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[Music]

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so I'll be taking a step back from

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multicellularity but not all the way

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back to prebiotic chemistry so right in

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the middle are all of us microbiology is

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trying to understand the diversity in

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this tiny cellular organisms and

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originally I guess I got involved in

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microbiology or interested out of

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curiosity but in the context of

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astrobiology understanding the whole

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diversity of how single cells can make a

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living is important to understand or or

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choose proper targets for exploration

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beyond ocean worlds

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I also interpret the findings from the

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data that we get back from our

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explorations and perhaps also in the

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future once we if we understand how the

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breadth of these organism on niches we

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can prevent an planet cross

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contamination by preventing organisms

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from one body going into another so in

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order to do these we need to understand

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what is what is the life on how

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microorganisms make a living essentially

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this is something that already explained

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but if you want something to if you're

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looking for something that is alive that

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grows reproduces and undergoes evolution

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that organism needs to be able to do one

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fundamental thing which is obtain energy

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and the difference with us that we

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depend on oxygen microbes prokaryotes

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can employ a wide variety of substrate

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to get energy and what that that shows

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the redox hour shows is that as long as

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microorganisms can figure out the

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biochemical strategy that allows them to

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take something to transfer electrons and

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harness the energy from the transfer of

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electrons from something that has a low

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redox potential to a higher one and

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transform that into an 18-2 ATP then

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then can they can use the energy to

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perform a wide variety of functions and

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survive and on earth we have

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huge number of Nicias tiny and big and

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all sorts of microorganisms employing

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different strategies and we had our task

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as microbiologist in a way is to

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understand each one of them and see the

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potential for each one of them even

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though if on earth they are in very

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small niches they could be predominant

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in other planets but we don't not only

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have this huge planet to explore life we

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have four other Earth's to explore

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respiratory diversity perhaps not in

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this geographical space dimension but in

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time and as Marcus already showed the

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earth our earth went through different

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transition periods over time that

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depending on on oxygen levels driven in

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turn by microorganisms and in each one

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of these scenarios the earth had

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completely different environments that

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probably select four different types of

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organisms at the beginning where there

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was no oxygen the atmospheric

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composition was also very different

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probably full of methane or hydrogen and

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so different microorganisms that perhaps

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are not very abundant now probably were

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very abundant at the beginning in the

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future so accordion worlds and also the

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oxygen that was drove different other

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electron acceptor levels such as iron

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and manganese and other substrates that

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microorganisms could incorporate for

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life and to develop different strategies

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to breed and generate energy and I am

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interested in particularly in iron and

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manganese and we know the broad trend of

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iron coming down from millimolar micro

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molar levels to nano molars in which

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nowadays iron becomes even limiting for

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life manganese is also probably

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following a similar trend and both can

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be used by bacteria to harvest energy

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and nowadays in there are very small

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pockets of the earth where these ancient

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conditions prevail and we call them some

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of them have very similar

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characteristics let's such as high iron

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no oxygen and we call these ancient of

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like the one picture that you see there

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so our strategy if you want to look at

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potential primitive mechanisms for life

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not only now but also in the past is to

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explore this ancient ocean analogues and

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search for the metabolisms that we that

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were interested in so what that's what

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we did we trying to understand the

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different forms of manganese cycling in

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the environment we took samples from

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Lake Madonna which is the original high

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iron very low carbon ancient ocean

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analog that has a manganese cycle as you

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can see there it's likely driven by

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microbes that oxidized and reduced

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manganese which ends up precipitating

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but geochemists have looked at this at

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this lake for a long time but and so

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it's very well characterized in terms of

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readouts it's we know that it's

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permanently anoxic and an iron manganese

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are our big electron donors acceptors

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but we don't know much about who is

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doing who's doing what and who is

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responsible for the manganese cycling so

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the strategy that we employed was to

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take sediments from this lake cultivated

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under three goe conditions a lot of iron

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very low oxygen with the selection

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pressure of adding only manganese as the

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electron acceptor and over time we hope

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that we would I say late organisms

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involved in manganese cycle we would

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have pure beautiful cultures cells and

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we would transfer them over time to get

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rid of all the settlements and and other

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components to obtain microbes that only

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use manganese so what what we saw after

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several transfers over around about a

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year is that I wish either this and this

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essentially shows the community

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structure who is there and what we use

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is the 16s the ribosome RNA genes as a

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phylogenetic marker to extract the whole

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the composition of the whole community

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and see who is there and the most

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important point of this figure is to

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show you that

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Murdo cyclists and we'll call the yes

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these two green and blue bars that you

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can see after a few transfers but not on

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the original inoculum were appeared and

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they are both from the same group of

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bacteria called beta Proteobacteria so

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we wanted to trying to understand why

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those were enriched in our conditions so

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we wanted to dig deeper into what were

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they doing there and and who they

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actually were so we took the whole comes

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the whole set of complete sample of DNA

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and proteins together and we did

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metagenomics and meta proteomics what

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this means is that on one hand we took

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the DNA the DNA is sequenced into very

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small small fragments are sequenced and

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then by informatica ly they're put

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together into bigger chunks eventually

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maybe making a complete genome if

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depending on the on your sample on the

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other hand we took the complete set of

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proteins also little peptides were

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passed through LC ms/ms and that pattern

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that is generated was compared back to a

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theoretical pattern from the translated

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nucleotides from the DNA and that way

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you can see which proteins are expressed

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and we did that with the help of

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brooklyn doctrine and from the

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university of washington so what we we

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saw that these declare amana that we saw

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were and reached also we could we were

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able to get the whole genome from the

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community and we compared it with known

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the terminus species we confirmed that

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this belongs to that genus and we

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compare the genomes their identity of

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the genomes to the two more to the one

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most closely related with the most

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likely closest relative and we found

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that they were 85 percent identical so

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to assign two different organisms into

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the same species you need at least 95

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percent so with 85 percent we concluded

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that this was a new genomics and genomic

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species we that was enriching our

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culture but we

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are still we weren't able to isolate it

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yet so we're calling it Candida to the

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terminus of Kolkata because it's still

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hidden in our samples it was hidden

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originally in Lake maternal and but but

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what we did but mentor proteomic reveal

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does I was that it was responsible for

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seventy-three percent of all the

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peptides that we found which means that

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even though we didn't really thought

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that this was a the most predominant or

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dominant member of the community based

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on that one genetic marker it was the

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most active and the rest of the DNA was

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probably from dead cells or or by us

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from the PCR or the method itself that

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bias is is bias depending on how many

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copies of that 16s RNA gene you have so

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then we I'm not going to go into a

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detail so don't worry but we were

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curious to see what attributes what

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forms that what lifestyle this bacteria

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has and we were here is about two things

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one their ability to transform metals

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and another thing that colder attention

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was a presence of a denitrification

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pathway that was active even though we

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had hadn't added any substrate to

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express this pathway but that's another

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story but back to the multi the multi

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humid I said these type cytochromes this

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is probably a case of structure

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determines function that just heard so

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the one thing that we rarely pay

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attention to

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besides the redox potential of different

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substrates that organisms use to make

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energy one has to consider the

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availability how the organism sees it

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and how available it is to them and even

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though manganese and iron are pretty

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good electron acceptors closer to oxygen

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than sulfate and sometimes nitrate they

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are they depend on depending on the

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redox state of the metal they're going

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to be found in solid or soluble form so

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when organisms

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reduce it or oxidize it they're going to

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either find it in a in an oxide form so

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it means that they cannot get it inside

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the cell like we do with oxygen or

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nitrate

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some other organisms or they're going to

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produce an outside which means they're

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going to that is going to precipitate

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and organisms probably do not benefit

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from precipitating things inside their

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cells so in both cases whether an

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organism's organism is oxidizing or

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reducing somehow very interestingly they

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use the same type of machinery in a way

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biochemical machinery they use this

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electron conduit that is made of

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cytochromes

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in the case of metal reducers it's

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called MTR no reduction and in the case

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of mine of metal oxidizers it's called

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MTO or Pio depending on whether the

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organism is phototrophic or not so we

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saw for the first time in this type of

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species that this organism had this gene

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this conduit that all these genes were

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together in one operon and we couldn't

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find it in any other declare Moniz as

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you can see from this graph these are

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two copies if you put them in the

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contact of a tree with closer organisms

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that also have this dis conduit which is

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important because if we had just tried

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to look at the AG on its own in the math

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the complexity of the community we

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wouldn't have known it belonged to the

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determiners with the reason we know it's

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a declare - units because we mapped it

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back to the metagenomic beam that we

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obtained and this this gene and the

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architecture of this operon is also

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present in two other beta parameters

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that are uncultured so this whole branch

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here are uncultured organisms that have

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this same operon also these this conduit

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is not present in the only other declare

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ramona's that are is unknown iron metal

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oxidizer under anoxic conditions so this

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is pretty unique and new - - these novel

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species another correlated somehow

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correlated operon that was that we saw

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is this cytochrome C rich opera as you

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probably saw from other talks the

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cytochromes are really good heme groups

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are really good at transferring

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electrons so whenever we see multi heme

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cytochromes in an organism we

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typically can very confidently say that

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they're doing some sort of election

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transfer outside of the South especially

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when there are Decca hims ten teams or

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more than ten so again we saw this under

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kitchen that was present only in these

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species this new species but not there

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well at the other determiners relatives

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again present in uncultured beta

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Protectorate rota cyclase Angelina

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Nadia's which means that they must be

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doing something that we haven't figured

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out yet many as some of these that are

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cultured and that have this operon have

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also been shown to be involved in metal

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cycling and we were also surprised to

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see this is just three examples of this

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whole genome nitrous oxide reductase is

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which means that is part of the DNA

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traffic Asian pathway it's not really

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important to the story but what is

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important is that these genome this

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strain as opposed to the other ones was

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highly enriched in sensors including in

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this nitrous oxide reductase operon

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which was pretty unique compared to the

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others which means it may use the one

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thing that I did not mention is that the

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reversibility of this conduit has been

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seen in many organisms for example metal

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reducers when you can reduce also

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electrodes if you instead of a metal you

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have an electrode but depending on the

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redox potential of the electron you can

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make the electrons go back so and many

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organisms change their metabolism

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depending on the redox potential of

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their environment so the high

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concentration of redox sensors in in

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metal reduction pathways coupled to

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nitrogen it could be an indication of

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the highly versatile flexible metabolism

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of these beta Porter bacterias in these

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unique environments so I'll just wrap it

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up by saying a couple of conclusions

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from from this short story is that doing

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this enrichment and putting the

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selection pressure allowed us to magnify

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the existence of this novel conduit

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which we will have seen by targeting the

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whole

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in situ even because the concentration

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of this population was very small the

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abundance at the same time because we

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enrich we were able to capture a genome

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by genomic meaning and that constraints

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how who we assigned this dysfunction to

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if we had just looked at a bunch of

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genes from the from the public from the

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community in situ we would have probably

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mapped it to one of different completely

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different species but now here we have

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it in the concept context of a whole

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organism and that's what we're looking

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for we're trying to understand the

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evolution of organisms big and this

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probably is the product of lateral gene

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00:16:10,160 --> 00:16:07,380
transfer but we were able to map it

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straight on to these one species and

365
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also thanks to my proteomics and meta

366
00:16:19,940 --> 00:16:17,430
meta genomics we can see that this whole

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opera not just one particular gene but

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different operands we could see them

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again expressed on different samples

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00:16:27,890 --> 00:16:26,250
from different locations so now even

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though we were not able to isolate one

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species we can collect all this

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information and perhaps that will guide

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us to further efforts in isolation or

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understanding what can they be doing as

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opposed to having one single cell in the

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00:16:45,740 --> 00:16:42,990
lab and I'm still working on trying to

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00:16:47,710 --> 00:16:45,750
isolate this beta protect area too

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00:16:50,090 --> 00:16:47,720
because we do need to characterize

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00:16:54,140 --> 00:16:50,100
biochemically everything that we see

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from our meta proteome original and I

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also would like to thank the full

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astrobiology community it's very

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00:17:04,430 --> 00:17:00,330
inspiring to be here and listening to

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00:17:06,380 --> 00:17:04,440
all it talks and Jen glasses lab but

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00:17:07,670 --> 00:17:06,390
also other mentors that I've been having

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00:17:10,819 --> 00:17:07,680
for the past two years like Chris