Petroleum Metabolism by Northern Gulf of Mexico Vibrios

D. Jay Grimes and Shuo Shen Vibrio 2014

Edinburgh, Scotland 4 April 2014

Petroleum

Dr. D. Jay Grimes

• Petroleum is a complex mixture of hundreds of compounds, predominately hydrocarbons – aliphatic, alicyclic, and aromatic

• The main chemical families are (ave. %): – Alkanes (paraffins), with methane being the smallest (30%) – Cycloalkanes (naphthenes) – smallest are cyclopentane

and cyclohexane (49%) – Aromatic hydrocarbons, characterized by the presence of

at least one benzene ring, including benzene, toluene, ethylbenzene, xylene (BTEX) and the polycyclic aromatic hydrocarbons (PAHs) (15%)

– Asphaltenes – PAHs complexed with N, S, O (6%)

Petroleum Degradation

Dr. D. Jay Grimes

• More than 75 genera belonging to the domain Bacteria are able to grow on hydrocarbons

• Microbes in the domain Archaea have also been shown to degrade or utilize hydrocarbons

• Many other microbes, including some cyanobacteria, can degrade hydrocarbons but cannot utilize them as sole sources of carbon and energy

• More than 100 genera of fungi have been shown to degrade hydrocarbons, as have a few algae

Petroleum Degradation by Vibrios

Dr. D. Jay Grimes

• Vibrios first reported to degrade oil by ZoBell in 1943 • Degradative ability firmly established by the extensive

work of Walker and Colwell (1974 & 79) and Grimes et al. (1984) demonstrated that Vibrios responded to hydrocarbon wastes in the Atlantic

• Growth of Vibrios on PAHs reported by Okpokwasili et al. (1986) and again by Beradesco et al. (1998)

• West et al. used a 123-trait NT study to show that 3 of 39 phenanthrene degraders from the Chesapeake were Vibrios and Geiselbrecht et al. (1996) did a similar study in Puget Sound

OH OH

COOH

CH3

-C-SCoA O

R- CH2 - CH2 -C-SCoA O

O R- CH CH-C-SCoA

R- CH -CH2 -C-SCoA O OH

R- C - CH2 -C-SCoA O O

CoASH

R-C-SCoA O

NAD + H + + NADH +

H2O

FAD FADH2

Fatty acyl CoA Acetyl CoA

TCA CO2

n-Alkane * Alcohol + H2O Aldehyde Fatty acid

COOH

CH3

OH

OH OH

Catechol

COOH NH2

OH OH HOOC

CH3

COOH

CH3

OH

COOH

OH

COOH

OH OCH3

COOH

OH

COOH

OH OH HO

Protocatechuate

Ring fission

Diverse Marine Microbes Utilize Various Hydrocarbon Biodegradation Pathways to Degrade the Compounds

Found in Crude Oil

Slide courtesy of Dr. Ron Atlas

*

*ring cleavage requires dioxygenases *requires a monooxygenase

Dioxygenases • Dioxygenases effect aromatic ring fission • Many Bacteria and Archaea possess dioxygenases • Eukarya and some Bacteria initiate ring fission with

cytochrome P450 enzymes • The Pseudomonas dioxygenase systems are among

the most highly studied • Several Vibrio spp. possess dioxygenases, including a

clinical strain – V. parahaemolyticus RIMD 2210633 • Vibrios evolved in the ocean – not in humans as

symbionts and pathogens • Oil seeps have existed on the ocean floor for eons

FISH

Vp

Dr. D. Jay Grimes

Ring Fission

Dr. D. Jay Grimes Pathways from: http://www.ence.umd.edu/~eseagren/bioAHC97.htm

*

* Vibrio parahaemolyticus RIMD 2210633 contains this dioxygenase, See: Grimes et al. 2009. Microb. Ecol. 58:447-460.

Beta-oxidation

Dr. D. Jay Grimes

Alkane + Reduced rubredoxin + O2 Primary alcohol + oxidized rubredoxin + H2O

Vibrios and Oil GCRL Microbial Response • DH (MC252) surface and deep

water samples and shoreline • Sediment samples • Oyster samples • Sargassum samples • Dioxygenase-positive Vibrios • Microcosms (oil & dispersants) • Community profiles (using 16S

rDNA) • Metagenomics and proteomics • Phylogeny of specific genes

(e.g., dioxygenases) • Vibrio identification

FISH

Vp

Dr. D. Jay Grimes

Trips •3 cruises on R/V Ferrel with Hazen’s group •1 cruise on the R/V Tommy Munro • Inshore sampling in small boats

Vibrios and Oil

Dr. D. Jay Grimes

• Examined 48 strains for the presence of dioxygenases and the ability to grow on Alaskan North Slope crude oil (ANS crude)* Examined each strain for the Rieske Fe-S

center Examined each strain for the presence of

naphthalene dioxygenases Dioxygenases will be sequenced and

compared Attempted to grow each strain in a salts

solution containing 200 ppm ANS crude Each ANS broth will be examined for

metabolites by Dr. Roger Prince

*ANS crude oil supplied by Dr. Roger C. Prince, ExxonMobile

Rieske Fe-S Region PCR (Ní Chadhain et al. 2006. Appl. Environ. Microbiol. 72:4078-4087)

The α subunit consists of 1) Rieske Fe-S center common to all

dioxygenases 2) Mononuclear Fe-containing catalytic domain 3) The Rieske degenerate primers are:: Rieske_F TGYMGNCAYMGNGG Rieske_R CCANCCRTGRTANSWRCA

Targeted region – 78bp 50bp

100bp

L 1 2 3 4 5 6 NC L

Lanes 1. LBNL VC4 (presumed V. diazotrophicus) 2. LBNL VC2 (presumed V. diazotrophicus) 3. LBNL 15A (presumed V. splendidus) 4. LBNL 2 (presumed V. splendidus) 5. P. aeruginosa ATCC 10145 6. LBNL TOP (presumed V. campbellii)

A random group of isolates that gave results in the first run were re-run using different PCR. These results were much cleaner. Lanes 1-4, 6 gave the correct PCR amplicon. It can be concluded that these isolates have a rhd protein. The exact dioxygenase is unknown since degenerate primers were used, however it is one that only targets nonpolar aromatic hydrocarbons. (N. Noriea, unpublished data)

Dr. D. Jay Grimes

Dr. D. Jay Grimes

nahAC is one of several naphthalene dioxygenases in bacteria, all of which contain the active Rieske Fe-S center. (Wilson, M.S., C. Bakermans, and E.L. Madsen. 1999.

Appl. Environ. Microbiol. 65:80-87.) 1 2 3 4 5 6 7 8 9 10

1 V. cholerae 1067 blood 2 V. cholerae ATCC 11623 3 V. cholerae ATCC 14035 4 V. cholerae ATCC 51394 5 V. cholerae ATCC MB12787 6 V. diazotrophicus LBNL VC2 7 V. mimicus CRAB1HL2 8 V. parahaemolyticus 2007-074 9 V. splendidus AF1012VSP.7-20-10

L base pair ladder

nahAC

Targeted region – 482bp (all 9 strains possess nahAC)

Forward: AC114F: CTGGCWWTTYCTCACYCAT Reverse: AC596R: CRGGTGYCTTCCAGTTG

Vibrios and Oil

Dr. D. Jay Grimes

Alaskan North Slope crude oil broth • 1/100th strength modified Bushnell and Haas* broth

DI water ……………… 1000 mL MgSO4 ……………….. 0.002 g CaCl2 …………………. 0.0002 g KH2PO4 ……………… 0.01 g K2HPO4 ……………… 0.01 g NH4NO3 ……………… 0.01 g Salts solution ……….. 1.0 mL

0.002 g MnSO4 0.0005 g ZnCl2 5.0 g FeCl3 100 mL DI water

• Added, per L of 1/100th B&H broth 200 ppm ANS crude oil 2 ppm Corexit

*Bushnell, L.D. and H.F. Haas. 1941. The utilization of certain hydrocarbons by microorganisms. J. Bacteriol. 41:653-673.

Vibrios and Oil

Dr. D. Jay Grimes

Alaskan North Slope crude oil broth • Monitored growth by

measuring turbidity • In all cases measurable

turbidity was visualized with the naked eye

• Will send mercuric chloride-preserved cultures to Roger Prince for GC-MS analysis of metabolites

Biolog turbidometer

Vibrios and Oil

Dr. D. Jay Grimes

Species and strain Source Rieske Dioxygenase Growth Vibrio alginolyticus FER 3-04 USM 8E water, MC252 Vibrio campbellii BAA-1116 ATCC Vibrio campbellii LBNL 2 water, MC252 Vibrio campbellii LBNL top B water, MC252 Vibrio cholerae 14035 ATCC Vibrio cholerae 11623TT ATCC Vibrio cholerae 51394S ATCC Vibrio cholerae MB12.7874 clinical Vibrio diazotrophicus LBNL VC 2 water, MC252 Vibrio diazotrophicus LBNL VC 4 water, MC252 Vibrio fischeri VF VL 52 squid (E. Ruby) Vibrio fischeri VF ES 114 squid (E. Ruby) Vibrio harveyi RS 85 SPL red snapper spleen, wild Vibrio harveyi RS 85 LS2 red snapper lesion, wild Vibrio harveyi AF 1070 VHA cobia, aquaculture Vibrio harveyi crab 2 LS2 blue crab lesion, wild Vibrio harveyi FER 3-10 USM 6E water, MC252 Vibrio harveyi FER 3-10 USM 7E water, MC252 Vibrio mimicus crab 1 HL 2 blue crab hemolymph Vibrio parahaemolyticus 2006-001 sediment Vibrio parahaemolyticus 2006-004 water Vibrio parahaemolyticus 2006-008 sediment Vibrio parahaemolyticus 2006-011 water

We will examine the following 48 strains for these 3 features:

Vibrios and Oil

Dr. D. Jay Grimes

Species and strain Source Rieske Dioxygenase Growth Vibrio parahaemolyticus 2006-013 sediment Vibrio parahaemolyticus 2006-018 sediment Vibrio parahaemolyticus 2006-023 sediment Vibrio parahaemolyticus 2006-034 sediment Vibrio parahaemolyticus 2007-058 oyster Vibrio parahaemolyticus 2007-061 oyster Vibrio parahaemolyticus 2007-062 water Vibrio parahaemolyticus 2007-067 sediment Vibrio parahaemolyticus 2007-071 sediment Vibrio parahaemolyticus 2007-074 water Vibrio splendidus LBNL 4A water, MC252 Vibrio splendidus LBNL 15 water, MC252 Vibrio splendidus LBNL 16A water, MC252 Vibrio splendidus AF 1012 VSP 07-20-10 sediment Vibrio vulnificus RS 85 SPL 3V red snapper spleen, wild Vibrio vulnificus crab 1 HL4V blue crab hemolymph, wild Photobacterium angustum RS 80 LIV2 red snapper liver, wild Ph. leiognathi RS 80 SPL1 red snapper spleen, wild Ph. damselae subsp. damselae RSQT Ph red snapper, aquaculture Shewanella algae BLACK sediment Shewanella algae sediment Escherichia coli 11775 ATCC Pseudomonas aeruginosa 27853 ATCC Staphylococcus haemolyticus AF1064 laboratory contaminant

Vibrios and Oil

Dr. D. Jay Grimes

Strain Rieske nahAC Growth Vibrio campbellii LBNL 2C + + + Vibrio campbellii LBNL top B + + - Vibrio cholerae 1067 clinical (blood) - + - Vibrio cholerae ATCC 11623TT - + + Vibrio cholerae ATCC 14035 - + + Vibrio cholerae ATCC 51394 - + - Vibrio cholerae ATCC MB12787 - + - Vibrio diazotrophicus LBNL VC 2 + + - Vibrio diazotrophicus LBNL VC 4 + + - Vibrio fischeri VF ES 114 - + + Vibrio parahaemolyticus 2007-074 + + + Vibrio splendidus AF 1012 - + - Vibrio splendidus LBNL 15 + + - Vibrio splendidus LBNL 16A + + - Vibrio splendidus LBNL 4A + + - Vibrio vulnificus RS 85 SPL 3V - + +

Vibrios and Oil

Dr. D. Jay Grimes

Tar balls discovered on Ship, Horn islands By MARY MARGARET HALFORD mhalford@sunherald.com A team from the Mississippi Department of Environmental Quality discovered as much as 300 pounds of tar balls on Horn Island and 10 pounds on Ship Island on Friday that the department says is related to the 2010 BP Oil Spill. March 21, 2014, updated 5 minutes ago Read more here: http://www.sunherald.com/2014/03/21/5434500/tar-balls-discovered-on-ship-horn.html#emlnl=Breaking_News#storylink=cpy

Questions?

This work was supported by: • NSF-NIH Ecology of Infectious Diseases • Gulf of Mexico Research Initiative

administered by the Northern Gulf Institute • BP contract to Terry C. Hazen

If you would like the references cited in this talk send an email to: jay.grimes@usm.edu