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you

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[Music]

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thanks Eric Eric for the invitation to

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talk today so yeah my lab studies these

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King the synthetic iron oxidizing

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bacteria and on the one up here standing

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and presenting today but this is really

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the work of Gerald Murray who just got

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his PhD in Kerstin kuso's lab at the

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university of llena and roman Barco

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who's a actually an NSF postdoc who

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splits his time between my lab and Ken

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Nealson slab at USC and I also like to

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thank NASA Astrobiology for funding and

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NSF and in case any of you were

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that little symbol there that's the

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alchemists sign for iron which is what

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we study so why iron first of all it's

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probably the most abundant

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chemosynthetic element on earth and

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probably on any other rocky planet that

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we know about well maybe I shouldn't say

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that being in an astrobiology meeting

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but anyway I'll say it anyway because

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I'm a microbiologist and I can say

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stupid things about geology and plants

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so so another thing is bio signature so

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I'm sure some of the member you've seen

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this paper that you recently came out in

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nature where they were claiming for

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these iron oxide bio signatures in 3.8

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or older Giga billion year old rocks or

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older from the Hudson Bay and you know

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very interesting I'm a I study modern

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systems I've never actually gone out in

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the field and collected a rock or looked

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at a rock slide under the microscope but

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when I look at these types of structures

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you know they are somewhat consistent

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they certainly are not totally

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consistent with anything we see in the

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modern world but you know after four

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billion years who knows so I guess I'm a

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hopeful hopeful agnostic and that this

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work will really be borne out but just

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to show that you can find micro fossils

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that are very well preserved

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in this slide here is 170 million year

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old earth crust where you see all these

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little filamentous structures that are

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about one or two microns in diameter

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this is about a three week old culture

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of an iron oxidizing bacteria that was

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grown in the lab and you can see there's

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a lot of similarity in those structures

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and when you look at the fine detail it

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becomes pretty clear that some of these

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types of structures clearly must have

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been formed by by microbes how closely

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related they are to the microbes we have

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today that's another question so we

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definitely see biogenic structures in

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nature that these organisms form and

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they can be small and large so on the

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left here is a intact microbial mat that

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we did a three dimensional imaging of

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with the confocal microscope and it was

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formed by this sheath forming bacterium

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here what's really cool about this is

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that that 50 micrometer or so layer at

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the leading edge of that mat is where

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all these sheaf forming bacteria are and

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all its red materials just the empty

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sheets that they leave behind so they

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really are geo engineers or ecological

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engineers they structure their

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environments and produce massive amounts

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of this amorphous very hydrate which is

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quite a reactive mineral to go to the

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other scale this is a the golden tower

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which we discovered a couple of years

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ago while doing submersible dives at the

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aschen event site and mariana that

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little bar down there is 10 centimeters

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so this structure is nearly 10 meters

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tall and it seemed as far as we could

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tell it was composed primarily a

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bacteria that looked like this form

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these long stalks so this is formed over

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a diffuse event flow where there was

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iron coming out and it just slowly built

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up over time as far as we could tell and

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form this massive structure in fact very

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interesting too that they undergo very

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little grazing and things like that by

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protozoa or shrimp so they don't undergo

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the same kinds of pressures that for

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example sulfur oxidizing mats too so

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they so they have the potential to

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really accumulate

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so what's going on how do these bacteria

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actually oxidizing iron and that's the

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primary topic I want to discuss today so

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this is a model that we came up with

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published last year which is based on

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both comparative genomics and some

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wholesale proteomics we did with an

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organism we've been studying in lab for

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a while Mary profundus roxton's

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so this is an example of extracellular

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electrons transfer because it cells they

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so they oxidize iron on the outer

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membrane because again they have the

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problem as soon a neutral pH as soon as

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you oxidize iron you immediately

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precipitate a faery hydride mineral if

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you do inside the cell you're going to

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fill yourself with insoluble iron oxides

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so we believe this process should occur

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at the outer membrane so there you have

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floor you have to get electrons from the

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outer membrane across the periplasm to

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the inner membrane to the electron

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transport chain and so we found in all

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the iron oxidizers both from freshwater

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and marine systems that we've looked at

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is this cyc to homolog should call cyc

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to pv-1 and we believe this could be the

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did the initial iron oxidase this is a

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it's a poor in cytochrome C complex and

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and then electrons would so that would

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be the initial electron acceptor and

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that electrons get transferred across

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the periplasm

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through we found some other cytochromes

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it could be potential shuttles whether

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your shuttles or could form a wire we

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don't really know and then there's an

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electron transport chain which has the

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different components interesting aspects

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of this are this alternative complex 3

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which we quite commonly find the

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military oxido reductase system and then

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most of these organisms use the

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cytochrome C VD 3 type terminal oxidase

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which is a high affinity iron oxidation

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that makes sense because we think these

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organs are generally micro silica so

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that's what we've known and most of the

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organs we've isolated have been obligate

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iron oxidizes I should

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say but recently relaxed not that recent

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now but a few years ago I isolated this

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new organism which were just in the

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process of publishing on and of calling

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it your CEO by Laura it represents a new

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genus in this group of Zeta

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proteobacteria Zeta proteobacteria are

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almost exclusively iron oxidizers that

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live in marine environments so isolated

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one strain from the tagged vent site in

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the mid-atlantic ridge and another one

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from the snail vents in the Marianas on

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the opposite sides of the world at least

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and it's kind of a nondescript organism

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in terms of them if you have a lot of

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the other iron oxidizers it's a little

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rod and it forms these very amorphous

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particulate iron oxides it doesn't make

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any of those structures that I was

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showing earlier but what's really cool

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about it is it grow not only on iron but

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also on hydrogen and so those

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organization on or enriched and isolated

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on zero valent iron which is both a

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source of iron and we use it as that but

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you also have to be careful because it

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can be a source of hydrogen as well and

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it turned out this organism grew really

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well in the enrichment and part of the

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reason was that because it was growing

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both on the iron and the hydrogen and

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when we separate those out and grow it

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on ferrous chloride or hydrogen you can

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see in the squares here is the hydrogen

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it grows a little bit better and in the

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circles here is growing on ferrous

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chloride and a pretty pretty decent cell

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yields and doubling times on the order

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of 15 to 20 hours if you don't add

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either hydrogen or iron there's no

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growth so we sequence the genomes of

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these two organisms and found that they

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did indeed have hydrogenase the

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hydrogenation subunits for uptake of

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hydrogen and then we compared so we have

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a bunch of single-cell genomes from

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others ada proteobacteria that we've

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isolated or sorry we haven't ISIL we've

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isolated the single cells and sequence

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the genomes and we found that they all

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belong to this single clade here which

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recalls 800 tu9 and they were different

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from these other isolates that we have

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which represent different clades within

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group and so that was interesting so it

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seemed like it was this one clade that

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we have that it seems to have the

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ability to it has these hydrogenases

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that are capable of promoting growth on

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hydrogen and so then we were asked the

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question of well how where are they

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found and so we scraped the short read

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archive to look specifically for this

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particular group and so we found about

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50,000 projects that have been done that

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are non-human associated there's about

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30,000 human associated projects in

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there and we found only about four or

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five projects where the this particular

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OTU is found and they were all at higher

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environments that makes us think these

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organisms although they can grow on

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hydrogen they may be more adapted for

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growing on iron so what can we learn so

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I meant to say that the beauty of having

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this organism is everything we've done

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before was done an obligate iron

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oxidizer so now we have an organism that

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can grow on both hydrogen iron we can at

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least do some comparative proteomics in

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this case and so this was work that

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Romans been doing and so I'm showing

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here is the proteome map so so we as I

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said we sequence the genome it's about

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99 plus percent complete but there are

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gaps in it so these shows the 13

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different contexts on the outside here

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which is the genome and then all the

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proteins that we identified are in this

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inner blue ring here and then proteins

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that are upregulated on hydrogen are

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shown in the green and on iron on in the

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red so basically we found about 42

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percent of the proteins and the

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proteomics experiment and about a

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hundred of them are upregulated on iron

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and about 1.5 or I'm sorry about 150

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were upregulated on hydrogen and it's

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easy for me to put this slide up here

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and talk about it but took Rome on

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several months to do this you had to

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grow all these cells in triplicate

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batches to get really robust statistics

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and the challenge with these we can't do

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genetics with them and the challenge

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with biochemistry is getting enough

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biomass and then you've got all these

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iron oxides around that you have to deal

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with the hydrogen actually was a lot

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easier of course because we didn't have

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the iron around but still it was it was

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a technical challenge to do this and so

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what did we find in terms of the

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comparative proteomics in terms of what

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was expressed and what wasn't so we

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found the hydrogenase operon on hydrogen

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was more highly expressed that was good

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we found that the phosphate that there's

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a transport system for phosphate which

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is highly expressed on iron this is

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actually nice internal control because

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as e self expire knock sides those iron

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oxides very strongly buying phosphorus

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and so the fact that they overexpressed

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these systems for taking up phosphorus

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is sort of makes sense there was also

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this thyroid auxin reductase which is

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involved and one of the things that's

280
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involved in is probably defense against

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reactive oxygen species again when you

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have iron around an oxygen you're going

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to get the potential for reactive oxygen

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species these lawyers had to deal with

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what was not differentially regulated

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with Rubisco there their ability to

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succeed too that's not surprising or the

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CVD 3 the AC t system also was expressed

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in both cases and the cyc 2 was also

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expressed about equivalently this was a

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little disappointing I guess because of

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course we were hoping to see this stuff

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regulated when they were growing on hot

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on iron and downregulated a hydrogen so

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the case was true for how the hydrogen

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is but not for the cyc 2 so I'll just

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come back to the model here so I mean it

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was good that we saw cyc 2 highly

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expressed in these because we'd only

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done this in a different species before

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so that's consistent with it being there

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we didn't see the cyc one we think

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there's a different system that they may

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use for shuttling electrons so you know

305
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what's going on here in terms of this

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well I can wave my hands and say they're

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probably constitutively expressing the

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cyc 2 and

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and the primary argument I could make

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sure that an ecological right but these

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organs really want to grow on iron and

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so they always have they're always ready

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to grow on iron but if some hydrogen

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comes along and they can adapt and take

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over and use that instead so with that

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I'm going to conclude again these

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neutral philic iron oxidizers are

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obligate and this utilized

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extracellular electron transfer and as I

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said cyc too seems to be constitutively

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expressed and I just talked about the

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the ecological reason for that but

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another thing that we're thinking about

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it would be really interesting now is

325
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perhaps your sea of Ivor is essential as

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a sentinel for the presence of hydrogen

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a diffuse flow events because we only

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see it in some communities so it would

329
00:14:20,140 --> 00:14:19,010
be interesting go out and see if those

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are the communities where as more

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hydrogen present so thanks thank you

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00:14:39,780 --> 00:14:32,480
Dave time for again probably one

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00:14:43,800 --> 00:14:42,240
hi David my name is Sandra um I was only

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thinking expand a little bit on the

335
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electron transport mechanism across the

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membrane a little bit so you know I mean

337
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we think that that there there's the

338
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cytochromes they're probably involved in

339
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shuttling electrons from the outer

340
00:15:04,410 --> 00:15:02,320
membrane or from that I mean we we're

341
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still I mean I should our models still

342
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based around a cyc - like proteins being

343
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that outer that does the initial contact

344
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with Fe - and oxidizes the FE 3 and that

345
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then those electrons are transferred and

346
00:15:21,300 --> 00:15:19,170
yeah so we don't find some of the

347
00:15:23,610 --> 00:15:21,310
canonical proteins that have been found

348
00:15:27,090 --> 00:15:23,620
for example in acidify Oh bacillus that

349
00:15:30,660 --> 00:15:27,100
are involved in that process you know we

350
00:15:32,550 --> 00:15:30,670
don't find necessarily the same systems

351
00:15:34,800 --> 00:15:32,560
it would be in some of the iron reducers

352
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which go in the other direction

353
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so you know whether this is some kind of

354
00:15:43,769 --> 00:15:40,589
a shuttle system where there's a soluble

355
00:15:46,889 --> 00:15:43,779
cytochrome that does that transfer or

356
00:15:48,300 --> 00:15:46,899
whether there's actually a he or chain

357
00:15:50,370 --> 00:15:48,310
of heenes or something like that but

358
00:15:52,889 --> 00:15:50,380
after the wire these organs have some

359
00:15:55,139 --> 00:15:52,899
fairly large gene groups in them but not

360
00:16:00,389 --> 00:15:55,149
as not as large as for example geo

361
00:16:03,269 --> 00:16:00,399
bacter we don't tend to find cytochromes

362
00:16:07,230 --> 00:16:03,279
that have say 30 or 40 your genes in

363
00:16:08,850 --> 00:16:07,240
them or cytokines and groups in them but

364
00:16:11,189 --> 00:16:08,860
they some of them have 10 to 20 but

365
00:16:12,900 --> 00:16:11,199
we're still that's we're trying to sort

366
00:16:14,340 --> 00:16:12,910
that out one thing I would say is I

367
00:16:16,439 --> 00:16:14,350
don't think there's any Universal

368
00:16:18,059 --> 00:16:16,449
mechanism here I think you know each

369
00:16:19,620 --> 00:16:18,069
system that each organism

370
00:16:20,550 --> 00:16:19,630
maybe not each organism but I think

371
00:16:24,329 --> 00:16:20,560
there's going to be quite a bit of

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modularity in this system thank you okay