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. Vibrio tubiashii
RE98

3. How do changes in the environment
influence Vibrio tubiashii physiology?

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

6. Low oxygen conditions
OD@600

7. Approach - Protein Expression
Protein Extraction
2D gels
ID differences

8. Low oxygen conditions

9. Low oxygen conditions
for audience orientation

10. Differential Expression

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. Low oxygen conditions

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. Control

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. V. tubiashii
V. tubiashii +
live oysters
V. tubiashii +
sterile oysters
Oysters
Experiment Two: Oysters

17. Approach - Gene Expression
RNA Extraction
Select Genes
qPCR

18. Approach - Protein Expression
Protein Extraction
2D gels
ID differences

19. Gene Discovery using NGS
Library Prep
Cluster Generation
Sequencing

20. Gene Discovery using NGS
226 million bp

21. Genes

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. 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. Approach - Protein Expression
Protein Extraction
2D gels
ID differences

25. 24 hour
exposure

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. 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. Summary - Low oxygen
• Low oxygen conditions alter protein
expression patterns
• Comparative genome analysis is effective to
assess physiological response in V. tubiashii

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. 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