... NAD-dependent malic enzyme. Urocanate hydratase.

1/3 Anoxic, 6hours13. Vibriobactin-specific 2,3- dihydroxybenzoate-AMP ligase. Regulatory protein luxO. NAD-dependent malic enzyme 1. NAD-dependent malic enzyme. CTP synthase.14. Thiamine biosynthesis protein thiI. GTP-binding protein engA. Soluble pyridine nucleotide transhydrogenase. Thiamine biosynthesis protein thiI.15. Probable endonuclease 4. Probable endonuclease 4. RNA polymerase sigma-32 factor. Probable endonuclease 4. Probable inorganic polyphosphate/ATP-NAD kinase. S-adenosyl-L-methionine-dependent methyltransferase mraW.16. Fatty acid metabolism regulator protein. ATP synthase a chain. Lipase. Fatty acid metabolism regulator protein.17. 2-dehydro-3-deoxyphosphooctonate aldolase. Diaminopimelate epimerase. Glucosamine-6-phosphate deaminase. Glucosamine-6-phosphate deaminase. Glucosamine-6-phosphate deaminase. N-acetylmannosamine kinase. 2-dehydropantoate 2-reductase. 2-dehydro-3-deoxyphosphooctonate aldolase.18. UvrABC system protein A. UvrABC system protein A.19. 20. Probable O-sialoglycoprotein endopeptidase. Formimidoylglutamase. tRNA pseudouridine synthase D. Homoserine O-succinyltransferase. Succinylglutamate desuccinylase. Putative ribosome biogenesis GTPase rsgA 2. Formimidoylglutamase. tRNA pseudouridine synthase D.21. Probable sucrose-6-phosphate hydrolase. DNA repair protein recN. Urocanate hydratase. DNA repair protein recN. Thiol:disulfide interchange protein dsbD. Thermostable direct hemolysin 2. Peptide deformylase 2.23. Ferric uptake regulation protein.


~ 23 upregulatedproteins

1/3 Aerobic, 6 hours1. Nuclease sbcCD subunit C.2.Autoinducer 2 sensor kinase/phosphatase luxQ.3. Microbial collagenase.4. Glutamate-ammonia-ligase adenylyltransferase. Glutamate-ammonia-ligase adenylyltransferase.5. Chaperone protein hscA homolog. Vitamin B12 transporter btuB.6. DNA polymerase III subunit alpha.7. Methionyl-tRNA synthetase. Methionyl-tRNA synthetase. UvrABC system protein B. Methionyl-tRNA synthetase. Methionyl-tRNA synthetase.8. Glutamyl-tRNA synthetase. GMP synthase [glutamine-hydrolyzing]. Membrane-bound lytic murein transglycosylase F. GMP synthase [glutamine-hydrolyzing].9. Aldehyde dehydrogenase. ATP synthase subunit alpha. UDP-N-acetylmuramate--L-alanine ligase. Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase. Aldehyde dehydrogenase. ATP synthase subunit alpha. UDP-N-acetylmuramate--L-alanine ligase.10. Dihydrolipoyl dehydrogenase. Regulatory protein luxO. 3-isopropylmalate dehydratase large subunit. Regulatory protein luxO.11. Regulatory protein luxO. Cytochrome c-552. Nicotinate phosphoribosyltransferase.12. Lipid-A-disaccharide synthase. 3-oxoacyl-[acyl-carrier-protein] synthase 2.13. Nuclease sbcCD subunit C.14. HTH-type transcriptional regulator malT.15. DNA-directed RNA polymerase subunit beta'. Glutamate-ammonia-ligase adenylyltransferase.16. Glycine dehydrogenase [decarboxylating]. HTH-type transcriptional regulator malT. HTH-type transcriptional regulator malT. HTH-type transcriptional regulator malT.17. ToxR-activated gene A lipoprotein. ToxR-activated gene A lipoprotein.18. UvrABC system protein A. HTH-type transcriptional regulator malT. UvrABC system protein A. Outer membrane protein U. Cell division protein zipA homolog. Outer membrane protein U. Cell division protein zipA homolog.

1/3 Aerobic, 6 hours 19. Phosphoenolpyruvate carboxylase.20. Regulatory protein luxO.21. Lipid-A-disaccharide synthase. Lipid-A-disaccharide synthase. Putative dioxygenase VC_1345.22. D-erythrose-4-phosphate dehydrogenase.23. Beta-hexosaminidase. N-acetyl-gamma-glutamyl-phosphate reductase. UPF0176 protein VC_1259.24. Phosphate import ATP-binding protein pstB 2. Diaminopimelate epimerase. Glutamate racemase. Pirin-like protein VC_A0969. 25. Carboxylesterase bioH. Vitamin B12 import ATP-binding protein btuD. Shikimate dehydrogenase. Uncharacterized exonuclease xni.26. Transaldolase. Dihydrodipicolinate synthase.27. Leucyl/phenylalanyl-tRNA--protein transferase. UPF0271 protein VV2_0273. UPF0135 protein VC_2093.28. Anthranilate synthase component 1. Glucose-6-phosphate isomerase. Glucose-6-phosphate isomerase.29.30. PTS system N-acetylmuramic acid-specific EIIBC component. L-arabinose isomerase. Histidine ammonia-lyase. Regulatory protein luxO.31. Glucose-6-phosphate isomerase. Phosphoenolpyruvate carboxykinase [ATP]. Phosphoenolpyruvate carboxykinase [ATP]. Peptide chain release factor 3.32. Peptide chain release factor 3.33. PTS system N-acetylmuramic acid-specific EIIBC component. Regulatory protein luxO.34. GTP-binding protein era homolog.35. UPF0229 protein VV1_2091. UPF0229 protein VV2350.36. UPF0294 protein VC0395_A1830. Vibriobactin utilization protein viuB. Vibriobactin utilization protein viuB. Putative phosphotransferase VVA0515. Putative phosphotransferase VV2_0006. UPF0294 protein VC_2238.

1/3 Aerobic, 6 hours 37. Aspartate carbamoyltransferase catalytic chain. UPF0276 protein VP3015. Recombination-associated protein rdgC. Porphobilinogen deaminase.38. Toxin coregulated pilus biosynthesis protein F. tRNA-modifying protein ygfZ. tRNA 2-thiocytidine biosynthesis protein ttcA. Toxin coregulated pilus biosynthesis protein F. tRNA 2-thiocytidine biosynthesis protein ttcA. tRNA 2-thiocytidine biosynthesis protein ttcA. tRNA 2-thiocytidine biosynthesis protein ttcA.39. Probable O-sialoglycoprotein endopeptidase. Homoserine O-succinyltransferase. 4-hydroxythreonine-4-phosphate dehydrogenase. 40. Autoinducer 2-binding periplasmic protein luxP. 3-ketoacyl-CoA thiolase. 3-ketoacyl-CoA thiolase.41. N-acetylglucosamine-6-phosphate deacetylase. Lipid-A-disaccharide synthase. Probable cysteine desulfurase. Sulfate/thiosulfate import ATP-binding protein cysA. Tryptophan synthase beta chain.42. Glucans biosynthesis protein G. Inner membrane protein oxaA. CTP synthase. Inner membrane protein oxaA. UPF0061 protein VC_1931. Glucans biosynthesis protein G.43. Catalase-peroxidase. GTP pyrophosphokinase. 1,4-alpha-glucan-branching enzyme. Catalase-peroxidase.44. 1-deoxy-D-xylulose-5-phosphate synthase. 3-octaprenyl-4-hydroxybenzoate carboxy- lyase. 1-deoxy-D-xylulose-5-phosphate synthase.45. DNA mismatch repair protein mutS. DNA mismatch repair protein mutS. DNA mismatch repair protein mutS. Autoinducer 2 sensor kinase/phosphatase luxQ. DNA mismatch repair protein mutS. DNA gyrase subunit B.46. Autoinducer 2 sensor kinase/phosphatase luxQ.47. Outer membrane protein U. Cell division protein zipA homolog. Outer membrane protein U. Cell division protein zipA homolog. Outer membrane protein U. Cell division protein zipA homolog.

Control

~ 47 upregulatedproteins

Control

biosynthesis response to endogenous stimulus

transport 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 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

Chart 6

Low Oxygen

Control

GO Annotations

Experiment Two: Oysters V.

tubi

ashi

i

V. tu

bias

hii +

liv

e oy

ster

s

V. tu

bias

hii +

st

erile

oys

ters

Oys

ters

Approach - Gene Expression

RNA Extraction

Select Genes

qPCR


Protein Extraction

2D gels

ID differences

Gene Discovery using NGS

Library Prep

Cluster Generation

Sequencing

Gene Discovery using NGS

226 million bp

0

5

10

15

0 hour 1 hour 24 hour

0

2

4

6

0 hour 1 hour 24 hour0

2

4

6


0

2

4

6

8

0 hour 1 hour 24 hour0

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10

15


0

2

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6


FlhG

ASPCheW

flagOMP

Rse

outer membrane protein Wflagellar biosynthesis protein FlhG

chemotaxis protein

flagellin

alkaline serine proteasesigma-E factor negative regulatory protein RseA


Protein Extraction

2D gels

ID differences

24 hour exposure

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

peptide ABC transporterbacterial 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

Summary - Low oxygen

• Low oxygen conditions alter protein expression patterns

• Comparative genome analysis is effective to assess physiological response in V. tubiashii

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

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

V tubiashii - NSA 2009

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