genomic dna extraction pcr amplification of 16s rdna separate mixture of 16s rdnas by cloning into...

Genomic DNA

extraction

PCR amplification of 16S rDNA

Separate mixture of

16S rDNAs by cloning into

plasmid vector and

transformation of host cells

ATTCGATCCG...

B. subtilis

B. cereus

M. luteus

P. aeruginosa

P. stutzeri

Sequence individual

cloned genes, compare

sequence to database, and

identify original bacteria

Bacteria in sample

Laboratory Methods

10

AY513646.1|Psychrobacter.alimentariusF282 CGO 1

F238 CGO 1F227 CGO 1F103 CGO 1RCAB016058.1|Psychrobacter.pacificensis

AA29 BRR 1RCF213 CGO 1RC

AB002657| 16S Mariana trench mudAJ619070.1|Beer bottling plant biofilmL01575.1|Thyasira.flexuosaAA04 CGO 1RC

A196 NIS 1AA53 BRR 1AA33 BRR 1AA61 BRR 1

AF384206|Ectothiorhodospira.spAJ404972|Alcalilimnicola.halodurans

A092 NIS 1RCA182 NIS 1A021 NIS 1RCAA31 NIS 1

L10949.1|Marine.aggregategi 19851156 gbgi 19851165 gbgi 19851177 gbgi 19851169 gbAF321239|Pseudomonas.sp.

F104 CGO 1PFU492830|Pseudomonas.fulgidaAF388206|Pseudomonas.spAF131104.Pseudomonas.putida.MTPSEIAM13|Pseudomonas.fragi

ABE308319|Azotobacter.beijerinAA77 NIS 1RC

AB125367.1|Pseudomonas.pachastrellaeAA92 NIS 1

AJ609270.1|Marinobacter.sedimentalisDA30 CGO 1DA16 CGO 1DA42 CGO 1DA55 CGO 1DA72 CGO 1DA79 CGO 1RCDA22 CGO 1RCDA38 CGO 1DA49 CGO 1DA37 CGO 1DA50 CGO 1F019 CGO 1RCF046 CGO 1RCF180 CGO 1F044 CGO 1F078 CGO 1F030 CGO 1RCF184 CGO 1F124 CGO 1RC

AY386337.1|Marine.oligotrophic.waterAA05 NIS 1RC

AB015519.1|deep sea sedimentsDA73 CGO 1

U70693.1|Continental shelf Cape haterasAF001650.1|Continental shelf off OregonCA36 CGO 1

gi 19851209 gbgi 19851368 gbgi 19851219 gbF145 CGO 1gi 19851230 gbgi 19851179 gb

gi 19851409 gbAA62 NIS 1

AA87 BRR 115282445.Aquifex.aeolicus.VF5

16S rRNA sequences identified as within the Class Pseudomonadales (2002 data)

Colors indicate what location and type of sample from which the sequences were obtained

Bamboo coral from Murray Seamount

Black coral from Murray Seamount

Bamboo coral Isidella from Warwick Seamount

Water collected at Murray Seamount

Caribbean coral (Rohwer et al., 2002)

Closest BLASTN match in Genbank

Biofilm from rock at Murray Seamount

Bamboo coral from Chirikof Seamount

Specific to Coral

Life as we know it can be divided into three broad groups, the Eucarya, Archaea, and Bacteria. The only group visible to us with the naked eye is the Eucarya, containing animals, plants, and fungi. The Bacteria and Archaea are single-celled microbes and are only visible with use of a microscope. However, despite their small size, they have important roles in many spheres, and interactions with

many larger organisms. These interactions can be either detrimental or beneficial to the eukaryote host.

What is TIGR?TIGR (The Institute for Genomic Research) is a private, not-for-profit research institute located in Rockville, Maryland. TIGR principally conducts large-scale genome sequencing projects; sequencing targets include many microbial species, but also plant, animal, and fungal organisms. The DNA sequence data obtained from these projects is used to derive genome-based insights into the biology of the target organism.TIGR has been participating in research cruises aboard R/V Atlantis, at the invitation of WHOI and NOAA OE, since 2002.

In Search of Bacterial Communities Inhabiting Deep-Sea Corals on Gulf of Alaska SeamountsKevin Penn and Naomi Ward, TIGR

AcknowledgementsWe thank the Captain and crew of the R/V Atlantis, as well as members of the Alvin group, for all their help during both the 2002 and 2004 expeditions. Special thanks to Randy Keller, Brad Stevens, Tom Shirley, Tom Guilderson, Amy Baco-Taylor, and Peter Etnoyer for taking us on board. Thanks also to WHOI and NOAA’s Ocean Explorer program (especially Catalina Martinez) for supporting our participation.

AcknowledgementsWe thank the Captain and crew of the R/V Atlantis, as well as members of the Alvin group, for all their help during both the 2002 and 2004 expeditions. Special thanks to Randy Keller, Brad Stevens, Tom Shirley, Tom Guilderson, Amy Baco-Taylor, and Peter Etnoyer for taking us on board. Thanks also to WHOI and NOAA’s Ocean Explorer program (especially Catalina Martinez) for supporting our participation.

Bamboo coral observed during Alvin dive 4035.We know that bacterial populations are associated with these corals, but their function (parasitic, or beneficial to

the coral) remains unclear.

Collection of bamboo coral specimen using Alvin’s manipulators

Bamboo coral specimen successfully transferred to the surface in Alvin’s basket

Collection of exopolysaccharide (mucus) produced by bamboo coral.

Coral mucus may protect the coral from desiccation, predators, and disease.

We know that the mucus contains many different species of bacteria.The mucus presumably provides shelter and nutrition to the bacteria, but is

not yet known whether the bacteria provide a benefit to their coral host.

We use 16S rDNA cloning and sequencing to determine community composition, without the need to cultivate bacteria in the lab.This approach has been shown to recover bacterial diversity much more extensively than cultivation-based methods.

16S rDNA sequences can be used to draw a family tree of relationships between different bacteria.

These data suggest that different coral species harbor specific groups of bacteria.

Seamounts visited during the 2004 expedition to the Gulf of Alaska